The Role of Nitric Oxide in Ischaemia Reperfusion Injury in the Rabbit Heart.
Thesis submitted by
Vanlata Chhotabhai Patel
For the degree of
Doctor of Philosophy
in the
Faculty of Physiology
University of London
The Hatter Institute
Department of Academic and Clinical Cardiology
University College London Medical School
London WCIE 6D
May 1996 ProQuest Number: 10042811
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ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 I like to dedicate this thesis with all my love to my Mum, Hitesh, Aaran and all my family. (ii)
Abstract
The Role of Nitric Oxide in Ischaemia Reperfusion Injury in the Rabbit Heart.
Background; Nitric oxide (NO), synthesised by a constitutive enzyme from L-arginine, is a highly reactive species which is produced continuously by endothelial cells. NO maintains the coronary microcirculation in a state of active vasodilatation, prevents platelet and leucocyte adherence, and preserves physiological vascular impermeability, however, in excess NO may be injurious. This may be important during the early reactive hyperaemia after ischaemia, which is mediated by a substantial increase in NO release.
Brief periods of ischaemia followed by reperfusion leads to an increased tolerance of the myocardium to a subsequent and sustained ischaemic insult, this phenomenon is termed ischaemic preconditioning (IP). The mechanism for IP in not clear, however, recent evidence appears to support an important primary role for adenosine released by ischaemic cells during the preconditioning period.
Aims: This thesis examines some aspects of the role of nitric oxide in the myocardium and during ischaemia, reperfusion and its involvement in ischaemic preconditioning.
Method: The experimental model studied was regional ischaemia followed by reperfusion both in the in situ and Langendorff perfused rabbit hearts. Infarct size was the endpoint assessed but haemodynamic parameters and several biochemical determinants of injury were also measured. The role of NO in ischaemia reperfusion injury was studied by the inhibition of L-arginine analogue which inhibits NO synthesis, Nitro-L-arginine methyl ester hydrochloride (L-NAME) was used.
Results : Rabbit hearts pretreated with L-NAME were more resistant to injury than controls both in situ and isolated perfused hearts. When hearts were treated with both L-NAME and 8-p-sulfophenyl theophylline an adenosine receptor antagonist (SPT) the protection seen with L-NAME was abolished. Ischaemic preconditioning (IP) prior to sustained ischaemia and reperfusion reduced the infarct ratio both in situ and isolated hearts, this was not augmented by pretreatment with L-NAME. All protection due to IP was lost in hearts pretreated with SPT. We postulate that the protective effects of L-NAME is mediated largely by the release of adenosine.
Conclusion: We have demonstrated that the infarct size limitation following inhibition of NO synthesis shares a common mechanism with that of IP and is dependent on the release of adenosine. These results have implications for the mechanisms of coronary autoregulation during ischaemia and reperfusion injury, when the physiological mechanisms are deranged. (iii)
Abstract...... (ii) Table of Contents...... (iii) List of Tables...... (xiii) List of Figures ...... (xiv) Abbreviations...... (xvii) Acknowledgements ...... (xx)
Table of Contents
Chapter 1: Introduction
1.0 Cardiovascular System and its Control 1.0 Introduction ...... 1 1.0.1 The H eart...... 1 1.0.1.1 Heart Beat Coordination ...... 1 1.0.1.2 Sequence of Excitation ...... 2 1.0.1.3 Control of Heart Rate ...... 2 1.0.2 The Vessels...... 2 1.0.2.1 Intrinsic and Extrinsic Control of Circulation ...... 2 1.0.3 Control of Systemic Arterial Pressure ...... 3 1.0.3.1 Afferent Pathway ...... 3 1.0.3.2 Cardiovascular Control Centre ...... 3
1.1 Myocardial Ischaemia and Reperfusion 1.1 Introduction ...... 4 1.2 Pathophysiology of Myocardial Ischaemia ...... 5 1.2.1 Ultra Structural Change s ...... 5 1.2.2 The Spread of Infarction ...... 5 1.3 Biology of the Ischaemic Myocardium ...... 6 1.3.1 Metabolic Changes During Ischaemia ...... 6 1.3.2 Ionic Changes During Ischaemia ...... 7 1.3.3 Biochemical and Ionic Changes During Ischaemia ...... 8 1.4 Pathophysiology of Myocardial Reperfusion ...... 9 1.5 Metabolic Changes During Reperfusion ...... 10 1.5.1 The Calcium and Oxygen Paradoxes ...... 11 1.6 Free Radical Generation ...... 12 1.6.1 Free Radical Toxicity ...... 13 1.6.2 Possible Role of Nitric Oxide in Ischaemia Reperfusion 15 1.6.3 Toxic Role of Free Radicals Generated ...... 15 (iv)
From Nitric Oxide (Peroxynitrite) 1.7 The Causal Mechanism Determining Cell Death During Reperfusion 17 1.7.1 Reperfusion Hyperaemia ...... 17 1.7.2 Reperfusion Arrhythmias ...... 18 1.8 Ultrastructural Changes ...... 18 1.8.1 No-Reflow Phenomenon ...... 18 1.8.2 Reperfusion-Induced Haemorrhage...... 19 1.9 Reperfusion of the Vascular Bed ...... 19 1.9.1 Vascular and Microvascular Injury Impairing Reflow ...... 20 1.9.2 Leucocyte Accumulation During Reperfusion ...... 20 1.9.3 Neutrophil Recruitment to Sites of Tissue Injury ...... 22 1.10 Discussion ...... 23 1.11 Conclusion ...... 23
2.0 Ischaemia, Reperfusion and the Model Used for both the In Vivo and In Vitro Experiments
2.1 Introduction to Collateral Circulation in Species ...... 24 2.2 Ischaemia and Reperfusion in the Animal Models ...... 24 2.2.1 The Definition of Ischaemia ...... 26 2.2.2 Induction of Myocardial Ischaemia ...... 26 2.2.3 Region at Risk ...... 27 2.3 Assessment of Infarct and Risk Size ...... 27 2.3.1 Introduction ...... 27 2.3.2 Mechanism for Staining ...... 28 2.3.3 Advantages of TTC Staining ...... 29 2.3.4 Analysis of Infarct and Risk Areas ...... 29 2.4 Discussion ...... 29 2.5 Conclusion ...... 31
Section 2.0: General Biology of Vascular Endothelium
1.0 Introduction ...... 32 1.1 Histology ...... 32 1.3 Control of Vascular Tone ...... 33 2.0 Control of Vascular Tone by Endothelium-Derived Relaxing Factor ...... 33 2.1 Biosynthesis of Nitric Oxide ...... 34 2.2 Biochemistry of Nitric Oxide ...... 35 2.2.1 Types of Nitric Oxide Synthase ...... 37 2.2.2 Other Roles for L-Arginine ...... 37 2.2.3 Effector Pathway for Nitric Oxide ...... 38 (v)
2.2.4 Deactivation and Inhibition of Nitric Oxide Under Both Physiological and Experimental Conditions ...... 38 2.2.4.1 Haemoglobin ...... 39 2.2.4.2 Superoxide ...... 39 2.2.4.3 Methylene blue ...... 39 2.2.4.4 Reducing Agents ...... 40 2.2.5 Analogues of L-arginine (Inhibition of NO Synthesis) ...... 40 2.3 The Constitutive Enzyme ...... 41 2.3.1 Physical Mediators of the Constitutive Enzyme for Nitric Oxide ...... 41 2.3.1.1 Shear Stress...... 42 2.3.1.2 Local Mediators ...... 42 2.3.1.3 Hormonal regulation ...... 42 2.3.1.4 Products of Coagulation and Platelet Products 43 2.3.1.5 Neurotransmitters ...... 43 2.4. Effects of Nitric Oxide on the Vascular Tone ...... 44 2.4.2 Assessing Nitric Oxide-Related Dilation ...... 45 2.4.3 Nitric Oxide Synthase in the Endocardial Lining ...... 45 2.4.3.1 The Physiological Roles of Endocardial Endothelium ...... 46 2.4.4 Inhibition of Platelet Adhesion ...... 46 2.4.5 Regulation of Leukocyte Interaction with Vessel Wall ...... 47 2.4.6 Mitogenesis and Proliferation of Vascular Smooth Muscle Cells...... 47 2.5 The Inducible Nitric Oxide Synthase ...... 47 2.5.1 Inducible Synthase in the Macrophages, Neutrophils and Lymphocytes ...... 48 2.5.2 The Formation of NO by the Inducible Synthase in Tissues and Cells...... 49 2.5.3 Inhibition of the Inducible NO Synthase ...... 50 2.6 Response to Over Stimulus of NO by Inducible NOS ...... 50 2.6.1 Gross Vasodilation ...... 50 2.6.2 Peroxynitrite Radical ...... 51 2.6.3 Cellular Toxicity ...... 51 2.6.3.1 Nitric Oxide Synthase in Septic Shock ...... 52 2.6.3.2 Antimicrobial Actions ...... 53 2.7.1 The Role of Nitric Oxide in Autoimmunity and Inflammation ...... 53 3.0 Control of Vascular Tone by Other Vasodilators and Constrictors ...... 54 3.1 Purines (Adenosine) ...... 54 3.1.1 Adenosine Receptors ...... 54 (vi)
3.1.2 Effector Pathway ...... 55 3.1.3 Physiology of Adenosine ...... 55 3.1.4 Adenosine During Early Ischaemia ...... 55 4.0 Prostacyclin ...... 56 4.1 Endothelium-Derived Hyperpolarizing Factor ...... 57 5.0 Abnormal Endothelial Function in Disease ...... 57 5.1 Ischaemic and Reperfusion Injury ...... 57 5.2 Atherosclerosis ...... 58 5.3 Hypertension ...... 58 5.4 Clinical Implications ...... 59 6.0 Conclusion ...... 59
Section 3.0: Endogenous Myocardial Protection
Section 3.0: Endogenous Mechanisms and Mediators of Myocardial Protection 60 1.0 Ischaemic Preconditioning ...... 60 1.1 Introduction ...... 60 1.2 Protocol Used for Triggering Ischaemic Preconditioning ...... 61 1.3 Other End Points ...... 62 1.3.1 Stunning ...... 62 1.3.2 Arrhythmias ...... 63 1.4 Mechanisms of Ischaemic Preconditioning ...... 63 1.4.1 Adenosine Antagonist and Agonist ...... 63 1.4.2 The Role of A1 Adenosine Receptor Activation ...... 64 1.4.2.1 Adenosine Ai Analogs ...... 64 1.5 A Possible Role for the Adenosine A3 Receptors ...... 66 1.6 Preconditioning can be Mimicked by Other Agents ...... 66 1.6.1 Noradrenaline ...... 66 1.6.2 Bradykinin ...... 67 1.6.3 Endothelium Derived Relaxing Factor (EDRF) ...... 68 1.7 Intracellular Signalling Pathway ...... 68 1.7.1 G -Proteins ...... 68 1.7.2 The role of Protein Kinase C ...... 69 1.7.2.1 Pharmacological Activation and Inactivation of PKC...... 69 1.7.2.2 Protein Kinase C Translocation ...... 70 1.8 ATP Sensitive Potassium Channel ...... 71 1.8.1 Experimental Evidence Showing that K atp is Associated with Cardioprotection ...... 72 1.8.2 Mechanism of Protection by Activation of K atp ...... 73 1.9 Conclusion ...... 73 (vii)
2.0 The Delayed Myocardial Protection ...... 74 2.1 Delayed Protection ...... 74 2.1.1 Second Window of Protection ...... 74 2.2 Heat Shock Proteins ...... 74 2.2.1 HSP families...... 75 2.2.2 Normal Function of HSPs Under Physiological Conditions ...... 75 2.2.3 Stresses Capable of Inducing HSP Synthesis ...... 76 2.2.4 Mechanism of HSP ...... 76 2.2.5 Function of Increased HSP Expression Following S tress...... 77 2.2.6 The Protection of HSPs in the Hearts ...... 77 2.3 Anti-Oxident Proteins ...... 79 2.4 Conclusion ...... 79
3.0 Endogenous Protection: A Double Edged Role of Nitric Oxide ...... 81 3.1 Toxicity of Nitric Oxide ...... 81 3.1.1 Nitric Oxide is a Free Radical...... 81 3.1.2 Impair Mitochondrial Function ...... 82 3.1.3 Experimental Evidence for the Toxic Role of Nitric Oxide ...... 82 3.1.3.1 During Ischaemia and Reperfusion ...... 82 3.1.3.2 Treatment with L-arginine ...... 83 3.1.3.3 Nitric Oxide Donors ...... 84 3.1.4 Experimental Evidence Showing Protection with Nitric O xide...... 84 3.1.4.1 Exogenous Nitric Oxide or L-arginine ...... 85 3.2 Conclusion ...... 86
Section 4.0: The Objectives of this Research Were ...... 87
Chapter 2: Experimental Models and Methods
Section 1.0 and 2.0: In Vivo and In Vitro Models
1.0...... Species U sed ...... 89 2.0...... Anaesthesia...... 89 2.1 Introduction to Anaesthesia Used ...... 89 2.1.1 Administration of Anaesthetic ...... 89 (viii)
2.1.2 Assessing and Maintaining Anaesthesia ...... 90
3.0 General Surgical Preparation ...... 90 3.1 Calibration of Transducers and Amplifiers ...... 90 3.2 Tracheostomy ...... 91 3.3 Thoracotom y ...... 91 3.4 Ligation of the Left Circumflex Artery ...... 92 3.4.1 Induction of Ischaemia and Reperfusion ...... 92 3.5 Temperature Control ...... 93 3.6 Haemodynamic Monitoring ...... 93 3.7 Assessment of Infarct and Risk Size ...... 93 3.7.1 Risk Area: Staining with Fluorescent Dye ...... 93 3.7.2 Infarct Area ...... 94 3.7.3 Sectioning ...... 94 3.7.4 Analysing Infarct and Risk Areas ...... 98
4.0 The Isolated Langendorff Perfused Heart ...... 98 4.1 The Principles of Retrograde Perfusion ...... 98 4.2 The Perfusion Medium ...... 101 4.3 Surgical Preparation ...... 101 4.4 Preparation of the Langendorff perfused heart ...... 101 4.4.1 Electrical Stimulation of the Heart ...... 102 4.4.2 Temperature Control ...... 102 4.4.3 Flow R ate ...... 103 4.4.4 Left Ventricular Developed Pressure (LVDP) ...... 103 4.4.5 Coronary Perfusion Pressure (CPP) ...... 104 4.4.6 Reduction of Tissue Oedema ...... 104 4.4.7 Measurement of Oxygen, Carbon Dioxide and pH ...... 104 4.4.8 Arrhythmias ...... 105 4.4.9 Ischaemia and Reperfusion on the Isolated Hearts ...... 105
Section 3.0: Biochemical Assays
3.1 Experimental Procedures ...... 106 3.2 Deproteinisation of Tissue Samples ...... 106 3.3 Adenosine Triphosphate Determination (ATP) ...... 107 3.3.1 Experimental Procedures For ATP Assay ...... 107 3.4 Tissue Creatine Phosphate Determination (CP) ...... 108 3.4.1 Experimental Procedures For CP Assay ...... 108 3.5 Tissue Lactate Determination ...... 109 3.5.1 Experimental Procedures For Lactate Assay ...... 109 (ix)
3.6 Adenosine Measurement in Coronary Effluent ...... 110 3.6.1 The Principles of High Performance Liquid Chromatography ...... 110 3.6.2 Mobile Phase Preparation ...... 110 3.7 Sample Collection For the Adenosine Assay ...... 110 3.8 Measurements of adenosine concentration ...... I l l
Section 4.0: Isolated Mitochondrial Function
4.0 Measurement of Mitochondria Function ...... 112 4.1 Purity and Functional Characterisation of Intact Mitochondria ...... 112 4.2 General Method For the Isolation of Mitochondria ...... 112 4.3 The Oxygen Electrode ...... 113 4.4 Surgical Preparation ...... 113 4.4.1 Isolation of Mitochondria From Rabbit Heart ...... 115 4.4.2 Mitochondrial Protein Determination ...... 117 4.4.3 Polarographic Determination of Mitochondrial Respiration... .117 4.4.4 Assessment of Mitochondrial ...... 118 5.0 Statistical Analysis of Data Analysis...... 120
Chapter 3: Effects of inhibition of nitric oxide in both the in vivo and in vitro hearts
1.0 Introduction ...... 121 1.2 A im s...... 121 2.0 Experimental Methods ...... 122 2.1 Exclusion Criteria ...... 124 3.0 Specific Methods for the In Vitro Model ...... 125 3.1 Exclusions ...... 125 3.2 In Vitro Experimental Groups ...... 126 4.0 In Vivo R esult ...... 126 4.1 Infarct and Risk Determination ...... 126 4.2 Haemodynamic Data ...... 130 5.0 In Vitro Results ...... 135 5.1 Infarct and Risk Determination ...... 135 5.2 Haemodynamic Data ...... 135 6.0 Discussion ...... 141 7.0 Conclusion ...... 144 (x)
Chapter 4: Biochemical Parameters Measured In Vivo and In Vitro
1.0 Introduction ...... 146 1.2 A im s...... 146 2.0 Experimental Model for the In Vivo Study ...... 146 3.0 Specific Experimental Models and Aims for the In Vitro Study ...... 147 3.1 Experimental Protocol ...... 147 4.0 In Vivo Result ...... 148 4.1 Lactate...... 148 4.2 ATP and CP...... 152 4.3 Haemodynamic Data ...... 152 5.0 In Vitro Results ...... 152 5.1 Results for Adenosine Release ...... 152 5.2 Results for Lactate Concentration ...... 153 5.3 Haemodynamic Data ...... 153 6.0 Discussion ...... 160 7.0 Conclusion ...... 170
Chapter 5: Investigating the Role of Adenosine Release Following Treatment With L-NAME in Proctection of the In Vitro Hearts
5.0 A im s...... 163 5.1 Specific Experimental Model ...... 163 5.1.1 Exclusions ...... 164 5.1.2 Experimental Groups ...... 164 5.2 Results...... 165 5.2.1 Infarct and Risk Volume Determination ...... 165 5.2.2 Haemodynamic Data ...... 169 5.3 D iscussion ...... 173 5.4 Conclusion ...... 174
Chapter 6: Ischaemic Preconditioning in the In Vivo and In Vitro Rabbit Heart. And Does Nitric Oxide Play a Role?
6.0 Introduction ...... 175 6.1 A im ...... 176 (xi)
6.2 Experimental Model for the In Vivo Study ...... 176 6.2.1 Exclusions ...... 176 6.2.2 Ischaemic Preconditioning Protocol ...... 177 6.2.3 Experimental Groups ...... 179 6.3 Experimental Model for the In Vitro study ...... 179 6.3.1 Exclusions ...... 180 6.3.2 Experimental Groups ...... 180 6.4 In Vivo Result ...... 181 6.4.1 Infarct and Risk Volume Determination ...... 181 6.4.2 Haemodynamic Data ...... 181 6.5 In Vitro Results ...... 184 6.5.1 Infarct and Risk Volume Determination ...... 184 6.5.2 Haemodynamic Measurements ...... 184 6.6 Discussion ...... 187 6.7 Conclusion ...... 188
Chapter 7: Perfusion of Hearts With Adenosine Prior to Ischaemia and Reperfusion, Does This Confer Protection to the Myocardium
7.0 Introduction ...... 189 7.1 Aim s ...... 190 7.1.1 Experimental Protocol for Different CPP Models ...... 191 7.1.2 Exclusions ...... 191 7.1.3 Experimental Groups ...... 192 7.2 Results ...... 193 7.2.1 Infarct and Risk Volume ...... 193 7.2.2 Coronary Perfusion Pressure(CPP) ...... 194 7.2.3 Left Ventricular Developed Pressures ...... 202 7.2.4 Adenosine Concentration in the Coronary Effluent ...... 205 7.3 Discussion ...... 207 7.4 Adenosine Dose Response Study ...... 207 7.4.1 Exclusions ...... 208 7.4.2 Experimental Protocol ...... 208 7.5 Results ...... 208 7.5.1 Infarct and Risk Volume ...... 208 7.5.2 Haemodynamic Data ...... 209 7.5.3 Coronary Perfusion Pressure ...... 209 7.5.4 Left Ventricular Pressure ...... 209 7.6 Discussion ...... 213 (xii)
7.7 Conclusion ...... 214
Chapter 8: Isolated Mitochondrial Function
8.0 Introduction (Mitochondrial Function) ...... 215 8.1 A im ...... 215 8.1.1 Experimental Protocol ...... 216 8.1.2 Exclusions ...... 216 8.1.3 Experimental Groups ...... 217 8.2 Results ...... 220 8.2.1 Effects of Extraction of Mitochondria From Ischaemic and Reperfused Hearts ...... 224 8.2.2.Haemodynamic.Results ...... 226 8.3 D iscussion ...... 226 8.3.1 Effects of ischaemia, reperfusion and treatment with L-NAME on mitochondrial function ...... 226 8.4 Conclusion ...... 228
Chapter 9: Final Summary and Future Work 9.0 Summary of Results ...... 229 9.1 The Design of Future Studies ...... 231
Chapter 10 References...... 232 (xiii)
List of Tables
Table 3T Infarct size and haemodynamic data for the in vivo hearts ...... 127 Table 3.2 Infarct size and haemodynamic data from the isolated perfused rabbit h eart...... 136 Table 3.3 Measurement of blood gases for the in vivo rabbit h earts...... 134 Table 4.1 Parameters measured for the in vivo lactate study ...... 151 Table 4.2 Lactate, ATP and CP measurements for the in vivo hearts ...... 154 Table 4.3 Parameters measured for the in vitro rabbit hearts ...... 159 Table 5.1 Infarct and haemodynamic data for the isolated perfused rabbit hearts 167 Table 6.1 Infarct and haemodynamic data from the in vivo rabbit hearts ...... 183 Table 6.2 Infarct and haemodynamic data from the in vitro rabbit hearts ...... 185 Table 7.1 Data for the isolated perfused rabbit hearts. Hearts perfused with normal or highcoronary perfusion pressures ...... 195 Table 7.2 Infarct and haemodynamic data for isolated perfused rabbit hearts with different concentrations of adenosine ...... 211 Table 8.1 Data from the isolated perfused rabbit heart models for the determination of mitochondrial function ...... 221 Table 8.2 Mitochondrial respiration in mitochondria isolated from fleshly excised hearts, ischaemic or reperfused hearts ...... 225 (xiv)
List of Figures
Figure 1.0 Basic reflex for the regulation of arterial blood pressure ...... 3(i) Figure 2.1 Determination of ischaemic and risk zones ...... 96 Figure 2.2 Schematic diagram showing how the infarct and risk areas are analysed ...... 97 Figure 2.3 The Langendorff apparatus ...... 99 Figure 2.4 Heart perfused on a Langendorff apparatus ...... 100 Figure 2.5 Diagram of the Clarke oxygen electrode ...... 114 Figure 2.6 Isolation of mitochondria from the myocardium ...... 116 Figure 2.7 A typical oxygen electrode tracing and how indices can be directly calculated from the trace ...... 119 Figure 3.1 Experimental protocol for sustained regional ischaemia and reperfusion in the in vivo rabbit heart ...... 123 Figure 3.2 Percentage infarct size for the in vivo hearts ...... 128 Figure 3.3 Determination of infarct and risk volume in the in vivo hearts ...... 129 Figure 3.4-Physiological trace for the in vivo rabbit heart ...... 131 Figure 3.5 Blood pressure trace for the in vivo rabbit hearts ...... 132 Figure 3.6 Mean heart rate trace in the invivo model ...... 133 Figure 3.7 Infarct and risk results for the isolated Langendorff perfused rabbit hearts...... 137 Figure 3.8 Coronary perfusion pressure recorded at time points for the in vitro rabbit hearts...... 138 Figure 3.9 Left ventricular developed pressure trace for the Langendorff perfused rabbit hearts ...... 139 Figure 3.10 Left diastotlic pressure trace for the Langendorff perfused rabbit hearts...... 140 Figure 4.1 Experimental protocol for the isolated rabbit hearts. Used for the determination of adenosine and tissue lactate concentration ...... 149 Figure 4.2 Myocardial lactate concentration for the in vivo hearts ...... 150 Figure 4.3 Blood pressure graph ...... 155 (xv)
Figure 4.4 Adenosine standard curve ...... 156 Figure 4.5 Adenosine concentration ...... 157 Figure 4.6 Lactate measurements from the in vitro hearts ...... 158 Figure 5.1 Experimental protocol for the isolated Langendorff perfused rabbit h eart...... 166 Figure 5.2 Infarct and risk data for the isolated perfused hearts treated with L-NAME and SPT...... 168 Figure 5.3 Coronary perfusion pressure recorded at time points in the in vitro experiments ...... 170 Figure 5.4 Left ventricular diastolic pressure trace for the Langendorff perfused hearts ...... 171 Figure 5.5 Left ventricular developed pressure trace for the L-NAME and SPT Langendorff perfused heart ...... 172 Figure 6.1 Ischaemic preconditioning protocol for both the in vivo and in vitro rabbit hearts ...... 178 Figure 6.2 Infarct and risk volume for the in vivo hearts ...... 182 Figure 6.3 Risk and infarct volume for the in vitro rabbit hearts ...... 186 Figure 7.1 Bar graph represents infarction as a % of the risk area for the isolated hearts perfused at normal and high coronary perfusion pressure ...... 196 Figure 7.2 Coronary perfusion pressure trace (normal) ...... 197 Figure 7.3 Isolated rabbit hearts perfused at normal coronary perfusion pressure.... 199 Figure 7.4 Coronary perfusion pressure trace (high) ...... 200 Figure 7.5 Coronary perfusion pressure for the isolated perfused rabbit hearts at high pressure ...... 201 Figure 7.6 Left Ventricular systolic pressure for isolated hearts perfused at normal coronary perfusion pressure ...... 203 Figure 7.7 Left ventricular systolic pressure for isolated hearts perfused at high coronary perfusion pressure ...... 204 Figure 7.8 Adenosine concentration measured in coronary effluent taken during both normal and high coronary perfusion pressure ...... 206 (xvi)
Figure 7.9 Isolated rabbit hearts perfused at high coronary perfusion pressure and determination for infarct size using different concentrations of adenosine 212 Figure 8.1 Global ischaemic protocol for the determination of mitochondrial function ...... 219 Figure 8.2 Typical oxygen electrode tracing of mitochondrial respiration from the experimental protocol ...... 222/223 (xvii)
Abbreviations
Ach acetylcholine ADO adenosine ADP adenosine diphospate AICA acadesine AMP adenosine monophospate ANOVA analysis of variance ATP adenosine triphosphate BP blood pressure BSC bovine serum albumin CCPA 2-chloro-N6-cyclopentyl adenosine cGMP guanosine 2’5 cyclic monophate CGRP calcitonin gene-related peptide receptors CGS21680 2[4-(carboxyethyl)phenethylamino]-5-N-ethylcarboxamine adenosine hydrochloride CP creatine phosphate CPK creatine phospokinase CPP coronary perfusion pressure DAG diacyl glycerol DOG 1,2-dioctanoyl-sn-glycerol DMA deoxyribonucleotide D-NAME No-nitro-D-arginine methyl ester D-NMMA NCj-nitro-D-arginine methyl ester ECG electrocardiogram EDNO endothelium derived nitric oxide EDRF endothelium derived relaxing factor EDTA ethylenediaminotetracetic acid ET endothelin FAD flavin adenine dinucleotide FFA free fatty acid FMN flavin mononucleotide FPLSD fischers protected least significance difference test G-6-P glucose-6-phosphate G6P-DH glucose-6-phosphate dehydrogenase GTN glyceryl trinitrate H4 biopterin pteridine tetrahydrobiopterin HbO haemoglobin LDH lactate dehydrogenase HK hexokinase HPLC high performance liquid chromatography (xviii)
HR heart rate HSF heat stress factor HSP heat stress protein IL interleukin-1 IL-2 interleukin-2 IP ischaemic preconditioning IP3 inositol 1,4,5-triphosphate I/R ischaemia and reperfusion I/R% infarct risk percentage IV intravenous K potassium
K atp ATP sensitive potassium channel KCl potassium chloride KDa kilo delton KHB krebs Henseleit buffer LAD left anterior descending coronary artery LDH lactate dehydrogenase LDL low density lipoprotein L-NAME No-nitro-L-arginine methyl ester L-NIO N-iminoethyl-L-omi thine L-NNA N G-nitro-L-argi nine LPS lipopolysacharides LVDP left ventricular developed pressure NA nitro-L-arginine NADP nicotinamide adenine dinucleotide phosphate (oxidized) NADPH nicotinamide adenine dinucleotide phosphate (reduced) NANC non-adrenergic non-clolinergic NBT nitroblue tétrazolium NK neurokinine NO nitric oxide NOS nitric oxide synthase NS non-significant OD optical density ONOO- peroxynitrite anion ONOOH peroxynitrous acid PCA perchloric acid PD N[dimethylamino ethyl] benzosulfonamide PG prostaglandin P 0 l2 prostacyclin Pi inorganic phosphate PKC protein kinase C (xix)
PMNs polymorphonuclear neutrophils R-PIA R(-) N6-(2-phenylisopropyl) adenosine RNA ribonucleic acid RPP rate pressure product SEM standard error mean SOD superoxide dismutase SPT 8p-sulfophenyl theophlline TNF tumor necrosis factor TTC triphenyl-tetrazolium chloride TTC-ve triphenyl-tetrazolium chloride negative TRIS [hydroxymethyl]amino methane uv ultraviolet VF ventricularfibrillation (xx)
Acknowledgements
I am very grateful to a number of individuals who have helped me during my research and the completion of my thesis. I am especially grateful to Professor D M Yellon together with Dr R G Woolf son for their boundless support throughout my time in the department and also while writing up my thesis. Without their close supervision and support my research would have been less productive.
I would also like to thank Dr S Page for her support throughout my research, and also like to thank Professor R Ferrari and also Dr E Pasini for inviting me to their research laboratories in Italy and making my stay most enjoyable. Lastly I would like to thank Mrs S Bush-Cavell who has been most helpful.
I would also like to thank the MRC for providing 3 years of financial support to submit a PhD.
Finally I would like to thank my husband for being very understanding and encouraging, especially as my thesis took much longer then expected. I would also like to thank my mum for all her support with my research, as well as looking after my son. It is to them that I dedicate this thesis. Chapter 1 1
Cardiovascular System and its Control
1.0 Introduction
The cardiovascular system forms a circle, so that blood is continuously being pumped out of the heart through one set of vessels (arteries) and returned to the heart via a different set (veins).
1.0.1 The Heart
The heart is a muscular organ composed primarily of cardiac muscle (myocardium). The muscles of the atria and ventricles are so arranged that when they contract they lessen the capacity of the cambers which they enclose. The contracting chambers pumps the blood to the arteries.
The myocardium is composed of layers of cardiac muscle. Whose cells are joined end to end at intercalated disks, within which are two types of membrane junctions: 1) desmosomes and 2) gap junctions. Certain areas of the heart contain specialised cardiac muscle fibers which are essential for normal excitation of the heart. They constitute a network known as the conducting system of the heart.
The heart receives a rich supply of sympathetic and parasympathetic nerve fibres, the latter contained in the vagus nerve. The postganglionic sympathetic fibers terminate upon the cells of the specialised conducting system of the heart as well as on ordinary myocardial cells of the atria and ventricles. The parasympathetic neurons also innervate the conducting system and the atrial myocardial cells but not the ventricular myocardium to any great extent.
1.0.1.1 Heart Beat Coordination
This is made possible by two factors: 1) the gap junctions and 2) the specialised Chapter 1 2 conducting system. Several areas of the conducting system of the heart possess the capacity for auto-rhythmicity and pacemaking. The sino arterial (SA) node is a mass of specialised myocardial cells embedded in the right atrial wall (Fozzard 1991).
1.0.1.2 Sequence of Excitation
From the SA node, the wave of excitation spreads throughout both atria, passing from cell to cell by way of the gap junction. In addition there are several bundles of fibers which conduct the impulse directly from the SA node, this being the atrioventricular (AV) node, located at the base of the right atrium. After leaving the AV node the impulse enters the ventricles via the bundle of His, this divides into right and left bundle branches, which in turn, branch into the Purkinje fibers, which then spread throughout much of the right and left ventricular myocardium.
1.0.1.3 Control of Heart Rate
A large number of parasympathetic and sympathetic fibers end on the SA node. Stimulation of the parasympathetic vagus nerves cause slowing of the heart. The effects of the sympathetic nerves are just the reverse, their stimulation increases the heart rate.
1.0.2 The Vessels
The vessels are made up of arteries and veins, contain varying degrees of smooth muscle and elastic tissue. The smooth muscle of the arteries acts as a low-resistance conduits conducting blood to the various organs, while the elastic component acts to maintain the blood pressure within the vessel during diastole. The veins are the vessels through which blood flows back to the heart. The force driving this venous return is the pressure difference between the peripheral veins and the right atrium.
1.0.2.1 Intrinsic and Extrinsic Control of Circulation
The circulatory system is regulated by the tone of the smooth muscle surrounding the arterioles primarily, and to a certain degree of the veins. This control of muscle tone is Chapter 1 3 achieved in two main ways:- 1) Intrinsic Control Individual organs posses completely inherent mechanisms (ie mechanisms independent of nerves or hormones) for altering their own arterior resistance. This thereby confers upon the organs the capacity to self regulate their blood flow. The regulators are, decreased O2 concentration and increased CO 2 and H+ ion concentrations.
2) Extrinsic Control Most arterioles receive a rich supply of sympathetic postganglionic nerve fibers. Which release noradenaline, this combines with alpha-adrenergic receptors on the vascular smooth muscle to cause vasoconstriction ( Vander 1987).
1.0.3 Control of Systemic Arterial Pressure 1.0.3.1 Afferent Pathway
Arterial pressure is controlled by receptors and afferent pathways which bring information into the central nervous system (see figure 1.0). The most important of these are the arterial baroreceptors, found in the carotid sinus and the aortic arch. These are nerve endings, being sensitive to stretch or distortion. Similar receptors are found in the large arteries, large veins, pulmonary vessels, and the cardiac walls.
1.0.3.2 Cardiovascular Control Centre
The cardiovascular control centre for the baroreceptor reflex is in the medulla. This centre receives input from the barorecptors. This input determines the outflow from the centre along axons which terminate upon the cell bodies and dendrites of both, the parasympathetic neurons to the heart, and the sympathetic neurons to the heart, arterioles, and veins.
The synaptic connection within the medullary cardiovascular centre are such that when the arterial baroreceptors increase their rate of discharge, the result is a decrease in sympathetic outflow to the heart, arterioles, and veins and an increase in parasympathetic outflow to the heart. A decrease in firing rate of the baroreceptors results in just the opposite pattern. Chapter 1 30)
Figure 1.0. Basic reflex for the regulation of arterial blood pressure
Cardiovascular centre in medulla Descending pathway
Efferent neurons jnrmathetics parasympathetics
Afferent neurons (Ârteriole?)Cv einsDCHeartI) (Heart]) \ / troK C Heart volum Artenal baroreceptors
Cardiac output Total peripheral I I resistance |
Artenal blood pressure (regulated variable Chapter 1 4
Myocardial Ischaemia and Reperfusion
1.1 Introduction
The aetiology of ischaemia is diverse in clinical medicine. Ischaemia can result from atherosclerosis, thromboembolism, and external pressure on vessels as a result of tumours. It can occur during surgery, when the blood flow to an organ must be interrupted, as in organ transplantation. Ischaemia, from whatever source or model, is accompanied by a lack of both oxygen and substrate, and therefore a lack of aerobic energy production. If ischaemia is prolonged it will eventually lead to cell death.
For many years cardiologists have tried to develop new therapeutic means of protecting the ischaemic myocardium. Of the various approaches tried, pharmacological manipulation has been exhaustively tested, but with little success and it is now realised that with the exception of prompt thrombolysis little can be done by drugs to salvage muscle and limit the ultimate size of the evolving infarct (Hearse, et al 1984a). Although the restoration of myocardial blood flow or 'reperfusion' may lead to tissue salvage, the early restoration of coronary blood flow has itself been associated with what has been termed ‘reperfusion injury* (Braunwald and Kloner, 1985). This concept underlies the importance of understanding the dynamic nature of ischaemia and reperfusion in order to exploit intrinsic cellular mechanisms to protect the heart from ischaemic and reperfusion damage. Chapter 1 5
1.2 Pathophysiology of Myocardial Ischaemia 1.2.1 Ultra Structural Changes
Myocardial infarction is a dynamic process in which cells make the transition from reversible injury to irreversible necrosis in a number of stages. Distinct changes in the ultrastucture of the myocytes can be seen as early as 10 seconds following coronary occlusion.
The development of ultrastructural defects in the mitochondria and the sarcolemma of the myocyte suggests irreversible injury (Jennings, 1969a, Schaper, 1984). The mitochondria swell, exhibit disorganised cristae and contain one or more amorphous matrix densities (Jennings, et al 1978). In addition, the sarcolemma exhibits interruptions that are especially prominent in regions of subsarcolemma oedema (Jennings, et al 1986). The oedema which provides the force to form the blebs is the result of the osmotic load as well as the declining level of high energy phosphate (~P). Plasmalemmal disruption can occur in which proteins and enzymes can leak from the myocytes to the extracellular space, while the electrolytes in the intra and extracellular space gradually come into equilibrium through a sarcolemma which has lost physiological barrier function. The ultrastructural changes of irreversible injury is reported to occur after 40 minutes in the dog (Reimer et al, 1977) following severe ischaemia.
1.2.2 The Spread of Infarction
Prolonged occlusion of a coronary artery will result in necrosis of the myocardium served by that vessel, but the extent of myocardial infarction will vary, depending upon the duration of ischaemia and availability of collateral circulation. The subendocardium is particularly vulnerable to ischaemia due to its greater metabolic demands and the reduced capacity of subendocardial resistance vessel to vasodilate in response to hypoperfusion (Downey, 1994). This region of the myocardium is thus the first to Chapter 1 6 show irreversible injury. As ischaemia continues, infarction extends from subendocardium to subepicardium within the regions supplied by the occluded vessel, the rate of spread of infarction being modified by the presence and density of collateral blood flow. As mentioned above experimental models in the dog heart have shown cellular necrosis to be apparent after 40 minutes of occlusion of a proximal coronary artery (Reimer, et al 1977).
1.3 Biology of the Ischaemic Myocardium 1.3.1 Metabolic Changes During Ischaemia
Ischaemia initiates a series of events that continue until the myocytes die. There is inadequate supply of important sjubstrates, such as oxygen and glucose which are necessary to support oxidative phosphorylation. In response p-oxidation of free fatty acid (FFAs) ceases, and as a result of the disinhibition of glycolysis there is a rapid switch from p oxidation to glycolysis from glucose and glycogen stores. Conversion to anaerobic glycolysis leads to the accumulation of lactate and protons, which inhibit acyl CoA carnitine transferase and several key enzymes regulating glycogenolysis and glycolysis. The accumulation of the end products of anaerobic glycolysis leads to the eventual shut down of ATP resynthesis from intermediate metabolic pathways, and the accumulation of acyl CoA, FFAs and triglycerides in the cytosol (Morgan and Neely, 1990). The accumulation of lactate and other glycolytic intermediates results in decreased tissue pH.
The imbalance between resynthesis of ATP by anaerobic glycolysis and ATP consumption does not lead to an immediate drastic fall in intracellular ATP (ATPi). Creatine phosphate (CP) acts as a reservoir of high energy phosphates and during the early phase of ischaemia this is able to transfer the high energy phosphate fiom CP to ADP so as to maintain ATP levels (Allen and Orchard, 1987). However, this leads to the rapid elevation of inorganic phosphate (Pi), and as the pool of CP is depleted during continued ischaëmia, ATPi levels eventually fall. Failure to regenerate adenine nucleotides leads to the accumulation of adenine nucleosides (adenosine, hypoxanthine. Chapter 1 7 xanthine and in particular inosine) in the ischaemic myocardium.
Irreversible injury of the cell occurs during prolonged ischaemia and is characterised by the metabolic changes stated above. As these changes progress, there is loss of membrane integrity and cytosketetal apparatus, leakage of lysosomal enzymes into the various cellular compartments, efflux of protein and enzymes out of the cell into the extracellular space, and equalisation of ions between intra- and extracellular spaces (Jennings and Reimer, 1992).
1.3.2 Ionic Changes During Ischaemia
During the first minutes of myocardial ischaemia there is a rapid efflux of potassium (K) from the cell, leading to a triphasic rise in extracellular [K] ( Dresdner, 1990). This early rise in K is mainly due an increase in K efflux, rather than any changes in K influx. A number of theories have been proposed to explain the initial K efflux during ischaemia ( Kleber, 1983, Noma, 1985). Intracellular Na has been shown in recent studies to rise progressively during ischaemia (Malloy, 1990). The mechanisms by which this occurs remain uncertain but currently it is believed that the Na/H exchanger is responsible for the net sodium influx during ischaemia (Molloy et al 1990).
During the early phase of ischaemia cell calcium does not change significantly, however, cytosolic calcium rises after 10 minutes of ischaemia. The increase in cytosolic calcium is probably due to reduced uptake of calcium into the sarcoplasmic reticulum, due to reduced activity of the ATP dependent Ca/Mg ATPase pump and increased calcium release from the sarcoplasmic reticulum (Orchard and Allen, 1987). Chapter 1 8
1.3.3 Biochemical and Ionic Changes During Ischaemia
Cytosolic calcium and calcium transients are increased in ischaemia but contractility progressively declines. This decline precedes significant reduction in (ATP)i and is believed to be due to a decrease in the sensitivity of the myofilaments to (Ca)i (Allen and Orchard, 1987). Possible mechanisms by which the sensitivity of the contractile apparatus to calcium is reduced during ischaemia include (i) Modification of the contractile or regulatory protein resulting in a reduction in the affinity to calcium (Solaro et al, 1976). (ii) Inhibition of the calcium activated contraction by the rise in (Pi) (Kentish 1986). (iii) Inhibition of calcium binding to troponin by acidosis, leading to impairment of actin-myosin bridge formation (Fuchs et al, 1970). Thus the accumulation of metabolites of anaerobic metabolism, protons and inorganic phosphate, are believed to play a mayor role in the depression of myocardial contractility which occurs in ischaemia.
Indeed the combination of low (ATP)i, CP and high (ADP)i is believed to be responsible for rigor crossbridge formation of myofilaments and the development of ischaemia contracture (Allen and Orchard 1987). Ischaemia and the resultant changes in Ca2+ status have considerable effect on the cardiac action potential, including a diminution of the resting membrane potential, a slowing of the rate of depolarisation, a reduction in the amplitude of the action potential and marked shortening of the duration of the action potential (Gettes and Casio 1992).
The shortening of the action potential in the ischaemic zone may result in an electrical gradient between ischaemic and normal tissue (in which normal action potential is preserved). This gradient may provide a substrate for arrhythmias due to local current flows (Janse and Kleber 1981).
The loss of contractility that occurs on interruption of coronary result in pressurised perfusion this stretches the myocyte sarcomeres and enhance tension development. Chapter 1 9
Thus the immediate fall in coronary perfusion pressure leads to a shortening of sarcomere length and subsequent fall in contractility. Also the accumulation of metabolites results in the decline in contractility. Most importantly protons and phosphate interfere in the interaction between [Ca]i and troponin, leading to a reduction in the calcium sensitivity of the contractile apparatus. And finely, the depletion of ATP, leads to changes in the function of calcium pumps and loss of energy source for cardiac contraction.
1.4 Pathophysiology of Myocardial Reperfusion
Restoration of blood flow to the ischaemic yet viable myocardium is essential to the survival of the tissue. However reperfusion itself leads to alterations in the cellular metabolic and ionic status which may precipitate or aggravate injury to the myocardium, and thus reperfusion has been termed the ‘double edged sword’ (Braunwald and Kloner, 1985). It should be appreciated that it is important to separate the consequences of injury arising from the ischaemic period from those occurring during reperfusion; the recovery from ischaemia cannot arise without reperfusion and reperfusion cannot occur without preceding ischaemia, both are inextricably entwined. However changes in the metabolic and ionic status of the myocardium do occur on reperfusion, and undoubtedly play significant roles in the formation of ‘reperfusion injury’.
Reperfusion provides the oxygen and metabolic substrates necessary for the recovery of reversibly injured myocardium. Irreversibly injured myocytes subjected to coronary reperfusion develop the ultrastructural appearance of ‘explosive swelling,’ including architectural disruption, contraction bands, and intramitochondrial calcium-phosphate granules. Reperfusion of myocytes already irreversibly damaged by ischaemia results in an acceleration of the necrotic process. It is generally accepted that ischaemia is the primary cause of irreversible injury to myocytes following coronary occlusion and that reperfusion of irreversibly injured cells hastens the necrotic process, perhaps by providing excess fluid and electrolytes to cells with damaged sarcolemma. Chapter 1 10
Introduction of flow to the mitochondria show the first signs of recovery by exhibiting normal dense matrix granules and an increased number of cristae. The restoration of structural integrity requires a long time in many models (Jennings and Yellon 1992a).
1.5 Metabolic Changes During Reperfusion
The restoration of myocardial perfusion enables the washout of metabolite accumulation in the cytosol and extracellular space and restores the supply of metabolic substrates. Reperfusion however also gives rise to an increase in (Ca)i. In irreversibly injured cells, the rise in (Ca)i is excessive and can lead to cell contraction and death. In reversibly injured cardiac myocytes the rise is small and reversible, but changes in the haemostasis of calcium as well as other ions may be responsible for a delay in full recovery and occurrence of undesired complications, such as arrhythmogenesis.
Reperfusion occupies a central role in tissue protection as, without it, no recovery is possible (Braunwald and Kloner, 1985, Fox, 1992). At the time of reperfusion the myocyte is loaded with lactate, protons and Na ions. Washout of lactate from the intracellular space leads to the co-export of protons, contributing significantly to the restoration of pH (Vandenberg, et al 1993). Proton efflux is also facilitated by the Na/H exchanger (Karmazyn and Moffat, 1993), while uptake of HCOs allows the formation of CO 2 from excess protons (Vandenberg, et al 1993). All of these processes allow the rapid recovery of cellular [pH]i, essential for the resumption of normal cellular activity. Chapter 1 11
1.5.1 The Calcium and Oxygen Paradoxes
The calcium paradox was initially described by Zimmerman et al (1966). It was described as a condition in which the sudden readmission of calcium to the perfusate, after a brief period in which hearts are perfused with calcium-free media, causes massive tissue disruption, enzyme release, the development of contracture, and marked reductions of high energy phosphate stores (Hearse, 1977 and Hearse, et al 1976). Since perfusion of hearts with calcium-free media even for brief periods leads to increased cell membrane permeability, the primary lesions in the calcium paradox may be the separation of the external lamina and surface coat of the glycocalyx, disruption of the sarcolemmal bilayer, and the loss of intracellular calcium during the period of calcium depletion (Hearse, et al 1976). These changes may predispose the cell to a massive influx of calcium and loss of the regulation of cell volume during reperfusion. The influx of calcium may activate the production of free radicals, activate phospholipases which damage the sarcolemma, or disrupt mitochondria. These changes can lead to contracture, cell swelling, and necrosis.
It has been known that the réintroduction of oxygen to hypoxic or anoxic myocardium may also be detrimental, leading to excess liberation of cardiac enzymes and contracture. Indeed, the effects of restoring calcium to the perfusate after a short period of calcium depletion (calcium paradox) or of restoring oxygen after a relatively long period of anoxia or ischaemia (oxygen paradox) resemble each other in many ways (Hearse, 1977 and Hess and Manson, 1984). In both conditions, the sarcolemma is damaged, intracellular vesicles develop, and mitochondria move to the cell border. In addition, ultrastructural changes in myofibrils and sarcoplasmic reticulum have been observed in the rat heart after 105 minutes of reoxygenation following this period of anoxia (Hearse, et al 1976 and Gauduel and Duvelleroy, 1984). Interestingly, Hearse et al (1977) found that reperfusion of ischaemic hearts with anoxic fluid resulted in no increase in damage and no significant increase in enzyme leakage. However, reoxygenation of the anoxic perfused rat heart causes an immediate and massive enzyme release, and sudden and major ultrastructural damage, both of which are Chapter 1 12 directly attributable to the resumption of oxidative metabolism.
In the calcium paradox, there is passive movement of calcium into the cytoplasm, explosive cell swelling, and calcium-induced myofibrillar contracture. In the oxygen paradox, it is likely that the oxygen-induced re-energisation of the electron transport chain triggers the uncontrolled uptake of calcium by the mitochondria from the cytoplasm. Reperfusion or reoxygenation of ischaemic myocardium causes contracture of myofibrils, disruption of the sarcolemma, and the appearance of intramitochondrial calcium phosphate particles. The uptake of calcium by myocytes during reperfusion probably occurs as a consequence of passive diffusion into the cytoplasm. Interestingly, reduction of calcium concentration in the reperfusate influences mechanical recovery favourably (Shine and Douglas, 1983).
Since ischaemic myocardium is not exposed to calcium free conditions in a clinical environment, the calcium paradox is probably not involved in ischaemia and reperfusion in this setting. However, events observed in the similar and probably closely related oxygen paradox suggest that restoration of aerobic metabolism may be a critical component of reperfusion-mediated events and might extend the severity of ischaemic damage and limit the value of reperfusion (Hess and Manson 1984, Hearse, 1984a).
1.6 Free Radical Generation
Free radicals are molecules with an unpaired electron which makes them highly reactive. The unpaired electron is denoted by a ‘dot* in formulae. These molecules are described as free radicals as they contain an odd number of electrons. As such they are very potent oxidising and/ or reducing agents (Fen ari, 1986a, 1985, Hess, et al 1984 and McCord, 1985). Superoxide (Q2-), singlet oxygen (102), and the hydroxyl radical
(OH*), are the major oxygen free radicals formed during ischaemia-reperfusion
(Loessor, et al 1991). In addition hydrogen peroxide (H 2O2), an oxidant and an Chapter 1 13
important source of free radicals, is generated during reperfusion (Hearse, et al 1976, Gauduel and Duvelleroy, 1984).
1.6.1 Free Radical Toxicity
The oxygen-derived free radical may play a fundamental role in cellular damage in a wide variety of pathophysiological processes, including injury due to radiation, inflammation, myocardial ischaemia and reperfiision (Meerson, et al 1982, Ferrari, et al 1985, Wems, et al 1986, Hearse, et al 1978 and Rao, et al 1983). It is well known that oxygen supplied at concentrations greater than those in normal air might damage plants, animals and aerobic bacteria (Furberg, 1984). There is a considerable number of reports that have shown that as much as 21% oxygen has slowly manifested damaging effects.
Oxygen derived free radicals cause cellular damage by reacting with polyunsaturated fatty acids and result in the formation of lipid peroxides and hydroperoxides, which in turn inhibit many membrane bound enzymes, initiate chain-propagating reactions capable of damaging the sarcolemma, and thereby cause the disruption of cellular integrity (Rao and Mueller, 1983). Free radicals are capable of various toxic activities such as the inactivation of sulphydryl enzymes, cross-linking of protein and DNA breakdown, leading to functional alterations and structural changes which may cause reversible or irreversible injury. (McCord, 1985, Tappel, 1973 and Freeman and Crapo, 1982).
Several mechanisms appear to be responsible for the formation of oxygen-derived free radicals during reperfusion. During ischaemia, ATP is broken down to AMP which is ultimately metabolised to hypoxanthine. In addition the elevation of cytosolic calcium concentration caused by ischaemia enhances the conversion of xanthine dehydrogenase to xanthine oxidase (McCord, 1985 and Chambers, et al 1985). According to this concept, when molecular oxygen is reintroduced to cells containing high concentrations Chapter 1 14 of hypoxanthine, this enzyme causes the release of 02- and H 2O2. Secondly, during myocardial ischaemia, there is loss of the enzymes, superoxide dismutase, catalase, and glutathione peroxidase (Rao, and Mueller, 1981, and Meerson, et al 1982), which could protect the heart from oxygen-derived free radicals. The autoxidation of catecholamines could provide another source of free radicals (Burton, et al 1984, Singal, et al 1982 and Loesser, et al 1991).
Within the myocardium the most important sites for the formation of oxygen radicals are the mitochondria. Myocyte mitochondria provide a large potential source of 02- and
H2O2 from leakage of electron carriers out of the electron transport chain, as a consequence of reduction of oxygen by ubisemiquinone or by NADH dehydrogenase (Turrens and Boveiis, 1980). It is known that superoxide radical generation is greatest when the components of the respiratory chain are in a reduced state as, for example, following ischaemia. Under these circumstances readmission of oxygen leads to an enhance of 0-2 (radical) production by electron transport components of the ischaemic cell. In addition activated phospholipase may degrade cell membrane phospholipids, releasing arachidonic acids that can accelerate the synthesis of free-radical intermediates by the cyclooxygenase and lipooxygenase pathways (Kontos, et al 1980). From the outside of the myocardial cell, the enzyme xanthine oxidase is the major source of oxygen free-radical formation.
Finally ischaemia triggers the activation of the complement system and generation of chemotactic factors. As a consequence neutrophils accumulate in the vascular space of reperfused ischaemic myocardium and cause damage through their ability to produce oxygen free-radicals (Englar, et al 1983, McCord and Day, 1978).
In the heart there exists a series of endogenous defence mechanisms which are able to protect the cell against the cytotoxic oxygen metabolites. They include the enzymes superoxide dismutase, catalase and glutathione peroxidase plus other endogenous antioxidents such as vitamin E, ascorbic acid and cysteine (Ferrari, et al 1986b). The Chapter 1 15
basic premise for the involvement of oxygen in reperfusion damage is that ischaemia and reperfusion have altered the defence mechanisms against oxygen toxicity.
1.6.2 Possible Role of Nitric Oxide in Ischaemia Reperfusion
It has been suggested that NO may be cytotoxic when present in substantial excess (Beckman et al 1990). This deleterious role of NO may be important during early hyperaemia and in the presence of activated leucocytes which express inducible NO synthase (Hibbs et al 1988 and Moncada et al 1991). (See chapter 1 section 2.0 for a summary of nitric oxide).
1.6.3 Toxic Role of Free Radicals Generated From Nitric Oxide (Peroxynitrite)
It has been proposed that, under many pathological processes including hyperoxia (Freeman and Crapo, 1981), xenobiotic metabolism (Mason, 1982), inflammation (Weiss and LoBuglio, 1982), and ischaemia/reperfusion (McCord, 1985) there is an increase in the production of free radical .When generated in excess, 02- and OH*
(hydroxyl) radicals are a key mediator of cell injury (Hogg, et al 1992). The mechanism generally invoked for hydroxyl radical production involves the presence of superoxide, hydrogen peroxide and either iron or copper ions, formation occurs via the Haber-Weiss reaction (Halliwell, 1987 and Menasche, 1987). Beckman and colleagues (1990) have suggested that generation of HO via the Haber-Weiss reaction (Beckman,
et al 1990) may be limited in vivo, and they have proposed that NO reacts with 02 in
pathological states to produce cytotoxic species via the reaction shown below; this reaction is not dependent on the presence of transition metal ions.(Beckman, et al 1990). This involves the production of peroxynitrite arising from the reaction of NO with superoxide; Chapter 1 16
N 0+ 02 <------> ONOO-...... (1)
ONOO-+H+ <------> ONOOH...... (2)
ONOOH <------> .N02 + OH...... (3)
N 02 + • OH <------> N03 + H+...... (4)
It is probable that the interaction between superoxide and NO can occur in vivo, and it has been suggested that the vascular endothelium has the ability to regulate the effects of NO by generating superoxide (Halliwell, 1989). The reaction will effectively compete with the dismutation of superoxide. Peroxynitrite has a pKa of 7.5 and half life of 1.9s (Hogg, et al 1992) and will be substantially protonated (reaction 2) at physiological pH, to produce peroxynitrous acid. One route for the decomposition of this molecule is homolytic decay (reaction 3) to produce hydroxyl radical and nitrogen dioxide. It has been reported by Beckman et al (1990) that a minimum of 25% of peroxynitrite forms hydroxyl radical and nitrogen dioxide under physiological conditions with the remainder recombining to form nitrate (Beckman et al 1990). At physiological pH peroxynitrite anion is sufficiently stable to diffuse over several cell diameters to critical cellular targets before becoming protonated and decomposing. Peroxynitrite initiates lipid peroxidation and reacts directly with sulfhydryl group at a thousand fold greater rate than hydrogen peroxide at pH 7.4 (Radi, et al 1990).
Peroxynitrite reacts with transition metals to form a powerful nitrating agent (N02+).
The peroxynitrite is a very reactive species and is highly toxic. In some pathological situations, macrophages and neutrophils, recruited to a site of injury, can be activated to produce both superoxide and NO as part of the inflammatory response (Marietta, 1988 and Hogg, et al 1992). Therefore, it is possible that the formation of peroxynitrite occurs in both normal and pathological state in vivo, on reperfusion of ischaemic tissue, acute inflammation, and in sepsis. All events that can stimulate the production of NO and superoxide. Chapter 1 17
1.7 The Causal Mechanism Determining Cell Death During Reperfusion
Restoration of flow, however, might result in numerous further negative consequences, thus directly favouring the occurrences of cell death (Hearse, 1977, Poole-Wilson, 1985, Mayler, 1981, Ferrari, et al 1986a,b). Different possible factors include: depletion of high energy phosphate (Jenning, et al 1978), loss of adenine nucleotide (Reimer, et al 1981) and of catecholamines (Gaudel, 1979), accumulation of calcium (calcium paradox) and oxygen paradox (Ferrari, et al 1986a,b and Shen , et al 1972), activation of phospholipase and of proteases (Ogunrd, 1980), detergent effect of acylesters (Vander-Vausse, et al 1980) and of lysophosphoglycerides (Katz, et al 1983), hastening of the necrotic process of irreversibly injured myocytes, cell tearing due to contracture (Ganote, 1983), myocardial cell swelling, the no-reflow phenomenon, haemorrhagic myocardial infarction and the generation of free radicals (Meerson, et al 1982, Ferrari, et al 1985 and Wems, et al 1986) which may damage ischaemic myocytes, and the prolonged postischaemic depression of ventricular function (stunning of the myocardium). Reperfusion of the ischaemic tissue may also induce a number of important cardiac electrophysiologic changes which in turn can cause a variety of arrhythmias some benign, others potentially lethal (Manning and Hearse, 1984). Often these factors are interrelated and obscure the distinction between cause and effect.
1.7.1 Reperfusion Hyperaemia
As soon as flow is restored, the damaged myocardium develops marked reactive hyperaemia. Coronary arterial flow increases by 400-600% and stays elevated for many minutes, especially if the period of reversible ischaemia exceeds 5-8 minutes. The oxygen content of the damaged tissue increases, thus the reperfused tissue clearly contains abundant O2, a potentially deleterious agent, throughout the period of reactive hyperaemia. However, it should be pointed out that the deleterious events associated Chapter 1 18 with reperfusion cannot be avoided by the prolongation of an ischaemic period even in the presence of protective interventions such as pharmacological or biochemical manipulation of the ischaemic cell. These interventions, in the absence of reperfusion, do not restore normal contractile function and are able only to delay, but not to avoid, the development of injury. When it is not beneficial it accelerates the rate of development of necrosis (Hearse, 1988, Hearse and Yellon, 1984).
1.7.2 Reperfusion Arrhythmias
A potentially dangerous consequence of reperfusion is the occurrence of reperfusion arrhythmias, which can range from non-threatening ventricular premature beats to ventricular tachycardia and fibrillation. Reperfusion arrhythmias occur within two phases: An immediate phase, within the first minutes of reperfusion, and a delayed phase occurring between 2 and 7 minutes after reperfusion (Kaplinski, et al 1979).
The aetiology of reperfusion arrhythmias is complex. There is a large body of evidence to suggest that oxygen free radical injury may play a significant role in the genesis of these arrhythmias in experimental models (Hearse, 1991). A number of studies have shown that administration of a variety of anti-radical agents attenuate the incidence of experimental reperfusion arrhythmias, while free radical generating systems (peroxides, xanthine-xanthine oxidase) can all precipitate arrhythmias (Manning and Hearse, 1984).
1.8 Ultrastructural Changes 1.8.1 No-Reflow Phenomenon
On reperfusion, coronary flow is absent in the area in which irreversible myocyte injury has occurred, this is known as the no-reflow phenomena (Kloner, et al 1974a). Several mechanisms have been proposed for the no-reflow phenomenon. Ultrastructural studies of zones of no-reflow show actual microvascular damage which could impede inflow into the ischaemic zone (Kloner, et al 1974a). These Chapter 1 19 microvascular abnormalities include localised zones of endothelial cell swelling termed ‘blebs,’ which appear to plug capillaries; increased permeability of capillaries with and without gaps in the endothelium leading to rouleaux formation of erythrocytes with subsequent stasis; plugging of the microvascular bed by leukocytes (Engler, et al 1983); microscopic zones of haemorrhage (Reimer, et al 1977); and myocyte swelling which can compress the adjacent microvasculature. In addition, contracture of myocytes is another possible mechanism by which ‘no-reflow’ could occur.
1.8.2 Reperfusion-Induced Haemorrhage
It has be observed repeatedly in experimental studies that infarcts produced by transient periods of ischaemia followed by reperfusion contain areas of haemorrhage (Reimer, et al 1977, Kloner, et al 1983). Reperfusion can convert a bland infarct into a haemorrhagic one and, more importantly, that haemorrhage, due to coronary reperfusion, may lead to the extension of myocardial necrosis (Bresnahan, et al 1974). Haemorrhagic infarction is caused by reperfusion of myocardium in which the damage of microvasculature allows the extravasation of blood. It is now clear that, in dogs, relatively short periods (<40 mins) of severe ischaemia cause necrosis of myocytes but usually not of vascular endothelial cells, and reperfusion of tissue in which the endothelium has been spared injury will not cause haemorrhagic infarction. However, long periods of severe ischaemia can damage not only myocytes but cause changes in the capillary endothelial cells as well (Kloner, et al 1980). The latter consist of localised zones of endothelial swelling which protmde into the vessel lumen, of gaps within the endothelium, and of loss of endothelial pinocytotic vesicles.
1.9 Reperfusion of the Vascular Bed
Removal of a coronary occlusion does not necessarily lead to full restoration of perfusion to myocytes in the previously ischaemic area. Reperfusion may lead to a progressive decline in flow following the initial minutes of reactive hyperaemia. The Chapter 1 20 explanations for this phenomenon include microvascular injury with endothelial cell swelling, impairment of endothehal endocrine function , and the occlusion of capillaries with platelets and neutrophils (Forman, et al 1989). Although endothelial cells are relatively resistant to hypoxia, ischaemia and reperfusion lead to impairment of the capacity to release nitric oxide with consequent reduced dilator activity (Boissel, 1992). Neutrophils are less compliant than red cells and have a larger cross sectional diameter (7.5p) than capillaries (5p). Furthermore, activated neutrophils are even less compliant, contributing to capillaiy plugging (Engler, et al 1986).
1.9.1 Vascular and Microvascular Injury Impairing Reflow
Mico-emboli have been demonstrated in the distal vasculature following ischaemia and reperfusion, and platelet aggregation leads to a direct constrictor response on the distal vasculature (via mechanisms including the release of serotonin and ADP). Reperfusion increases the efflux of macromolecules from the intravascular space to the extra vascular space. Neutrophil activation and endothelial dysfunction involve activation of the complement cascade with release of superoxide radicals and cytokines.
1.9.2 Leucocyte Accumulation During Reperfusion
Polymorphonuclear neutrophils (PMNs) are known to begin accumulating in irreversibly injured myocardium within the first 60 minutes after reperfusion, after as little as 40 minutes of preceding ischaemia (Mullane, et al 1992). Neutrophils are seen to adhere and stick to the capillary endothelium; thereafter they migrate from the capillaries into the extra vascular space. Finally, they adhere to the irreversibly injured myocytes. This process is time dependent, shortly after reperfusion the number of neutrophils sticking to the endothelium is low but steadily increases with time.
These accumulating PMNs play an important role in the digestion of necrotic myocytes and participate actively in the healing process, through which the infarct is replaced by Chapter 1 21 scar tissue. However, PMNs also have the potential to cause injury to viable myocytes (Go, et al 1990). Neutrophils accumulate rapidly in the intramyocardial vessels and myocardium, maximal infiltration occurs in the subendocardium, the region of greatest ischaemic injury, but significant accumulation can occur in the subepicardial tissue (Mullane and Young, 1992).
The deleterious effects of neutrophils accumulated within the ischaemic-reperfused myocardium (Go, et al 1990) have been attributed to the generation of oxygen-derived free radical species (McCord, 1985), plugging of the microvasculature by adherent and aggregation neutrophils, and adherence and emigration of neutrophils in post-capillary venules (Kubes and Granger, 1992). Adherence of stimulated neutrophils to endothelial cells release cytotoxic oxygen-derived free radicals and microvascular embolization.
Leukocyte sequestration after ischaemia reperfusion has been demonstrated in many organs, and attenuation of leukocytes accumulation is tissue protective (Horgan, et al 1992, Ma X , et al 1991 and Vasthere, et al 1990). However there is considerable controversy as to whether neutrophil accumulation and activation in viable post ischaemic tissue contribute to reperfusion injury. Although some studies using anti neutrophil monoclonal antibodies (Mullane, et al 1992, Simpson, et al 1990) have demonstrated a reduction in infarct size in experimental models of myocardial infarction, other similar studies have failed to demonstrate infarct size reduction with anti-neutrophil antibodies (Reimer, et al 1983 and Tanaka, et al 1993).
However, the activated neutrophil is capable of promoting tissue injury, by release of oxygen fi-ee radicals, hydrogen peroxide, proteolytic enzymes and cytokines; additional endothelial damage resulting in swelling and also necrosis may cause recurrent ischaemia. Thus PMNs have significant potential for lethal damage in the myocardium at risk. Chapter 1 22
1.9.3 Neutrophil Recruitment to Sites of Tissue Injury
Neutrophil recruitment to sites of tissue injury is mediated by multiple adhesion molecules, which are sequentially required for the attachment of the cells to vascular endothelium and their subsequent extravasation into the surrounding tissue (Smith, et al 1991b, Lasky, 1991). Leukocyte endothelial cell interactions involves at least two classes of adhesion molecules that are expressed on the surface of neutrophils. These include L-Selectin which is constitutively functional on non activated leukocytes, and the B2-integrins (GDI 1/CD 18 family of glycoproteins), which are unregulated when neutrophils are activated (Gallatin, et al 1983, Von Andrian, et al 1993 and Lefer, et al
1994). L-Selectin has low-affinity binding properties that are thought to cause neutrophil rolling. Firm adhesion is mediated by Bi-integrins. These consist of a family of heterodimers, each of which has a common B-subunit designated CD 18, and one of three known as the a-subunits designated GDI la and GDI Ic.
P-Selectin, another member of the selectin family of adhesion glycoproteins, located in the Weibel-Palade bodies of the vascular endothelium and in the a-granules of the platelets {McEver,. 1991), is rapidly translocated to the cell surface after stimulation with thrombin, histamine, oxygen-derived free radicals, or hypoxia reoxygenation (Geng, et al 1990, Palluy, et al 1992, McBver, 1991). This adhesion glycoprotein is believed to play a significant role in early leukocyte endothelial interaction (ie rolling of leukocytes along endothelial surface) and recruitment of leukocyte to injured tissue.
Recent studies of the leukocytes and endothelial adhesion molecules involved in the acute inflammatory response indicate a complex pattern of neutrophil endothelium interaction that precedes emigration of leukocytes from the vasculature into the surrounding tissue. Leukocyte emigration is now known to be a multi step process involving sequential activation of various adhesion molecules (Lefer, et al 1994). Chapter 1 23
1.10 Discussion
Ischaemia of the myocardium leads to depletion of high energy phosphates and accumulation of sodium and calcium which result in loss of contractility and increase in resting tension. Myocardial reperfusion may be described as a double edged sword. Although it clearly exerts deleterious effects on severely ischaemic areas (injured cells may cause serious cardiac arrhythmias) when myocardial reperfusion is carried out relatively early in the course of ischaemia, its effects are usually beneficial. Reperfusion also permits the washout of accumulated metabolites including lactate, phosphate and protons, permitting the resumption of normal cellular metabolic activity. It is intriguing to consider that myocardial salvage may be optimised if the conditions under which reflow occurs can be modified, such as by enrichment of the reperfusion medium with substrate with reduction of its calcium concentration and also reduce free radicals generation. In addition to injury to the myocytes, the endothelium is also vulnerable to ischaemia-reperfusion injury and may result in low-flow and impairment of vasodilatory reserve.
1.11 Conclusion
It therefore appears that reperfusion of ischaemic tissue is able to salvage reversibly injured myocardium, but can also lead to further deterioration of irreversibly injured tissue. Chapter 1 24
2.0 Ischaemia, Reperfusion and the Model Used for both the In Vivo and In Vitro Experiments
2.1 Introduction to Collateral Circulation in Species
The coronary circulation comprises epicardial conductance vessels, penetrating intramural arteries, intramural arterioles and capillaries, and venules and veins which converge to form epicardial coronary veins.
Anastamotic connections between epicardial and intramyocardial vessels can occur to a variable degree between species and between individuals (Maxwell, 1987). Maxwell et al (1987) has recognised that a wide spectrum of collateral flow exists between various mammalian species, which must always be taken into account in the study of pathophysiology and control of regional ischaemia and myocardium infarction. A study by Schaper et al (Schaper, 1984) demonstrated an inverse correlation between infarct volume and collateral blood flow. Therefore in species such as the rabbit, pig and rat which exhibit zero or minimal collateral flow, rapid and complete infarction may occur (Harken, et al 1981,Flores, et al 1984, Schaper, 1984, Maxwell, et al 1987). In dogs and cats, infarction proceeds at a slower rate since they possess significant collateral flow (Maxwell, et al 1987 and Schaper, 1984). Interestingly, the guinea pig has a high percentage of collateralisation and does not exhibit any infarction at all following epicardial coronary occlusion (Flores, et al 1984, Schaper, 1984, Maxwell, et al 1987).
2.2 Ischaemia and Reperfusion in the Animal Models
So where in this spectrum of collateral flow does the human heart lie? It is believed that the level of collateral flow during regional ischaemia in man is dependent on the age and pathology of each individual. Sudden occlusion of a major artery in a young man Chapter 1 25 may produce fully transmural infarction, as the low collateral flow of the undiseased heart is unable to support ischaemic myocardium. This situation would be best mimicked experimentally by animals exhibiting zero or minimal collateral flow such as the pig, rabbit, ferret or baboon. In contrast, atherosclerosis and coronary artery disease generally may provide sufficient stimulus for the induction of collateral growth, which may reduce the size of infarction following occlusion of an artery. This would match the collateral status of the dog. The difficulty of extrapolating from the dog, rabbit or any other species to the human heart needs no elaboration. In addition to vital differences in collateral supply there also exists undoubtedly differences in basal metabolism, pharmacological responsiveness and vulnerability to ischaemic injury. Add to these the complications arising from the major surgery and trauma associated with most large animals studied, the presence of anaesthesia, and the fact that co-existing disease frequently seen in the human are rarely stimulated in experimental studies, then it takes as quoted by Schaper et al (Schaper W. 1984) *a major leap of faith* to extrapolate current animal studies to the human heart. Animal studies are, however, essential and it is felt that these leaps of faith are necessary, placing a high demand on todays investigators to apply more cautious and well considered interpretations.
The importance and variability of collateral flow has only relatively recently been appreciated. Therefore, many experimental studies completed in the early eighties, which did not account for collateral flow, are subjected to inaccuracies. Collateral flow artifacts can be avoided by using analysis of variance on the infarct size, with collateral flow (measured by radio labelled microspheres) as a covarient. This determinant does not apply when assessing experiments in animals, such as the pig or rabbit, which have a negligible collateral flow. Maxwell et al (1987) reported that in the rabbit, ischaemic areas could easily be identified as a very sharp boundary between the non-ischaemic and the ischaemic areas of myocardium. Both these observations enable clear and easy analysis of the risk zones in each heart and highlight a major consideration for choosing rabbits as the species for our study. Even so, it is not only collateral flow that accounts for the variability between animals. For example, the rabbit, with a very limited collateral circulation, also displays unexplained variability in infarct size unaccounted Chapter 1 26 for by any haemodynamic parameter. Why this occurs is still unknown .
2.2.1 The Definition of Ischaemia
Schaper et al (1988) has defined myocardial ischaemia as a situation of severely reduced regional myocardial blood flow which is insufficient to provide for the metabolic needs of that tissue (definitions on myocardial ischaemia see ‘End of year editorial’ 1994). In ischaemia, still remaining blood has a normal oxygen tension, in contrast to the situation of hypoxia, where a normal flow rate persists but the oxygen tension of the perfusate is significantly reduced.
Ischaemia provokes tissue injury that in the first stage is only slight, but increases in severity with time. Initially this injury is still reversible and therefore, is capable of complete recovery upon reperfusion. With time, tissue damage results in irreversible cell death and therefore, the structural and functional properties of the myocardium do not recover upon reperfusion. As long as 24 hours after induction of ischaemia, canine hearts showed individual variability in the speed and development of myocardial infarction. It is concluded that the severity of injury increases with the duration of ischaemic insult. The differences between individual animals caused by significant variations in collateral blood flow and rate of oxygen consumption may also determine infarction. Time, therefore, appears to be only one of the factors influencing the speed and development of myocardial necrosis as was reported by Schaper et al (1984).
2.2.2 Induction of Myocardial Ischaemia
Ischaemia can be acute occlusion of the coronary arterial flow. Induced ischaemia can be global or regional. Total global ischaemia is created by shutting off completely the flow of perfusate, for instance by cross-clamping the aorta in a Langendorff-perfused hearts or arterial cross-clamp in a working heart model. Partial global ischaemia is created by reducing the flow rate of the perfusate. Chapter 1 27
Regional ischaemia is induced by occluding a certain branch of the coronary artery. The left ventricle is the most powerful heart chamber, being the one that ejects the blood into the systemic circulation; the myocardial wall being the thickest in the left ventricle. Therefore, in most instances the left anterior descending coronary artery (LAD) is the one to be occluded, because this usually leads to the creation of a well-defined ischaemic zone in the left ventricular wall, upwards from the apical region. In perfused hearts LAD occlusion is typically carried out by ligation of the artery with silk ties fastened around the artery. Other techniques, include inflated balloon occluders placed around the vessel or utilising metal pellets released from catheters.
2.2.3 Region at Risk
When a coronary artery is occluded in an experimental model, the infarction will develop only within the region of the occluded artery, and none of the adjacent well- perfused tissue will be involved. This region supplied by the occluded coronary artery is, therefore, called the ‘region at risk* (Miura, et al 1988). This risk zone is potentially salvageable, provided irreversible damage has not occurred. Thus, the size of the region at risk is another major determinant of the size of the eventual infarct.
2.3 Assessment of Infarct and Risk Size 2.3.1 Introduction
Electron microscopy may detect areas of necrosis as early as 20 minutes (Fishbein et al. 1981, Nachlas and Shnitka, et al 1963) after coronary occlusion, however, due to sampling problems and high cost, this method does not lead itself readily to the quantitation of infarct size.
The present methodology using Triphenyl tétrazolium chloride represents a technique whereby infarct size can be reliably determined with relatively low variability. Chapter 1 28
Triphenyl tétrazolium chloride (TTC) and nitroblue tétrazolium (NET) are used for the gross histochemical staining technique. Infarct size determination using these histochemical stains have shown to be reliable and simple to use (Lie, et.al 1975, Klein, et.al 1981 and Warltier, et al 1981). The colourless tétrazolium salts form coloured precipitates (TTC-red and NBT- blue) in the presence of intact dehydrogenase enzyme systems, requiring a short incubation.
2.3.2 Mechanism for Staining
Tétrazolium salts are chemically characterised by a ring structure, which contains one carbon and four nitrogen atoms, one of which is quartemary. The reduction is combined with opening of the ring, which leads to the coloured formazan. Tétrazolium salts are reduced by redox systems having a lower redox potential than the dye itself.
In the areas of necrosis where reflow has been established after ischaemia the intracellular enzymes are rapidly washed out (Klein, et.al 1985). Therefore the areas of necrosis lack dehydrogenase activity and fail to stain (Vivaldi, et al 1985, and Fishbein, et al 1981). The biochemical mechanism for tétrazolium staining has clearly been explained by Klein et.al (1981), who have shown that the viable myocardium reduces the TTC salts to a formazan pigment. This is achieved by the oxidation of NADH by diaphorases to form the formazan stain. Normal and risk areas of the myocardium will react with the TTC salt to form the formazan pigment this will stain the myocardium brick red, while the infarcted myocardium will not take up the stain, as a result of washout of the cofactors, dehydrogenase and diaphorases, on reperfiision.
A number of studies have been carried out to assess the accuracy of the TTC technique for quantitating and comparison of infarct size with other techniques (Kloner, et.al 1981, Warltier, et al 1981 and Vivaldi, et.al 1985). The accuracy of staining with TTC as a macroscopic means for identification and quantitation of myocardial infarcts after coronary occlusion in the dog was assessed by Fishbein et.al (1981) where they reported TTC staining to correlate well with histological techniques. Chapter 1 29
2.3.3 Advantages of TTC Staining
Shorter experiments can be designed for specific studies, avoiding some complications of long-term anaesthesia and instrumentation, and in avoidable spontaneous changes in heart rate and blood pressure or arrhythmias, which could complicate interpretation of results. Infarct size determination using TTC staining is accurate and reliable, since it is easier to planimeter the infarct in one complete section.
Other methods utilising enzymatic alterations in tissue or morphological studies by light and electron microscopy pose technical problems such as rapid freezing in liquid nitrogen and /or careful preparation of numerous specimens. While TTC is economical and infarct size can be quantitated as early as 3-6 hours after coronary occlusion (Fishbein, et.al 1981).
2.3.4 Analysis of Infarct and Risk Areas
Once the infarct is delineated grossly a number of techniques are available for estimating infarct size. All have advantages and disadvantages but seem to be of acceptable reproducibility and accuracy. The only difficulties that may arise when measuring infact size may be due to patchy infarction, such infarcts are only known to occur in the global ischaemic model (Fishbein, et al 1981). However, more sophisticated methods of quantitation such as those based on electronic image analysis would probably be helpful in such circumstances.
2.4 Discussion
There is a wide spectrum of collateral flow in many different species that are used in the laboratory. Collateral flow can be determined using radiolabelled microsphere technique during myocardial ischaemia. In animal experiments collateral flow is divided into four different groups these are high (guinea pig), significant (dog and cat), minimal (rat) and Chapter 1 30 zero collateral flow (ferret, baboon, rabbit and pig). Therefore differences in collateral flow found in the species may explain some of the contradictory results obtained with studies of the pharmacological limitation of myocardial infarction.
Pathologic demonstration of myocardial infarction is necessary for validating newly developed radioisotopic and computed axial tomographic techniques of infarct imaging, for assessing the effects of pharmacologic interventions on myocardial infarct size (Kloner, et al 1978 and Rude, et al 1979) and for diagnosing myocardial infarction in cases of sudden death (Fallon, 1979). Therefore reliable techniques for assessing the presence and extent of myocardial infarction shortly after coronary occlusion are important. Histologic methods are useful, but the myocardium must be ischaemic for 12 to 24 fours before definite signs of necrosis are present (Jennings, 1969 Jennings, et al 1973). The TTC staining has recently been used to delineate infarcted myocardium ( Knight, 1965, Lie, et al 1975, Kloner, et al 1978 and Rude, et al 1979) at times earlier than the development of histologic necrosis (Lie, et al 1975 and Fallon, 1979). TTC technique is an accurate method for determining infarcts grossly. Results of TTC staining compete well with histologic evidence of necrosis 6 hours after occlusion and with ultra structural evidence of necrosis as early as 3 hours after occlusion. Also the areas that were shown to be necrotic by light and electron microscope failed to stain with TTC, these regions unstained by TTC always showed numerous mitochondrial amorphous dense bodies, marked intracellular edema, and disrupted sarcolemmal membranes; features that have been associated with iireversible myocardial cell damage (Jennings, et al 1969, Jennings, et al 1973 and Kloner, et al 1974). The loss of TTC staining depends on the time course of irreversible myocardial injury, as well as the kinetics of cofactor diffusion from necrotic myocardium. For example because of potential differences in collaterals, (Vivaldi, et al 1985) myocardial damage following coronary artery occlusion in the rat probably develops more rapidly than that in man, and like wise, the time course of TTC staining defects evolution is probably more rapid in rat.
The TTC technique allows quantitation of myocardial necrosis after only a few hours of Chapter 1 31
coronary occlusion and is of considerable value. Time consuming and costly histologic or ultra structural studies are not necessary as a routine. Shorter experiments can be designed for specific studies, avoiding some complications which could complicate interpretation of results. Early quantitation with TTC should be helpful in experimental studies of the progressive events of infarct evolution, as well as in evaluation of the effects of interventions during the very early phases of ischaemic injury.
2.5 Conclusion
It is concluded that TTC reliably aids quantitation of infarct size at 24 hours and locates early irreversibly injured myocytes. Although TTC may detect an infarct as early as 30 hours, intensity of staining is maximal by 6 hours after occlusion. Chapter 1 32
Chapter 1 Introduction Section 2.0: General Biology of Vascular Endothelium
1.0 Introduction
Vascular endothelium forms a continuous lining to the inner surface of all blood vessels and separates circulating blood from vascular smooth muscle and surrounding tissue. It is not merely a passive diffusion barrier, but represents a fully functional organ with a variety of biological functions. Such functions include control of vascular tone, inhibition of blood coagulation, vascular wall metabolism, transcapillary permeability of solutes and water, and remodelling of the underlying vascular tissues (Gerlach, et al 1985 and Hammersen, et al 1985). The endothelium has therefore become the focus of great interest
1.1 Histology
The endothelium forms a monolayer of cells with similar morphology lining the lumen of arteries, veins and capillaries alike (Belloni and Tressler, 1990). The endothelium is composed of flat, squamous-type cells with a central nucleus which are linked by specialised desmosomes, though in some tissues such as the liver and glomerulus the layer is fenestrated. The rounded to slightly ellipsoid nucleus is surrounded by a dense perinuclear cytoplasm, rich in organelles. The cytoplasmic organelles consist of rough endoplasmic reticulum, ribosomes and scattered mitochondria. Also present in Chapter 1 33 endothelial cells, are rod-shaped Wiebel-Palade bodies which represent the intracellular storage site of Von Willebrand Factor (De Groot, et al 1989 and Wagner, et al 1982). Along both luminal and intimai surfaces of the endothelial cell membranes pinocytotic vesicles are abundant. Microfilaments, intermediate filaments and microtubules constitute the three major fibrous protein systems of the endothelial cells (Kalnins, et al 1981). The free surface of the endothelial cell is not smooth, particularly in the area overlaying the nucleus where there are microvilli which contain ribosomes. Lying over the plasma membrane is a highly organised meshwork of glycocalyx (Luft, 1966). The strategic location of endothelial cells at the interface between the intravascular and interstitial compartments dictates that interactions may occur in both luminal and abluminal directions.
1.3 Control of Vascular Tone
Endothelial cells produce vasodilators and vasoconstrictors, which are important in local and systemic control of vascular tone. Two potent vasodilators, synthesised by vascular endothelial cells are prostacyclin (PGI2) and endothelium-derived relaxing factor (EDRF). There is also evidence for an additional vasorelaxant factor, endothelium-derived hyperpolarizing factor (Southerton, et al 1987, Komori and Suzuki, 1987).
2.0 Control of Vascular Tone by Endothelium-Derived Relaxing Factor
In 1980, Furchgott and Zawadzki were the first to demonstrate that the vascular relaxation induced by acetylcholine was dependent on the presence of the endothelium and provided evidence that this effect was mediated by a labile factor, later termed endothelium-derived relaxing factor (EDRF). It was later shown that the properties of EDRF are identical to those of nitric oxide (NO) (Azuma, et al 1986, Radomski et al 1987a,b and Moncada, 1992 review). Ignarro and colleagues (1987a/b) have presented Chapter 1 34 both biological and chemical evidence to show that the EDRF from endothelial cells in both bovine pulmonary arteries and veins is NO. Arterial endothelium seem to release larger amounts of NO than veins (Luscher, et al 1988a/b, Yang et al 1991, Vallance et al 1989a, 1989b). Nitric oxide released by the endothelial cells is a chemically unstable compound with a very short half-life of only 6 seconds (Gryglewski, et al 1988). It reacts readily with oxygen to produce almost inactive compounds (Radomski, et al 1987c, Palmer, et al 1987). It is rapidly inactivated by superoxide anion (0-2), haemoglobin and methylene blue (Martin et al 1985a, Moncada, et al 1986), and its actions on vascular smooth muscle and platelets are potentiated by superoxide dismutase (SOD) (Palmer, et al 1987^
Many potent vasodilator agents such as acetylcholine, bradykinin, calcium ionophore A23187, histamine, thrombin, serotonin, substance P and adenine nucleotides require the endothelium to be intact to have such effects (Furchgott, 1983). This endothelium dependent vasodilation is mediated by nitric oxide.
2.1 Biosynthesis of Nitric Oxide
In 1988 Palmer et al showed the amino acid L-arginine was the precursor for the synthesis of NO by vascular endothelial cells. They observed that the release of EDRF from cultured endothelial cells induced by bradykinin was diminished by depriving the cells of L-arginine. The release of EDRF could be restored by L- but not D-arginine. (Palmer, et al 1988a). Using radio-labelled amino acid, it has been shown that NO is generated from the metabolism of L-arginine to L-citrulline (Palmer and Moncada, 1989). The synthesis of EDRF from endothelial cells is catalysed by a cytosolic enzyme, which has now been called NO synthase (NOS). This enzyme converts L- arginine to NO and generates equal amounts of citruUine (Mayer, et al 1989, Mulsch, et al 1989, Moncada and Palmer, 1990). Chapter 1 35
2.2 Biochemistry of Nitric Oxide
During the past few years, a family of NO producing enzymes, the NO synthase (NOS) have been purified, cloned and partially characterised (Nathan 1992, Marietta, 1993 and Mayer, 1993). All NOS isozymes described so far are biochemically similar and catalyze an NADPH-dependent conversion of L-arginine into L-citrulline and NO. The NADPH-derived reducing equivalents, which are required for reductive activation of molecular oxygen, are shuttled through the enzyme-bound flavins FAD and FMN to a thiol-ligated, catalytically active prosthetic heme group. Thus, NOS appear to represent self-sufficient cytochromes P-450 with the reductase and oxygenase domains located within one single polypeptide chain (Sheta, et al 1994). However, NOS is unique among cytochromes P-450 requiring the pteridine tetrahydrobiopterin (H4 biopterin) as cofactor, which remains bound as a prosthetic group to NOS during enzyme purification (Mayer, et al 1991 and Pollock, et al 1993). Understanding is still limited concerning the mechanism of L-arginine oxidation and the role played by the heme and H4biopterin in oxygen activation. There is evidence that H4 biopterin may be both an allosteric effector and a redox-active cofactor of NOS (Back,, et al 1993 and Klatt, et al 1994). The diagrams on the next pages shows the synthetic and effector pathways for nitric oxide. Chapter 1 36
Nitric oxide/cyclic GMP signal transduction pathway
Hormones Receptor and other \ygands Arginine or ^ arginine-like Precursor 1 Inhibitory Arginine Analogs Calcium/calmo ÎL-NAME, L-NMMA) dulin Magnesium NADPH Tetrahydrobio pterin EDRF/NO FMN, FAD Synthase Thiols (Types i-iv) V J
EDRF (Guanylyl Cyclase-Activating-Factor Endogenous Nitrovasodilator)
Oxidising or reducing agents and t Vy^cavenger^ Active Inactive <£) Soluble Soluble _ Guanylyl Guanylyl Cyclase Cyclase
OTP cyclic GMP Chapter 1 37
At sub saturating concentrations of L-arginine or H4biopterin, reductive activation of molecular oxygen uncouples from the NO synthesis, leading to NOS-catalyzed generation of superoxide and H 2O2 (Heinzel, et al 1992 and Culcasi, et al 1994). This uncoupled oxygen activation may be of particular relevance to the pathophysiology of NO, because NO and superoxide rapidly react to form peroxynitrite, a molecule which exhibits cytotoxic properties, presumably due to generation of hydroxyl radical-like species at physiological pH (Beckman , et al 1990 and White, et al 1994).
2.2.1 Types of Nitric Oxide Synthase
Recently it has become apparent that there are at least two types of NO synthase: a constitutive enzyme and an inducible enzyme. The constitutive enzyme is Ca2+ calmodulin dependent, requires NADPH as coenzyme and two molecules of tightly bound flavin per molecules of enzyme. The inducible enzyme is Ca^+ independent, is activated by cytokines and synthesises substantially greater quantities of NO for longer periods (Moncada, et al 1991).
It has also been demonstrated that platelets produce NO and the L-arginine: NO pathway acts as a negative feedback mechanism to regulate their aggregation. The formation of NO from L-arginine in platelets is also Ca2+ dependent and requires the presence of NADPH (Radomski, et al 1990b). Isoforms of NO synthase are currently being cloned and their molecular masses range from 125 to 160 kDa (Bredt, et al 1991).
2.2.2 Other Roles for L-Arginine
When investigating the existence and role of NO formation in different organs, it will be necessary to take into account that L-arginine, besides being the precursor of this pathway, is also a constituent of proteins, participates in amino acid metabolism, and is required for the biosynthesis of polyamines. Chapter 1 38
There are, however, a number of other biological actions of L-arginine which may be mediated by the L-arginine: NO pathway. Administration of L-arginine induces the release of growth hormone (Merimee, et al 1965), prolactin (Rakoff, et al 1973), insulin (Dupre, et al 1968) and glucagon (Palmer, et al 1975) in a number of species both in vivo and in vitro. L-arginine also stimulates the release of pancreatic somatostatin (Utsumi, et al 1979), pancreatic polypeptide (Weir, et al 1979), adrenal catecholamines (Imms, et al 1969) and vasopressin (Eriksson, et al 1982). There is circumstantial evidence to suggest that at least one of these actions of L-arginine may be mediated via the formation of NO, since rats starved for 48 hours show significantly reduced insulin release in response to glucose and also have lowered endogenous levels of cGMP. Furthermore, analogues of cGMP potientate the insulin response to glucose in pancreatic islets from starved rats, but not in those from fed rats (Laychock, et al 1983). L-arginine increases skin allograft rejection (Rettura, et al 1979a) and tumour regression (Rettura, et al 1979b) and decreases tumour recurrence in animals (Seifter, et al 1980). These actions could be a result of enhancement NO generation by macrophages or may be due to the thymotropic effect of L-arginine.
2.2.3 Effector Pathway for Nitric Oxide
The vascular responses elicited by NO are due to the activation of soluble guanylate cyclase leading to the accumulation of guanosine 3;5;-cyclic monophosphate (cGMP) in the target cells (Gruetter, et al 1981). The target of NO is the haem moiety of soluble guanyl cyclase, to which it binds avidly leading to increased cGMP levels. cGMP- dependent phosphorylation of the myosin light chain is responsible for mediating the vasodilating effects of NO.
2.2.4 Deactivation and Inhibition of Nitric Oxide Under Both Physiological and Experimental Conditions.
Nitric oxide’s actions as an inter and intracellular signal depend on its having only a Chapter 1 39 limited period of action. The limited half-life of NO is an advantage in terms of its roles as a local vasodilator and in the respiratory burst, but at the same time the variety of possible reactions makes it difficult to identify precisely the factors most important to its half-life in vivo. Unlike other physiological transmitters, there is no proposal that a specific deactivating enzyme or factor even exists for NO. Nitric oxide reacts readily with oxygen, Hb02, and perhaps many normal constituents of cells. These reactions are favoured by the fact that NO is a free radical.
2.2.4.1 Haemoglobin
Haemoglobin acts as an inhibitor of endothelium dependent relaxation (Ignarro, et al 1987a,b and Beny, et al 1989). It inhibits the increase in cGMP associated with relaxation, its inhibitory action can be readily reversed by washout from the arterial preparation. Haemoglobin inhibits endothelium derived relaxation by reacting with NO before the latter gains access to the guanylate cyclase of the smooth muscle cell (Ignarro, et al 1987a,b, and Beny, et al 1989}. Myoglobin is also a potent inhibitor, but methemoglobin is a relatively weak inhibitor (Ignaixo et al 1980, Ignarro and Gruetter, 1980 and Gruetter, et al 1981).
2.2.4.2 Superoxide
The superoxide anion (02 ) generated from the reduction of 02 is responsible for the rapid inactivation of NO. An other potent inhibitor of endothelium dependent relaxation is 6-anilino-5,8-quino-linedione (LY83583), which is not a reducing agent, but on interaction with vascular tissue it stimulates the formation of 02-, which then can inactivate NO (Gryglewski, et al 1986 and Moncada, et al 1986).
2.2.4.3 Methylene blue
Methylene blue inhibits guanylate cyclase, and thereby inhibits both endothelium Chapter 1 40 dependent relaxation of arteries and also the increase in cyclic GMP (Gruetter, et al 1981, Ignarro, et al 1982, Martin et al 1985, Wood and Ignarro 1987, Palmer and Moncada, 1989). Since then the compound has been frequently used as an alleged specific inhibitor of soluble guanylyl cyclase in various biological systems (Mayer, et al 1993).
2.2.4.4 Reducing Agents
A number of reducing agents (hydroquinone, dithiotheritol, phenidone, and pyrogallol) are inhibitors of endothelium dependent relaxation, and it has been shown in bioassay that these agents inactivate NO in its transit from endothelial cells to smooth muscle (Marklund and Marklund, 1974, Hutchinson, et al 1987 and Misra 1974).
2.2.5 Analogues of L-arginine (Inhibition of NO Synthesis)
Nitric oxide is synthesised from the guanidino nitrogen atom(s) of L-arginine. Many analogues of L-arginine have been characterised as inhibitors of endothelial NO synthase (Rees, et al 1989a). The inhibitors are stereospecific and have different potencies but are usually reversible by excess L-arginine (Palmer, et al 1988a, Rees, et al 1989a and Gold, et al 1989b). NO inhibitors do not affect the responsiveness of target cells to other sources of NO, such as sodium nitroprusside or glyceryl trinitrate (Rees, et al 1989b). This implies a simple competitive inhibition at the active site of the NO synthesising enzyme and other investigations of the inhibition characteristics in vitro have reached the same conclusion. A number of variations between species in the sensitivity of tissues to the inhibitors and reversibility by L-arginine may be due to cell specific differences in the turnover of the compound via diffusion, transport or metabolism.
Other L-arginine analogues have been reported to be inhibitors of NO synthesis in vitro and in vivo, including No nitro-L-arginine (L-NNA), N-iminoethyl-L-omithine (L- Chapter 1 41
NIO) and Ng nitro-L-arginine methyl ester (L-NAME), all these compounds essentially reproduce the actions of L-NMMA in vascular tissue both in vivo and in vitro (Ishii, et al 1990 and Moncada, 1992).
2.3 The Constitutive Enzyme
In normal blood vessels, NO is produced in the endothelial cells, by nitric oxide synthase, which is expressed constitutively and releases low or intermediate amounts of NO for short periods of time. The activation of endothelial cells by hormones, neurotransmitters, physical and chemical stimuli triggers the production of NO, possibly following an increase in the intracellular concentration of free calcium (Moncada, et al 1991). Nitric oxide then diffuses towards the lumen, at the interface between the blood and the vessel wall, there it will inhibit the activation and adhesion of blood cells (eg platelet, neutrophils (Assoian, et al 1986). Nitric oxide will also diffuse towards the underlying smooth muscle causing relaxation of the smooth muscles resulting in vasodilation (Gruetter , et al 1979 and Garg, et al 1989) and prevent the proliferation of smooth muscle cells.
2.3.1 Physical Mediators of the Constitutive Enzyme for Nitric Oxide
The continuous release of NO is mainly brought about by a stimulatory effect of the blood flow on the endothelial cells. Increases in blood flow induce endothelium- dependent dilatation (Rubanyi, et al 1986a) which is mediated by the constitutive enzyme for NO (Pohl, et al 1991) and /or by endothelium-derived vasodilator prostaglandins (Koller and Kaley, 1990). Chapter 1 42
2.3.1.1 Shear Stress
An increase in blood flow is associated with increased shear stress acting on the endothelial cells, which is the stimulus for NO formation (Pohl, et al 1986 and 1991, Cooke, et al 1990 and Melkumyants, et al 1987). This adaptation leads to coordinated dilation in arteries of all size (Griffith, et al 1987). Furthermore, the shear induced release of NO plays an important role in the control of myogenic blood vessels constriction. Myogenic constriction occurs in response to increases in transmural pressure and contributes significantly to autoregulation of blood flow. It has been shown that inhibition of NO or NO synthesis, by abolishing the negative feedback, shear-induced release of NO (Kuo, et al 1990 and Pohl, et al 1991), enhances myogenic constriction of blood vessels. Impaired endothelial function is associated with reduced vascular conductivity under conditions of increased pressure (Kostic and Schrader, 1992).
2.3.1.2 Local Mediators
A number of substrates produced and released in the tissues (arachidonic acid, bradykinin, histamine, substance P) can evoke endothelium-dependent relaxation. The endothelial cells may contribute to the vasodilatation during local inflammation (arachidonic acid, bradykinin, histamine) or accompanying axon reflexes (substance P).
2.3.1.3 Hormonal regulation
Substances circulating in the blood may evoke the release of endothelium-dependent responses. For example, the endothelial cells of a number of blood vessels contain a adrenoceptors that can cause the release of endothelium-derived relaxing factor when activated by a2-adrenoceptor agonist (Miller and Vanhoutte, 1985). This could contribute to the vasodilator effect that circulating catecholamines have in certain Chapter 1 43 vascular beds (eg coronary, splanchnic), like vasopressin causing endothelium- dependent relaxation in cerebral arteries but not in peripheral blood vessels (Katusic, et al 1984). This raises the possibility that endothelium-dependent responses to vasopressin may help to favour the redistribution of blood flow in the cerebral circulation.
2.3.1.4 Products of Coagulation and Platelet Products
ADP and thrombin were the first blood constituents shown to trigger endothelium- dependent responses (Furchgott and Zawadzki, 1981, DeMey et al, 1982b). It was later shown that aggregating platelets and, among the products they release, adenine nucleotides and serotonin, can also evoke endothelium-dependent relaxations (Cohen, et al 1983, Azuma, et al 1986 and Vanhoutte, 1988). The fact that platelets (both from animals and humans) can evoke the release of NO explains why the presence of the endothelium considerably reduces the ability of aggregating platelets to evoke contraction of isolated blood vessels (Cohen, et al 1983, Houston, et al 1986, Shimokawa, et al 1987). This must contribute to the protective role of the endothelium against intraluminal platelet aggregation and thrombus formation, whereas the absence of the endothelium favours the occurrence of vasoconstriction in response to platelet products.
2.3.1.5 Neurotransmitters
In large blood vessels, the acetylcholine released from cholinergic nerves cannot reach the endothelial cells. However it was reported by Bumstock G (1987) that certain endothelial cells contain the enzyme choline acetyltransferase, in addition to containing ATP, serotonin, or substance P. This has raised the possibility that these endothelial cells could act as sensors such that, when triggered, they could release substances, such as Ach, which in turn could act on the neighbouring endothelial cells to release endothelium-derived relaxing factor, causing inhibition of the underlying vascular Chapter 1 44
smooth muscle (Bumstock, et al 1987).
2.4. Effects of Nitric Oxide on the Vascular Tone
Endothelium-dependent relaxation has been demonstrated in vitro in a number of blood vessels of various species, including human. In many cases, at least part of this relaxation is inhibited by NG-methyl-L-arginine (L-NMMA) or other analogues of L- arginine in an endothelium-dependent way, which shows that relaxation is mediated by endothelium-derived NO. Nitric oxide synthase inhibitors have been shown to increase mean systemic arterial blood pressure in anaesthetised rabbits and rats (Rees, et al 1989a,b). Nitric oxide is released into coronary circulation of experimental animals under basal conditions and following stimulation of endothelium with agonists (Amezcua, et al 1988, Kelm and Schrader, 1990, Woditsch and Schror, 1992 and Berti, et al 1993). Constitutive nitric oxide synthase in coronary endothelial cells is probably the predominant source of NO in the heart, but NO release in de- endothelialised hearts has not yet been measured. NO synthesis in cultured endothelial cells can be inhibited by L-NMMA (Palmer and Moncada, 1989 and Mayer, et al 1989). An endothelium-dependent constriction of rabbit aortic rings by L-NMMA has been reported, which also could be reversed by an excess of L-arginine (Palmer, et al 1989a,b). An intravenous infusion of L-NMMA but not D-NMMA increases blood pressure and this rise in blood pressure is reversed by L-arginine but not D-arginine (Rees, et al 1989a, Aisaka, et al 1989). These findings suggest that EDRF is released constitutively from some vascular beds. In man, vasodilatation induced by bradykinin or acetylcholine can be inhibited by L-NMMA, and infusion of L-NMMA into the brachial artery induced vasoconstriction (Vallance, et al 1989a,b). In contrast, intravenous L-NMMA does not constrict the dorsal hand vein (Vallance, et al 1989a,b). These data suggest that the continuous basal production of NO exists only on the arterial vessels (Vallance, et al 1989a,b). Chapter 1 45
2.4.2 Assessing Nitric Oxide-Related Dilation
A strong correlation has been shown between the impairment of constitutively expressed endothelium-dependent relaxation of vascular smooth muscle and the level and duration of hypertension. Inhibition of NO synthase increases blood pressure as a result of resistance vessel vasoconstriction (Moncada, et al 1991). Several groups have reported that endothelium-dependent vasodilation is reduced in hypertensive animals models but not in man (Winquist, et al 1984, Luscher, et ale 1988a, Otsuka, et al 1988). This leads to reduced resting organ blood flow, which is most prominent in skeletal muscle, the intestinal vascular bed and in the renal circulation (Rees, et al 1989b). Elevated pressure and reduced organ blood flow are maintained as long as NO synthesis is inhibited (Hussain, et al 1992).
As NO is not the only a factor able to dilate arteriolar vascular smooth muscle, the degree to which it controls coronary flow in vivo can be difficult to establish. For example, the failure of flow to decrease after administration of an agent blocking NO production could reflect compensation for the cessation of NO production by another vasodilating mechanism rather than the absence of an NO effect (Chu, et al 1991). One approach for assessing NO-related component of vascular reserve in vivo has been to compare the flow increase produced by acetylcholine with that produced by GTN or other NO donors (a non-EDRF-dependent vasodilating agent) in the same vascular bed (Zeiher, et al 1991 and Treasure, et al 1990).
2.4.3 Nitric Oxide Synthase in the Endocardial Lining
Endocardial endothelium, the monolayer of cells lining the heart chambers, is ontogenetically similar to vascular endothelium. Brutsaert and colleagues (1988) were the first to demonstrate that endocardial endothelium was an important modulator of the performance of the subjacent myocardium (Shah, et al 1989 and 1990). This modulation of myocardial contraction by the endocardial lining was shown to resemble Chapter 1 46
the modulation of coronary vascular smooth muscle contraction by the coronary endothelial lining.
2.4.3.1 The Physiological Roles of Endocardial Endothelium
The physiological roles of endocardial endothelium-derived factors have yet to be clarified. In view of its short half-life, NO released from the endocardium endothelium is unlikely to penetrate the myocardium sufficiently for its contraction abbreviating effects significantly to influence global performance. However, there may be important regional effects where the myocardium is thin like in the atria, part of the right ventricle and in the foetal heart. The main role of NO released by the endocardial endothelium may therefore be a local one to inhibit platelet adhesion and thrombus formation in the heart chambers (Henderson, et al 1992 and Smith, et al 1992). The contraction prolonging factor endocardin, which is much more stable, could influence myocardial contraction both by direct diffusion and by being carried in the blood from the heart into the coronary circulation.
2.4.4 Inhibition of Platelet Adhesion
Endothelium-derived NO also inhibits the adhesion of platelets to the blood vessel wall and their aggregation and, possibly, cardiac contractility; these effects are mediated by activation of guanylate cyclase that results in the accumulation of cyclic GMP (Azuma, et al 1986, Dusting, et al 1987, Shimokawa, et al 1987, Furlong, et al 1987 and Radomski, et al 1987a).
The effect of NO is strongly synergistic with that of prostacyclin (Dusting, et al 1987 and Radomski, et al 1987b). Thus, the combined secretion of NO and prostacyclin by the endothelial cells at the interface constitutes a powerful antiaggregatory barrier. This NO is a pluripotent molecule that keeps the vasculature in a relaxed state and protects the intima from platelet aggregates, but may also be an important protective factor Chapter 1 47
against vascular damage produced by adherent white cells and against smooth muscle proliferation.
2.4.5 Regulation of Leukocyte Interaction with Vessel Wall
Nitric oxide is also involved in the regulation of leukocyte interaction with vessel wall, since it inhibits leukocyte activation in vitro and in vivo (Kubes, et al 1991). NO can inhibit leukocyte adhesion to vascular endothelium by either interfering with the ability of the leukocyte adhesion molecule CD11/DC18 to form an adhesive bond with the endothelial cell surface or by suppressing CD ll/CD 18 expression on leukocytes as they roll along the endothelial surface (Kubes, et al 1991).
2.4.6 Mitogenesis and Proliferation of Vascular Smooth Muscle Cells
Furthermore, NO has been shown to inhibit mitogenesis and proliferation of vascular smooth muscle cells, apparently by a cGMP-mediated mechanism (Garg and Hassid, 1989). By inhibiting the proliferation of vascular smooth muscle and their production of matrix molecules, NO could protect against a later step in atherogenesis, fibrous plaque formation.
2.5 The Inducible Nitric Oxide Synthase
Injuries (eg mechanical, chemical, viral, bacterial) of the vascular system causes the release of mediators such as interleukin-Ip (IL) and tumour necrosis factor (TNF) by inflammatory cells. These mediators induce a second isoform of NO synthase in most vascular cells (eg endothelial cells, fibroblasts, smooth muscle cells (Nathan, 1992)). The induced NO synthase causes the production of larger amounts of NO for longer period of time from L-arginine, than that produced by constitutive type (Nathan, 1992). Chapter 1 48
A high output synthetic pathway that produces nanomolar concentrations of NO could be cytotoxic, whereas a low-output pathway producing picomolar concentrations could act as a signal transduction system (Hibbs, et al 1987,1988 and Nathan, 1991).
It has recently been reported that inducible NO synthase is present in neutrophils (McCall, et al 1991), hepatocytes (Curran, et al 1989), endothelial cells, vascular smooth muscle cells (Radomski, et al 1990a, Rees et al 1990b, Beasley, et al 1991) and fibroblasts (Wemer-Felmayer, et al 1990). NO in the induced form is mediated through binding to an iron atom, the iron-sulphur cluster -dependent enzymes involved in mitochondrial electron transport and cis-aconitase (Drapier, et al 1988 and Hibbs, et al 1988). The production of NO in response to injury is likely to play a pivotal role in the host’s defence against intruders. NO is able to kill foreign organisms and abnormal cells, (Granger, et al 1990 and Hibbs, et al 1988) at these increased levels.
Recent observations indicate that constitutive and inducible NO synthase isoforms are not confined to separate cell types but occur in the same cell. For example, macropages can express large amounts of the inducible, cytosolic calcium-independent enzyme activity, but also contain some membrane-bound calcium-dependent NOS (Forstermann, et al 1993).
2.5.1 Inducible Synthase in the Macrophages, Neutrophils and Lymphocytes
It is clear that the inducible NO synthase is highly regulated by cytokines. While IFN- Y, IL-1, IL-6 and TNF are inducers of the enzyme, alone or in combination, in different systems (Drapier, et al 1988, Beasley, et al 1991, Denis, 1991 and Schini, et al 1991a). It is likely that in different systems, different combinations of these cytokines determine the biological outcome in terms of the production of NO.
The macrophage NO synthase was also found to be one of very few eukaryotic Chapter 1 49
enzymes containing both FAD and flavin mononucleotide (Stuehr, et al 1991). The NO pathway in macrophages has been proposed to be a primary defence mechanism against tumour cells and intracellular micro-organisms, as well as pathogens (Hibbs, et al 1990a,b). However, the biological significance of NO production by neutrophils remains to be elucidated. It is possible that expression of NO synthase in neutrophils may contribute to their cytotoxicity (Shalaby, et al 1985, Djeu et al 1986 and Kumaratilake, et al 1991 )
Lymphocytes, when stimulated with interleukin-2 (IL-2), syntheses NO (Kirk et al 1990). Also human peripheral blood mononuclear cells syntheses NO during mitogenic proliferation, and NO appears to promote lymphocyte DNA synthesis (Efron, et al 1991). Increasing evidence suggests that NO might play a role in inflammation either through its vasodilator properties or through the modulation of leukocyte behaviour (Hughes, et al 1990, lalenti, et al 1992).
2.5.2 The Formation of NO by the Inducible Synthase in Tissues and Cells
Apart from the constitutive nitric oxide synthase enzyme, which is present in endothelial cells, it has been shown that the inducible isozyme may also be localised in endothelial cells or in other cells of the vascular wall (Radomski, et al 1990b, Rees, et al 1990c Knowles, et al 1990b). Recent evidence has demonstrated the existence of an inducible NO synthase enzyme in the coronary circulation and the myocardial cells (Schulz, et al 1992). The inducible NO synthase enzyme is expressed in the presence of endotoxin or cytokines, and plays a role in the depressed cardiac function observed in endotoxic shock or in acute myocarditis or allograft rejection. Exposure of various cells types to bacterial lipopolysacharides (endotoxin) results in the induction of an L- arginine dependent NO pathway which appears to be a major factor associated with the hyporeactivity of arteries of a large range of vasoconstrictor agents (Stoclet, 1993). In agreement with this, inhibition of NO synthesis can restore peripheral vascular Chapter 1 50 resistance in experimental animals and man (Petros, et al 1991). Whether the coronary circulation also contains inducible NOS without prior exposure to endotoxin, is not clear. It has also been reported by Moncada et al (1992) that the isolated Langendorff perfused rabbit hearts treated with LPS have a dilated coronary circulation and show a hyporesponsiveness to vasoconstriction. Both phenomena are dependent on the induction of an NO synthase in the coronary vasculature (Smith, et al 1991a). The inducible isozyme may be localized in the endothelial cells or in other cells of the vascular wall.
2.5.3 Inhibition of the Inducible NO Synthase
Production of NO by activated macrophages is effectively inhibited by L-NMMA (Ding, et al 1988), and this inhibition can be competitively reversed by L-arginine (Hibbs, et al 1987a). NO in the macrophage is inhibited not only by L-NMMA, but also by L-canavanine (lyenger et al 1987). Also it has been shown that L-NIO is a potent, rapid in onset and irreversible inhibitor of the inducible isoform. (McCall, 1991).
Glucocorticoids inhibit the expression of the inducible form of NO synthase in endothelial cells, vascular tissue and macrophages after stimulation with LPS or IFN-y (Radomski, et al 1990a, Ree et al 1990b).
2.6 Response to Over Stimulus of NO by Inducible NOS 2.6.1 Gross Vasodilation
Overproduction of NO by the inducible NO synthase within vascular wall contributes to the profound hypotension and resistance to vasoconstrictor agents that characterises endotoxic shock. The mechanism of induction of NO by endotoxin probably involves the cytokines interleukin-1 and tumour necrosis factor (TNF). Induction of NO can take Chapter 1 51
several hours; corticosteroids can inhibit this induction but only if given before or at the same time as the first exposure to endotoxin (Moncada, et al 1991: review). Competive inhibition of NO synthase with L-arginine analogues reduces the hyporesponsiveness of isolated vascular tissues to pressor agents and the hypotension induced by bacterial infection or cytokines in animals and man (Nava, et al 1991 and Petros, et al 1991).
2.6.2 Peroxynitrite Radical
It is likely, that in some situations NO might interact with O 2 derived radicals to generate molecules that might enhance its cytotoxicity. There is evidence that such an interaction occurs between NO and superoxide anions to generate peroxynitrite radical, which when protonated will release OH- and NO2 (Beckman, et al 1990 and Hogg, et al 1992) (see chapter 1 section 2 for a more detailed account of the peroxynitrite radical).
2.6.3 Cellular Toxicity
Several groups have shown a cytostatic/cytotoxic effect of macrophage-derived NO on both microorganisms and tumour cells (Hibbs, 1987 and 1988). A high output synthetic pathway that produces nanomolar concentrations of NO could be cytotoxic, (Hibbs, 1987, 1988, Moncada, et al 1989 and Stuehr, et al 1989). The cytotoxic properties of NO demonstrated in experiments with tumour cells in which NO binds to an iron atom in the iron-sulphur cluster at the catalytic site of certain mitochondrial enzymes (Drapier, et al 1988), leading to intracellular iron loss. High levels of NO impair the function of mitochondrial and other FeS-containing enzymes (Drapier, et al 1988 and Geng, et al 1992), inhibit aconitase and ribonucleotide reductase, inhibit DNA synthesis and cause cytostasis (Hibbs, et al 1988 and Lepoivre, et al 1992).
It is clear that NO released by the inducible enzyme for long periods is itself cytotoxic. Indeed, NO is not only cytotoxic for invading microorganisms but can be cytotoxic for the cells which produce it and for neighbouring cells (Lepoivre, et al 1989 and Palmer, Chapter 1 52 et al 1992).
The cytotoxicty of NO has been demonstrated in rat pancreatic cells (Kroncke, et al 1991) and in hepatocytes that are killed by NO generated by Kupffer cells (Biller, et al 1989 and Curran, et al 1989). Also it has recently been reported that brain damage occurring after cerebral infarction is due to the excessive release of NO by a minority of NO-resistant neurons, and that further neuronal damage is preventable by inhibition of NO synthase activity (Gaily, et al 1990, Nowicki, et al 1991 and Buisson, et al 1992).
In some cells NO is cytotoxic and in others cytostatic (Hibbs, et al 1990a/b), suggesting that the sensitivity to NO may vary from one cell to another. The reasons for this are not clear. However, some selectivity may reside in the amounts of NO produced; a high output synthetic pathway that produces nanomolar concentrations of NO could be cytotoxic, whereas a low-output pathway producing picomolar or femtomolar concentrations could act as a signal transduction system (Hibbs, et al 1987 and 1988).
2.6.3.1 Nitric Oxide Synthase in Septic Shock
Septic shock in response to generalised infections with Grem-negative, but also Grem- postive bacteria is seen in immuno compromised patients. It is characterised by peripheral arteriolar vasodilatation, hypotension, microvascular damage and inadequate tissue perfusion leading to organ dysfunction and a mortality. The mechanism by which endotoxaemia causes the above vascular effects is not completely understood. A number of mediators are elevated in septic shock and have been implicated in its pathophysiology. These include platelet-activating factor, thromboxane Az, prostanoids, and cytokines such as interleukin-1, tumour necrosis factor-a and interferon-y. A number of studies reported in patients with serious septic shock in whom intravenous injection of L-NMMA (Petros, et al 1991) or L-NAME (Petros, et al 1991) caused a rapid and dose-dependent correction of hypotension in these patients. Chapter 1 53
In agreement with this, inhibition of NO synthesis can restore peripheral vascular resistance in experimental animals treated with endotoxin (Petros, et al 1991).
2.6.3.2 Antimicrobial Actions
Nitric oxide contributes to the cytotoxic and antimicrobial actions of macrophages activated by various immunological stimuli (Hibbs, et al 1990b). Macrophages activated with TFN-y and LPS have been shown to have a powerful cytostatic effect on the fungal pathogen (Granger , et al 1986) and protozoan, the microbiostatic effect being dependent on L-arginine, and inhibited by the presence of L-NMMA. Macrophages similarly activated can also kill the extracellular helminth in vitro and the intracellular protozoa by means of NO in vitro and in vivo (Mauel, et al 1991).
2.7.1 The Role of Nitric Oxide in Autoimmunity and Inflammation
Evidence exists that NO has an immunoregulatory role besides its action as in immune defence molecule at the endothelial layer; NO modulates leucocyte adhesion, an important process in tissue inflammation (Kubes, et al 1991). L-arginine supplementation enhances natural killer cell and lymphokine-activated killer cell activity, in vivo and in vitro (Park, et al 1991). The suppression or reduction in allogenic and mitogenic T-cell proliferation by macrophages was recently found to be mediated by NO release (Hoffman, et al 1990). A higher activation state of macrophages is often seen in autoimmune diseases (Rothe, et al 1990).
Increasing evidence suggests that NO might play a role in inflammation either through its vasodilator properties or through the modulation of leukocyte behaviour (Hughes, et al 1990, lalenti, et al 1992). The origin of NO in the inflammatory process is not clear, but it is likely to come from at least the blood vessels, the neutrophils and the Chapter 1 54 macrophages.
3.0 Control of Vascular Tone by Other Vasodilators and Constrictors 3.1 Purines (Adenosine)
Adenosine is an endogenous nucleoside which possesses numerous physiological properties, several of which are thought to be beneficial to the ischaemic myocardium (Belardinelli, et al. 1989, Newby, et al. 1990, Pantely and Bristow, 1990 and Stiles, 1991). Adenosine also has a powerful dilator action this being very important in the regulation of blood flow (Nees, et al 1985 and King, et al 1990).
3.1.1 Adenosine Receptors
Adenosine receptors are classified as Ai which inhibit adenyl cyclase, and A 2 which stimulate the adenyl cyclase system. The A i receptors in the heart which has been identified in cardiac tissue by several authors (Hosey, et al 1984 and Clemo, et al 1987) are found on cardiomyocytes and vascular smooth muscle cells (De-Rosiers, et al 1987 and King, et al 1990). The A2 receptors are found on the endothelium and vascular smooth muscle (Nees, et al 1985, King, et al 1990 and Romano, et al 1989).
Adenosine produces multiple effects on the heart. The negative chronotropic and dromotropic action and the inhibition of the inotropic response to catecholamines are mediated by A i, receptors, which have been identified in cardiac tissue. Adenosine has a powerful coronary dilator action and is thought to be of major importance in the regulation of coronary blood flow. Such action is mediated by the A 2 (Nees, et al 1985 and King, et al 1990). Chapter 1 55
3.1.2 Effector Pathway
Adenosine traverses the cell membrane via a nucleoside carrier system that represents a reversible process of simple and facilitated diffusion. Adenosine receptors are integral membrane proteins responsible for binding extracellular adenosine and initiating the transmembrane signal to activate a second messenger system. The activation of effector systems such as adenylate cyclase, phospholipase C, or an ion channel produces a series of biochemical events that result in physiological changes with the cell.
3.1.3 Physiology of Adenosine
Endogenous or exogenous adenosine is removed by phosphorylation by adenosine kinase to AMP, degradation to inosine by adenosine deaminase or washout in the circulation. The short half-life of adenosine on release from the cell makes it highly improbable that adenosine acts as a circulating hormone but rather as a local regulator.
Adenosine can protect the heart (Bumstock, 1980) from the deleterious effects of an inadequate blood flow and oxygen supply. All cells under stress release adenosine, which then can feed back in an autocrine manner to modulate the function of the cell. Endogenous adenosine originates from the extracytoplasmic hydrolysis of adenine nucleotides (ATP, ADP, AMP) by ectonucleotidases or, to a greater extent, from cytoplasmic degradation of nucleotide precursors.
3.1.4 Adenosine During Early Ischaemia
Adenosine has been shown to decrease the release of noradrenaline from sympathetic nerves during early ischaemia and to reduce the positive inotropic effect of catecholamines mediated via the adenyl cyclase system. Both of these effects would reduce oxygen demand during ischaemia and decrease the rate of ATP depletion. Chapter 1 56
Adenosine has also been shown to increase glucose transport during ischaemia and by this mechanism may increase myocardial energy production. Finally adenosine has been shown to inhibit superoxide anion production from neutrophils, inhibit platelet aggregation and microthrombi formation in the capillaries, and possibly reduce the no reflow phenomenon. Which of these actions of adenosine, if any, is involved in preconditioning is unknown.
4.0 Prostacyclin
Prostacyclin is synthesised in many tissues though largely by endothelial cells. Arachidonic acid is the precursor for prostacyclin synthesis. It is released from membrane phospholipids by two pathways: phospholipase A2 and phospholipase C. Prostacyclin is a potent inhibitor of platelets aggregation in vitro (Gorman, et al 1977, Tateson, et al 1977) and has strong anti thrombotic effects in vivo (Higgs, et al 1977). This action of prostacyclin is mediated by the stimulation of adenylyl cyclase in platelets (Gorman, et al 1977). Prostacyclin has been demonstrated to produce relaxation in most vascular smooth muscle (Bunting, et al 1976), however, there are exceptions such as rabbit aorta or rabbit femoral artery on which prostacyclin has no effect (Forstermann, 1990). Prostacyclin increases renal blood flow which may be responsible for its natriuretic action (Bolger, et al 1978, Hill and Moncada, 1979).
Prostacyclin and its stable analogues suppress the accumulation of cholesterol esters in vascular wall and macrophages, and inhibit the release of growth factors from endothelial cells, macrophages and platelets (Kappus, 1986, Willis, et al 1986). Prostacyclin potentiates streptokinase-induced thrombolysis in vivo and appears also to facilitate the endogenous production of tissue plasminogen activator (Musial, et al 1986).
Cytoprotective effects of prostacyclin have been shown in several cell types such as platelets (Blackwell, et al 1982), cardiac myocytes (Escoubet, et al 1986) and Chapter 1 57 hépatocytes (Bursch and Schulte-Hermann, 1987), and histoprotection has been observed also in ischaemic skin ulcers (Belch, et al 1983) and gastrointestinal ulcers (Miller and Jacobson, 1979).
4.1 Endothelium-Derived Hyperpolarizing Factor
Before 1980, when EDRF was recognised, it was known that acetylcholine could hyperpolarize vascular smooth muscle (Kuriyama and Suzuk, 1987, Kitamora and Kuriyama, 1979). There is evidence that some agents such as acetylcholine, that induce endothelium-dependent relaxation, also produce endothelium-derived hyperpolarizing factor, EDHF (Bolton and Clapp, 1986 and Mekata, 1986). In contrast to EDRF, the nitrovasodilators do not hyperpolarize vascular smooth muscle. Therefore it is proposed that EDRF involves two factors, one which activates guanylate cyclase in smooth muscle and the other which hyperpolarizes the membrane (Bolton et al, 1986).
5.0 Abnormal Endothelial Function in Disease 5.1 Ischaemic and Reperfusion Injury
Reperfusion of ischaemic tissue may terminate ischaemic injury, but further myocardial injury occurs during the reperfusion phase. Whereas the deleterious effects of ischaemia and reperfusion on myocardial contractile function have been recognised for some time (Opie, 1992), recently a number of studies also point to the potentially detrimental effect of ischaemia and reperfusion on endothelial cell function (Lefer et al 1991 and Sobey and Woodman, 1993). Endothelial dysfunction occurs early in myocardial ischaemia. Ischaemic episodes cause decreased endothelium dependent vasodilation in isolated vessels and in intact vascular beds (Tsao and Lefer, 1990a), together with enhanced coronary artery constrictor responses. This is a complex process in that it can be minimised by early reperfusion, but paradoxically may be at least in part caused as a result of reperfusion (Kin, et al 1992). The reperfusion that Chapter 1 58 follows transient coronary occlusion is associated with neutrophil influx with adhesion and injury to endothelial cells (Brady, et al 1992 and Tsao, and Lefer, 1990). (For further experimental evidence for the role of endothelium during ischaemia and reperfusion see chapter 1 section 3.0).
5.2 Atherosclerosis
Endothelium-dependent relaxations are often decreased in atherosclerotic arteries in response to ACh and other vasoactive substances, even though responses to endothelium-independent vasodilators remain fully active (Freiman, et al 1986 and Bossaller, et al 1987). The reduced NO release may predispose to thrombosis and vasospasm (Luscher , et al 1993 and Chester, et al 1990). Elevated vascular tone resulting from impaired relaxation (a dynamic coronary stenosis) may become crucial if an additional local injury occurs (Vanhoutte, 1988 and Clarke, et al 1990). The damaged endothelium regenerates quickly but afterwards may show paradoxical reactions to vasorelaxants, resulting in vasoconstriction. Endothelial dysfunction has also been observed in the coronary circulation of hypercholesterolaemic animals and humans (Drexler, et al 1991) and in the human forearm vasculature (Creager, et al 1990 and Chowienczyk, et al 1992). Both low density lipoprotein (LDL) and oxidised LDL inhibit the relaxation response to exogenous NO, and oxidised LDL also inhibits endothelium dependent responses (Jacobs, et al 1990 and Plane, et al 1992).
5.3 Hypertension
A decrease in endothelium dependent relaxation has been demonstrated in vessels from hypertensive animals (Luscher, et al 1988a) and in a variety of experimental models of hypertension (Winquist, et al 1984). Inhibition of NO synthase causes a rise in mean arterial blood pressure of approximately 40% in animal models, suggesting that normal blood pressure control involves the continuous release of NO (Rees, et al 1989). Combined with the evidence above that NO is a major component of the normal control Chapter 1 59
of blood pressure, it is tempting to assume that disturbances in the biochemical sequences of NO synthesis and activities to the vascular site may be involved in certain forms of hypertension or may be one of the fundamental factors in the process of hypertension development. Some forms of hypertension may also be due to a relative deficit of NO as a consequence of increased liberation of constricting factors, such as endothelin, by the endothelium cells.
5.4 Clinical Implications
The findings show that NO is a major determinant of blood vessels tone, platelet and neutrophil function, and that a lack of NO may play a role in the genesis of vasopasm (Vanhoutte, et al 1989) suggests that the L-arginine-NO pathway is a target for drug treatment and prevention of hypertension, shock and atherosclerosis. These observations, together with evidence, for example, of a role for NO in excitatory neurotransmission, the renal response to vasopressin, the control of protein synthesis in hepatocytes and possibly cell-to-cell communication in the adrenal gland high light the potential therapeutic significance of the L-arginine-NO pathway.
6.0 Conclusion
In the last decade a central role for the endothelium in the regulation of vasomotor tone has been defined with both endothelium-derived constrictor and dilator substances being identified. NO is released under physiological conditions by a constitutive, Ca2+ dependent enzyme in response to receptor stimulation.
In the cardiovascular system the release of vasoactive factors acts as a general adaptive mechanism whereby the vascular endothelium responds to changes in its environment and regulates blood flow and blood pressure through an action on the vascular smooth muscle and it may also play a role in the control of vascular smooth muscle proliferation. Chapter 1 60
Section 3.0: Endogenous Mechanisms and Mediators of Myocardial Protection
1.0 Ischaemic Preconditioning 1.1 Introduction
Classical ischaemic preconditioning was detected first by Murry et al (1986), in experiments designed to deplete myocytes of ATP using multiple brief episodes of ischaemia separated by periods of reperfusion. The aim for the study was to deplete ATP to very low levels without allowing the tissue to accumulate a significant load of ischaemic catabolities. It was hypothesised that ATP depletion would accumulate during each ischaemic episode and that the products of ischaemic metabolism would wash out during reperfusion. Intermittent reperfusion did not wash out the osmotic load, but four 10 minute episodes of ischaemia did not result in a cumulative loss of ATP over that which occurred during the first 10 minutes of ischaemia (Reimer, et al 1986).
The experiments of Murry et al (1986) and others have suggested that the metabolic alterations of preconditioning are associated with an increased tolerance to a sustained episode of ischaemia (Geft, et al 1982). In experiments in which the heart was preconditioned with four 5 minute episodes of ischaemia, each separated by 5 minutes of reperfusion, Reimer et al (1986) showed that the intermittent reperfusion appeared to maintain ATP at a level similar to that seen after one occlusion (Reimer, et al 1986). In addition significantly less infarct was observed in the preconditioned myocardium than in control hearts (Reimer, et al 1986). This protection was present even though the tissue had been exposed to 60 minutes of ischaemia compared to 40 minutes of ischaemia in the control hearts. Brief periods of ischaemia and reperfusion prior to the Chapter 1 61 longer ischaemic insult protected the myocardium, reducing the infarct volume to 25% from that seen in the control hearts (Murry, et al 1986).
However, the protection that is oberved as a expression of ischaemic preconditioning is lost when the sustained ischaemic insult is extended. For example no protection was seen in dogs following an ischaemic insult of 90 minutes, whereas at 60 minutes there was still marked protection (Nao, et al 1990). In addition, as reported by Murry and colleagues, the preconditioning effect was developed fully after 5 minutes of reperfusion, but was attenuated if the period of reperfusion was extended to 120 minutes (Murry, et al 1991).
1.2 Protocols Used for Triggering Ischaemic Preconditioning
Preconditioning can be induced using a number of different protocols and occurs in a variety of species. Initially in the dog, four 5 minute coronary occlusions were used (Murry, et al 1986) but single occlusions of 2.2, 5, or 15 minutes have been shown to be equally effective (Murry, et al 1991, L i, et al 1990, Auchampach et.al, 1993). In addition a single 5 minute or 2(x2) minute occlusion are sufficient to precondition rabbit (Cohen, et al 1991) or rat (Li, et al 1992b, Alkhulaifi, et al 1992 and Yellon , et al 1992c) myocardium. Multiple occlusion protocols of 3(x3) minutes or 5 (x3) minutes occlusions (Liu and Downey, 1992 and Li, et al 1992b) have been used to successfully precondition the rat, and in the pig two 10 minute occlusion periods have been shown to precondition myocardium (Schott, et al 1990).
Preconditioning has been demonstrated by many groups using different protocols and is associated with both a preservation of function and an anti-arrhythmic action in either both the in vivo and the in vitro models of myocardial ischaemia (Walker, et al 1992). The protection achieved with preconditioning has also been observed in humans with coronary artery disease (Muller, et al 1990 and Yellon, et al 1993). One such study observed in patients undergoing two sequential 90-second balloon angioplasties of the left anterior descending coronary artery demonstrated that the second episode of Chapter 1 62 ischaemia caused less chest pain, less ST-segment elevation, and less myocardial lactate production than the first (Deutsch, et al 1990). However, this study and others (Cribier, et al 1992) may not only be examining preconditioning but also the effects of recruitment of preformed collaterals, which may attenuate injury during subsequent ischaemic episodes. A report by Yellon et al (1993) showed preconditioning in the human myocardium prior to coronary artery bypass surgery. This study demonstrated a significant effect on the ability of sublethal ischaemia to delay the loss of adenosine triphosphate, in patients following a specific ischaemic preconditioning protocol consiting of two 3 minute periods of global ischaemia with 2 minutes of intermittent reperfusion.
1.3 Other End Points
Ischaemia reperfusion is associated with various other markers of myocardial injury. These include contractile dysfunction, arrhythmias, effects on autonomic nerve conduction and vasomotor function.
1.3.1 Stunning
The mechanical dysfunction that persists after reperfusion despite the absence of irreversible damage has been termed ‘stunning’. It is clear that preconditioning of isolated hearts subjected to global ischaemia results in greater recovery of functional parameters such as left ventricular developed pressure (Cave, et.al 1992 and Asimakis, et.al 1992). Studies from isolated hearts have shown that if functional recovery and infarct size are examined in the same hearts, the improved recovery of preconditioned hearts correlates with infarct size limitation. It is likely, therefore, that most of the benefit is the result of a reduction in infarct size (Walker, et al 1993 and Jenkins, et.al 1995). Chapter 1 63
1.3.2 Arrhythmias
Several investigators have examined the incidence of ischaemia and reperfusion induced arrhythmias following preconditioning (Vegh, et al 1992, Lawson, et al 1993 and Shiki, et al 1986). In a study using the rat heart a dose-dependent reduction in the frequency of ischaemia induced arrhythmias was demonstrated in vitro (Lawson, et.al 1993). An earlier study, again in the rat, was able to show a protective effect of ischaemic preconditioning in terms of a reduced incidence of reperfusion-induced arrhythmias (Shiki, et al 1986).
1.4 Mechanisms of Ischaemic Preconditioning
There are several theories concerning the identity of the signal that triggers intracellular events leading to protection. One of the most popular involves the generation of adenosine by ischaemic tissue. Adenosine is rapidly released from cells under stress, including ischaemic myocytes (Bellardinelli, et al 1989), and is thought to act as a local regulator of cell function via a feedback pathway (Stiles, 1991). During ischaemia, adenosine levels reach the macro molar range (Van Wylen, etal 1990), and its ability to induce cardioprotection against ischaemia-reperfusion injury has been demonstrated (Yao, et al 1994 and Ely, et al 1985). More direct evidence for its involvement in mediating the effects of preconditioning has been derived from several studies (see below).
1.4.1 Adenosine Antagonists and Agonists
Liu et al (1991) reported that treatment with adenosine receptor blockers, 8-p- sulphophenyl theophylline (SPT) and N[dimethylamino ethyl] benzosulfonamide (PD 115,199), 5 minutes before ischaemic preconditioning blocked the protection against subsequent sustained ischaemia and reperfusion in the rabbit hearts (Liu et al 1991, Armstrong, et al 1994 and Tsuchida, et al 1994a). This role for adenosine is supported Chapter 1 64 by several subsequent reports (Kitakaze, et al 1993a,b, Olafsson, et al 1987 and Toombs, et al 1992a) which shown that preconditioning can be triggered by exogenous adenosine or certain adenosine receptor agonists (Liu, et al 1991, Lasley, et al 1990, 1992, Norton, et al 1992, Toombs et al 1992a, Thornton et al 1992a,b).
1.4.2 The Role of Ai Adenosine Receptor Activation
Acadesine (AICA riboside), dipyridamole, and R75231 (Tsuchida, et al 1993a and Itoya, et al 1992), are all agents which augment the adenosine levels in tissue during ischaemia. Normally two minutes of ischaemia is not sufficient to induce the preconditioning phenomenon in the rabbit hearts, however hearts treated with adenosine promotors caused significant reduction in infarct size, mimicking preconditioning in these hearts (Tsuchida, et al 1993a and Itoya, et al 1992). There was no differences in infarct size in hearts not preconditioned but all lowered the threshold for preconditioning. Therefore it was concluded that the threshold for protection was lowered as a result of increased adenosine release during the preconditioning ischaemia.
1.4.2.1 Adenosine Ai Analogs
Three adenosine analogs have been examined: R-PIA, which is about 100-fold selective for Al receptors; 2-chloro-N6-cyclopentyladenosine (CCPA), which is 10,000-fold A i selective, and 2-[4-(2-carboxyethyl)phenethylamino]-5’-N-ethylcarboxamine adenosine hydrochloride (CGS 21680), a 170-fold Az-selective agonist. All of the animals used in these studies were subjected to a 30 minutes coronary occlusion followed by 180 minutes of reperfusion (Kitakaze, et.al 1993a,b,c and Auchampach, et.al 1993). All agonists were given over 5 minutes by intravenous infusion, beginning 15 minutes before a 30 minutes occlusion. Both R-PIA and CCPA reduced heart rate by about 30%, an effect of the A i agonist (Tsuchida, et al 1993c, Liu, et al 1991, Thornton , et al 1992a, Jon, et al 1991, Tsuchida, et al 1992). System hypotension results in reduction of heart rate, which results in hypotension as was seen by the Ai agonists. Chapter 1 65
The A2 agonist CGS 21680 had no effect on heart rate but caused a fall in arterial pressure to a level comparable to that seen with R-PIA (Thornton, et al 1992a and Tsuchida, et al 1993a,c). The cardiac output in these animals was increased, suggesting that the hypotension was due to A2-mediated peripheral vasodilation rather than cardiac depression (Cox, et al 1994). Both R-PIA and CCPA were very protective with significant infarct size reduction. When the A2-selective agonist, CGS 21680, was infused 15 minutes prior to the 30 minutes occlusion, no reduction in infarct size, was seen (42%), (Jon, et al 1991). It has also been observed that protection induced in preconditioning can be enhanced by inhibitors of nucleoside transport (eg dilazep, draflazine), (Van Belle, et.al 1993).
There is now evidence suggesting that adenosine (Al) may be involved in the mechanism of preconditioning in a number of species including the rabbit, dog and the pig. Intracoronary infusion of R-PIA, will limit infarct size in vivo in all these species (Grover, et.al 1992 and Van Winkle, et al 1994), and several adenosine A i antagonists also have been shown to block the protective effect of preconditioning in all these species (Kiakaze, et al 1993 and Auchampach, et al 1993). The role of adenosine receptors in preconditioning has also been shown to be involved in human myocardium. Using isolated superfused strips of human atrial muscle Yellon and Baxter ( 1995) showed that the protection conferred using preconditioning could be blocked by addition of an adenosine Al receptor antagonist SPT. In turn the specific adenosine receptor agonist R-PIA has been shown to mimic the effects of ischaemic preconditioning in the human muscle preparation. ChaptCT 1 66
Whether the protection from myocardial necrosis by ischaemic preconditioning is mediated by adenosine in the rat heart has yet to be firmly established. In a study using recovery of post-ischaemic contractile function as the end point, preconditioning could be prevented by the use of an adenosine Ai antagonist and induced by adenosine (Murphy, et al 1991). However, Liu and Downey (1992, 1993) reported that, while transient intracoronary infusion of an Ai receptor agonist did not limit infarct size, an adenosine receptor antagonist could not block the salvage effect of preconditioning in the rat heart. However, it would seem unlikely that adenosine is an endogenous mediator of ischaemic preconditioning in the rat (Asimakis, et al 1993 and Liu and Downey, 1992 and 1993). Thus there appears to be species differences in the mechanisms of ischaemic preconditioning.
1.5 A Possible Role for the Adenosine As Receptor
Liu and co-workers (1994b) have recently cloned and characterised an adenosine receptor, the As receptor. It is thought to resemble the Ai receptor in recognising substituted adenosine as agonists and it binds prototypical Ai receptor antagonists poorly (Liu, et al 1994b and Linden, et al 1993, Zhou Q-Y, et al 1992). They reported that ischaemic preconditioning is not exclusively mediated by the adenosine Al receptor but may involve the As receptor in rabbit heart. However, further work needs to be done on the As receptor and its role in the myocardium.
1.6 Preconditioning can be Mimicked by Other Agents 1.6.1 Noradrenaline
An alternative hypothesis for signal generation involves the observation that sympathetic neurotransmitter release and consequent a adrenoceptor activation can mimic the protective effects of ischaemic preconditioning. In a study using the rat heart, levels of noradrenaline were found to increase following the period of transient Chapter 1 67 ischaemia used for preconditioning (Baneijee, et al 1993). Noradrenaline release in the heart following a bolus of tyramine mimicked preconditioning in the rabbit and this protection could be blocked by either a Al receptor blocker or SPT {Thornton et al 1993a and Liu, et al 1994a). In addition, noradenaline administered exogenously, in place of the period of transient ischaemia, induced the same degree of protection against a subsequent global ischaemic insult. This effect was abolished in hearts from animals that were pretreated with reserpine to deplete endogenous stores of noradrenaline. Furthermore noradrenaline induced preconditioning was stimulated by phenylephrine and blocked by an al adrenoceptor antagonist. These findings have been confirmed by Bankwala, et al 1994 using an in vivo rabbit heart model. However, a subsequent study, again using the isolated rat heart, found that sympathetic neurotransmission was not the dominant mechanism of preconditioning (Weselcouch, et al 1995).
1.6.2 Bradykinin
The possible involvement of bradykinin in ischaemic preconditioning has been investigated recently by Downey’s group (Goto, et.al 1995). They used both in vivo and isolated rabbit heart models and examined the preconditioning effect by subjecting hearts to 30 minutes of regional ischaemia followed by reperfusion, using infarct size as the end-point They found that bradykinin administered to isolated hearts mimics the effects of ischaemic preconditioning but that neither administration of a nitric oxide synthase inhibitor or a prostaglandin synthase inhibitor affected the development of protection. However, polymixin B and staurosporin, both inhibitors of protein kinase C, abolished the effect. Protection induced by bradykinin was also inhibited by HOE 140, a bradykinin B2 receptor inhibitor. However, this inhibition could be overcome by amplifying the preconditioning stimulus using four cycles of 5 minute ischaemia with 10 minutes of intervening reperfusion. HOE 140 did not abolish the protection induced by an ischaemic preconditioning protocol. In contrast, in the in vivo heart, HOE 140 will abolish the effect of ischaemic preconditioning if given prior to the stimulus. From these observations it seems likely that bradykinin formed in vivo from blood-borne kininogens is released during ischaemic preconditioning and is associated with the Chapter 1 68 development of the protection observed
1.6.3 Endothelium Derived Relaxing Factor (EDRF)
The potential role of mediators derived from the endothelium has also been investigated. Recently, Vegh et al proposed that the L-arginine nitric oxide pathway could be involved in ischaemic preconditioning (Vegh, et al 1992a,b). They found in mongel dogs, the attenuation of ischaemia- and reperfusion-induced arrhythmias by preconditioning was blocked by pretreatment with L-NAME. Furthermore, intracoronary administration of methylene blue also prevents the effects of released NO on soluble guanylate cyclase (Vegh, et al 1992a,b). The protective effect of NO could involve dilation of micro vessels or inhibition of platelet adherence to endothelial cells, both of which could contribute to the anti-arrhythmic and anti ischaemic action of preconditioning (Vegh, et al 1992a). Furthermore, they suggested that the protection by NO generation during preconditioning involved a stimulation of guanylate cyclase and a subsequent elevation of guanosine 3’:5*-cyclic monophosphate (cyclic GMP) in the myocardium. An elevation of cyclic GMP has been suggested to be anti-arrhythmic (Opie, 1982a and Billman, 1990). In addition bradykinin is released early in ischaemia (Kitakaze, et.al 1993c) and has been postulated as a trigger for the release of NO and prostacyclin.
1.7 Intracellular Signalling Pathway 1.7.1 G-proteins
The mechanism by which the agonist mentioned above interact with the cell to cause the generation of an intracellular message is thought to involve coupling of the agonist receptor to G-proteins that span the cell membrane. Evidence in support of this hypothesis has been derived from experiments in which pertussis toxin is used to inhibit the action of G (G)i proteins. There is already considerable evidence that adenosine receptors are coupled to inhibitory Gi proteins (Stiles, et al 1991 and Iwase, Chapter 1 69 et al 1992), and that blockade using pertussis toxin, attenuates protection induced by ischaemic preconditioning in the rabbit (Thornton et al 1993b). Similarly the muscarinic (M2) receptor stimulation by carbachol mimics preconditioning in the rabbit (Thornton 1993b,c). The M2 receptor is known to be coupled to the same pertussis toxin-sensitive G-protein (Bimbaumer, 1990, Graziano and Gilman, 1987 and Kubalak, et al 1991). Another possible trigger for this response might be the alpha-adrenergic receptor, which is also coupled by Gi (Fedida, et al 1993, Talosi and Kranias, 1992). In addition reports by Kitakaze et al (1993c) have shown that alpha agonists can induce preconditioning. In rats the situation is less clear, with reports both for the involvement of pertussis sensitive G-proteins in the anti-arrhythmic effects of preconditioning (Piacentini, et al 1993), and against involvement in infarct size limitation (Liu, et.al 1993a).
1,7.2 The Role of Protein Kinase C
Adenosine (Al) has also been reported to cause phospholipase C activation (Kohl, et al 1990, Schiemann, et al 1991 and Stiles, 1992). Phospholipase C activity leads to the production of two second messengers, diacylglycerol (DAG) and D-myo-inositol 1,4,5-triphosphate (IP3). DAG, in turn, activates protein kinase C (PKC) Capogrossi, et al 1990, Kishimoto, et al 1990 and Yuan, et al 1987). Activation of PKC is thought to play a key role in the intracellular signalling pathway and has been studied in several species.
1.7.2.1 Pharmacological Activation and Inactivation of PKC
In a series of experiments using the isolated rabbit heart (Ytrehus, et al 1994) pretreatment of rabbits with pertussis toxin attenuated the preconditioning effect (Thornton 1993c) suggesting that the adenosine receptors are coupled to the effector by a pertussis toxin sensitive Gi-protein. Recently it has been shown that administering a PKC blocker, staurosporine (Liu, et al 1994a and Ytrehus, et al 1994) or polymyxin B Chapter 1 70
(Casnellie, 1991, Tamaoki, 1991 and Ytrehus, et al 1994) just prior to the sustained ischaemia abolished the protective effect of preconditioning. In turn both physiological and pharmacological activation of PKC predominantly using phorbol esters or oleoyl acetyl glycerol pretreatment are to known induce translocation of the enzyme is isolated cells (Kraft, et al 1983 and Rona, 1985), brain, and heart (Yuan, et al 1987) mimicking ischaemic preconditioning by modulate the adenyl cyclase system (Strasser, et al 1992, Naghshineh, et al 1986, Toews, et al 1987, Yoshimasa , et al 1987 and Ytrehus, et.al 1994). The involvement of PKC has also been demonstrated in the rat (Speechly-Dick, et al 1994a), a species in which adenosine does not appear to be a mediator. It would seem possible, therefore, that PKC may provide a unifying link in an effector pathway that may be activated by different agonists in several species.
1.7.2.2 Protein Kinase C Translocation
Activation of PKC is thought to involve translocation to the cell membrane, since inhibition of microtubular activity with colchicine prevents the development of protection following ischaemic preconditioning in the rabbit heart (Liu, et.al 1994a). According to the translocation theory, activation of adenosine receptors during the first occlusion stimulates a lipase to produce diacylglycerol which in turn activates membrane bound PKC and causes cytosolic PKC to translocate into the membrane. This brief episode of ischaemia would result in some protein phosphorylation but more importantly, primarily initiate the translocation process since most of the PKC in the quiescent myocyte is cytosolic. With reperfusion PKC will remain in the membranes for an extended time. With the onset of the second ischaemic episode kinase activity could then begin at once since PKC is now in the membranes (Liu, et al 1994a). The protective state afforded by ischaemic preconditioning lasts for about one hour in rabbits (Van Winkle, et al 1991), dogs (Murry, 1991), pigs (Sack, 1993) and rats (Li, et al 1992b,c). Loss of the preconditioning protection may simply reflect the return of PKC back to the cytosol. However, it should be stressed, that there is no direct evidence to confirm the physical presence of PKC in the cell membrane following preconditioning. Chapter 1 71
It is not known how the presence of activated PKC in the cell membrane leads to the changes in cell metabolism necessary to lead to the observed preservation in ATP levels associated with protection. The most popular theory, however, involves the ATP sensitive K"*" channel (as shown below).
1.8 ATP Sensitive Potassium Channel
It was found that the outward ‘injury current’ observed in myocardial ischaemia
(Noma, 1983) was due to K+ efflux through channels opening as a result of falling levels of ATP. It has been postulated that opening of the channel is responsible for the shortening of the action potential duration observed during ischaemia and the consequent reduction in Ca2+ influx that may lead to reduced myocardial contractility and sparing of ATP. Gross and Auchampach (Gross, et al 1992, Auchampach, et al 1991 and Grover, et al 1992) showed that blockade of the K atp channels with glibenclamide abolished the protection of preconditioning in an in situ canine heart. Toombs et al (1993) used an in situ rabbit model and reported blockade of the protective effect of ischaemic preconditioning with intravenous glibenclamide. This effect has been repeated (Auchampach, et.al 1992 and Natsuto, et al 1992) using a more specific blocking agent (5-hydroxydecanoate). However, a K atp channel opener (pinacidil), did not confer protection (Thornton et al 1993b).
Experimentally, Katp channels are inhibited by much lower concentrations of ATP than those measured during early ischaemia (Nichols, et.al 1991a), and although only minor increases in channel conductance (<1%) are necessary for a significant effect on action potential duration, the fall in ATP levels during ischaemia is insufficient to explain the observed reduction in action potential duration (Nichols, etal 1990b,c). Therefore, it is unlikely that a direct link exists between ATP levels during ischaemia and Katp channel activity. One explanation might be provided by the action of an intermediary agent that is released during ischaemia causing activation of intracellular pathways leading to Chapter 1 72 phosphorylation of the Katp channel. Adenosine has been shown to perform this role and several investigators have attempted to demonstrate a convincing link by examining the cardioprotective effect of adenosine in the presence of K+ channel blocker (Yao, et al 1994 and Pearson, et al 1978).
1.8.1 Experimental Evidence Showing That K a tp Channel are Associated With Cardioprotection
Studies from several different laboratories have indicated that pharmacological activation of myocardial Katp is associated with profound cardioprotective effects both in vivo and in vitro models of myocardial ischaemia (Toombs, et al 1992b, Grover, et al 1990 and Cole, et al 1991). Daut and coworkers (1990) showed that hypoxia and adenosine produced coronary vasodilation via glibenclamide-sensitive potassium channels in isolated guinea pig hearts. These results suggest that Ai receptor stimulation may result in activation of Katp channels in ischaemic cardiac muscle. It has been reported that adenosine or the selective Ai receptor agonist cyclopentyladenosine (CPA) activated the K atp channel in neonatal rat or guinea pig ventricular myocytes via a Gi protein mediated process (Richardt, et al 1987 and Kirsch, et al 1990). They further showed that the protection afforded by intravenous adenosine could also be blocked by glibenclamide. Similarly Grover et al (1992) showed in an in situ canine model that glibenclamide could block the protective effect of the Al adenosine agonist R(-)N6-(2-phenylisopropyl) adenosine. Recent studies from Yellon’s group (Speechly-Dick, et.al 1995b) have shown using the isolated human atrial trabeculae that the K atp channel is involved in the mediation of ischaemic preconditioning in man via activation of PKC. The protection can be abolished using chelerythrine (a PKC inhibitor), and can also be mimicked, using 1,2-dioctanoyl-sn- glycerol (DOG, a PKC agonist). The importance however is the result demonstrating that the protection induced by DOG could be abolished by glibenclamide which directly implicates that K atp channel as a possible end effector through which protection is manifested Chapter 1 73
1 .8 .2 Mechanism of Protection by the Activation of K a tp
The mechanism by which Katp channel activation protects the myocardium during a subsequent ischaemic period is currently not fully understood. Activation of the K atp channel has been shown to result in a shortening of action potential duration, a decrease in calcium influx, a rapid loss of of contractile function, and preservation of intracellular ATP (Yao, et al 1993a,b and D’Alonso, et al 1992). Previous studies in dogs (Murry, et al 1990) and pigs (Kida, et al 1991) demonstrate that ischaemic preconditioning leads to a decrease in ATP degradation and a reduction in the accumulation of toxic cellular metabolites. Hence it remains possible that the adenosine and KATP theories of preconditioning are actually complementary. ADO release during ischaemia may alter the gating characteristics of the Katp channel such that the apparent potency of the Katp openers is enhanced.
1.9 Conclusion
Since the initial report of the phenomenon in 1986 there has been been explosive growth in the understanding of preconditioning, it effects on the heart and its underlying mechanisms. Preconditioning also has a powerful anti-arrhythmic effect. The anti-arrhythmic protection provides the only firm evidence to date that preconditioning can protect against reversible as well as irreversible myocardial injury. Preconditioning limits infarct size in all models studied. It would appear that a common mechanism must be operative, but, no one theory proposed to date has been confirmed in all species.
The protection seen with preconditioning has led us to appreciate that the heart may have an endogenous means to adapt to ischaemia. The reasons why brief period of ischaemia should delay myocyte death remains unknown. Therefore understanding these adaptive changes may eventually lead to improved therapy for the ischaemic heart in man. Chapter 1 74
2.0 The Delayed Myocardial Protection 2.1 Delayed Protection
Classical preconditioning describled above is a short lived phenomenon with protective benefits waning within 2 hours of reperfusion (Van Winkle, et al 1992). However, there is evidence for the possibity of a delayed, but perhaps longer lasting protection, known as the second window of protection (Marber, et al 1993, 1994, Baxter G et al 1994a, 1994b, 1995c).
2.1.1 Second Window of Protection
The mechanism of the delayed ( or second window of protection) myocardial protection is unknown, but it may be due to changes in the gene expression of some protective substances. Experimental evidence so far implicates two classes of cytoprotective proteins, namely heat shock proteins and endogenous antioxidant proteins (Yellon, etal 1995a).
2.2 Heat Shock Proteins
Heat shock proteins (HSPs) expression is increased when a cell is confronted with a sudden increase in temperature (Craig, 1985 and Lindquist, et al 1988). This was originally described by Ritossa (1962) when he raised the temperature of Drosophila embryos to 5 degrees above their normal range. This heat shock produced a profound change in the polytene chromosome, with a few new puffs forming very quickly and old puffs regressing. Subsequently, it was shown that the appearance of these puffs coincided with the synthesis of a small number of proteins, the heat stress proteins (HSPs) (Tissieres, et al 1974). Initially it was thought that HSPs were effective in preventing the unfolding of proteins only at high temperature (Lindquist, et al 1988). It Chapter 1 75 is now clear that HSPs also serve important physiological functions and that many of them are present and active in normal cells (Lindquist, et al 1988).
2.2.1 HSP families
The proteins encoded by these genes were called heat shock proteins (HSPs) and were identified by their subunit molecular weights as measured by sodium dodecyl sulfate- polyaclamide gel electrophoresis. They fell into three size classes: four had molecular weights between 20 000 and 30 000, two had molecular weights in the range with a molecular weight of 83 000. But the 70 kilo Dalton (kDa) protein was identified as the major HSP (Lindquist, 1988).
2.2.2 Normal Function of HSPs Under Physiological Conditions
The importance of many HSPs is based on their capacity to associate with other proteins in a way that modifies destiny and function of the latter (Craig, 1985, Lindquist, et al 1988).
HSP 90 binds to steroids receptors, preventing their interaction with nuclear DNA until the steroid hormone is bound (Sanchez, et al 1987 and 1986). In the absence of HSP 90, steroid receptors interact with DNA independent of the presence of steroid hormones.
Members of the HSP 70 and HSP 60 families play a major role in folding, unfolding and translocation of polypeptides as well as in assembly and disassembly of oligomeric protein complexes (Reading, et al 1989, Chirico, et al 1988 and Deshaies, et al 1988). Many proteins are translocated between intracellular compartments in an unfolded state (Eiler, et al 1986). HSP 70 and HSP 60 cognates are involved in the unfolding of cytoplasmic proteins and their subsequent translocation into mitochondria, chloroplasts. Chapter 1 76 or the endoplasmic reticulum (Chirico, et al 1988). Once inside these compartments, members of the HSP 60 and the HSP 70 families facilitate refolding of the proteins and their subsequent assembly into oligomeric complexes (Reading, et al 1989, Chirico, et al 1988 and Deshaies, et al 1988).
Nucleotide sequences of complementary DNAs and genes for the major HSP 70s have been determined for species as diverse as bacteria and humans, and they show a high homology.
2.2.3 Stresses Capable of Inducing HSP Synthesis
Although heat stress has been the most extensively characterised, many other stresses are capable of inducing HSP synthesis. These include uncouplers of oxidative phosphorylation, inhibitors of electron transport components, glucose deprivation (Lee, et al 1981 and Rodenhiser, et al 1986), calcium ionophores (Welch, et al 1983), glucose (Pouyssegur, 1977), amino acid analogues (Kelley, et al 1978 and Uney, et al 1988 and Lindquist, 1986), steroids hormones, heavy metals, cadmium (Kelly, et al 1978), ethanol (Rodenhiser D I et al 1986), sodium arsenite (Rodenhiser, et al 19861, 2-mercaptoethanol (Whelan S A et al 1985), 2,4-dinitrophenol (Weitzel G U et al 1985), DNA and RNA viruses, hypoxia (Guttman, et al 1980), anoxia and /or ischaemia and generators of free radicals such as hydrogen peroxide (Polla, 1988), have all been reported to induce HSPs in vivo or cell cultures in vitro.
2.2.4 Mechanism of HSP
The activation of heat shock genes both by whatever means involves a similar mechanism of which a specific transcription factor, heat stress factor (HSF) is activated by the stressful stimulus, and then binds to the promoters of the heat shock genes and stimulates their transcription.(Hammond, et al 1982). It is assumed therefore that the heat shock response is a general homeostatic mechanism that protects cells and indeed Chapter 1 77
the entire organism from the effects of environmental stresses,
2.2.5 Function of Increased HSP Expression Following Stress
When a cell is heat-shocked, disassembly of oligomeric complexes and the unfolding of polypeptides occurs and it is probably a major task for HSPs to prevent or reverse these events. Once refolding becomes impossible, HSPs may accelerate the removal of denatured proteins. The presence of denatured proteins inside a cell induces HSP synthesis via a positive feedback loop (Anathan, et al 1986). These features have sometimes been called "house keeping functions* of HSPs (Ang, etal 1991).
A wide variety of direct and indirect evidence suggests that HSP play a critical role in the defence of the cell against damaging agents (Yellon, et al 1992a). For example a 30 minutes incubation at 430C has observed to result in the synthesis of HSPS of 110, 90, 70 and 65 kDa in mouse lymphocytes (Rodenhiset, et al 1985) and HSPs of 107, 70, 68 and 27 kDa in rat liver cells (Landry, et al 1982). Such cells survive a subsequent heat shock better than untreated cells. Moreover, such cells also exhibit improved survival when exposed to other stressful stimuli such as ethanol or anoxia, while prior treatment with these agents induces HSP expression, as if HSP synthesis had been induced by elevated temperature. Once increased, the HSPs function to protect cellular proteins from dénaturation, or if damage has occurred, promote disaggregation and allow refolding back to an active conformation.
2.2.6 The Protection of HSPs in the Heart
It has been shown that heat stress causes a significant elevation in the levels of the inducible 70kDa stress protein over a number of hours, reaching a maximum between 24 and 72 hours. The inducible 70kDa has been detected in neonatal and adult heart tissue of a wide variety of species including dogs, rabbits and rat (Currie, 1983a, 1987 and Yellon, 1990) Chapter 1 78
The ability of prior heat stress to protect the myocardium against a subsequent oxidative stress, ischaemia and reperfusion, has been demonstrated by several groups (Currie, et.al 1988, Yellon, et.al 1992b and Donnelly, et.al 1992). Reports by Currie et al (1988) and Donnelly et al (1992) shown that by raising the body temperature of anaesthetised animal to 420C for a period of 15 minutes protects the heart against an ischaemic insult (Currie, et al 1988),the heat stress response was associated with enhanced post ischaemic ventricular recovery in the rat heart following subsequent ischaemia and reperfusion. Reperfusion injury was significantly reduced in the heat shock hearts, as measured by creatine kinase release (Currie, 1988 and 1990). Interestingly it has also been reported that ischaemia results in the increased synthesis of 70kDa, with a decreased synthesis of most cellular proteins as reported in heat shock (Currie, 1987, Knowlton, 1991 and Dillmann, et al 1986). These proteins are also elevated 24 hours following ischaemic preconditioning which coincides with the time course of the ‘second window* of protection (Marber, et.al 1993). It has already been shown that a relationship exists between the amount of HSP expression and the degree of protection observed (Marber, et al 1994 and Hutter, et.al 1994). Direct evidence for the causal role of HSPs in delayed protection has recently emerged from experiments using the technique of gene transfection (Williams, et.al 1993, Mestril, et.al 1994 , Heads, et.al 1994a and Heads, et.al 1994b). Further support has come from studies involving the use of a transgenic mouse that over expresses HSP70i resulting in significant myocardial protection (Marber, et.al 1994, Radford, et.al 1994 ).
Heat stress pretreatment has also been shown to improve function in the hypothermically preserved kidney transplant (Perdrizet, et al 1989). The effect of transient hyperthermia has been studied in isolated cells (Weitzel, et al 1985), isolated organs, and intact animals (Currie, et al 1983a,b and Donnelly, et al 1992). It is apparent in each study that a consequence of exposure to hyperthermia is the induction of HSP expression. There are also increases in cytosolic calcium (Drummond,et al 1986 and Stevenson, et al 1986), and intracellular antioxidant activity (Currie, et al 1988) as well as decreased in pH (Weitzel, et al 1985 and Drummond, et al 1986), Chapter 1 79
associated with transient thermal stress. It is apparent that these changes can affect cellular survival.
2.3 Anti-Oxidant Proteins
During ischaemia and reperfusion reactive oxygen species are generated, and are thought to be responsible for much of the resultant cellular injury. Myocardial cells are equipped with endogenous antioxidant systems which scavenge excess free radical and limit damage. It has recently been demonstrated that a number of stresses including sub lethal ischaemia (Hoshida, et.al 1991 and Hoshida, et.al 1993), and cytokine administration (Matsuda, et.al 1988), can all increase the activity of endogenous antioxidants. Hoshida et.al (1993) reported that the activity and content of manganese- superoxide dismutase is increased in the dog heart 24 hours after multiple brief coronary occlusions. Several studies have suggested a role for endogenous catalase in protection against hydrogen peroxide injury. In the pig heart catalase activity was observed to increase 24 hours after ischaemic preconditioning using multiple coronary artery occlusions (Flack, et.al 1991).
2.4 Conclusion
Stress proteins are induced in the heart by various physiological and pathological conditions. The means of protection seen with heat stress or any other stresses that results in the increase in HSPs production is not clear. However, it is possible that the expression of HSP on their own plays a protective role with respect to their ability to cause folding and unfolding as well as translocation and the assembly and disassembly of protein complexes. Another mechanism of protection following heat shock was identified by Currie et al (1988), who reported that the protective effect following heat shock could be due to the direct release and increase in the levels of the endogenous antioxidant, catalase. Chapter 1 80
The ability of the heart to survive a subsequent stress has now been demonstrated by a number of different laboratories using different species and different end points. It has also been shown that the stress proteins are also elevated 24 hours following ischaemic preconditioning which synchronise in time course with the ‘second window’ of protection (Marber, et.al 1993). This influences the resistance of the heart to ischaemia and may offer an endogenous pathway to myocardial protection. Such a pathway represents an obvious means for therapeutic interventions. Chapter 1 81
3.0 Endogenous Protection With Nitric Oxide: A Double Edged Role of Nitric Oxide
3.1 Toxicity of Nitric Oxide 3.1.1 Nitric Oxide is a Free Radical
Endothelial cells, neutrophils and macrophages produce both superoxide (O 2 ) and nitric oxide (NO )(Moncada, et al 1988). Nitric oxide is a free radical which reacts at or near diffusion limited rates with the oxygen radical superoxide (O 2-) to produce a series of exceedingly reactive and toxic species. Because nitric oxide contains an unpaired electron and is paramagnetic, it rapidly reacts with superoxide to form peroxynitrite anion (ONOO-) in high yield (Blough and Zafirions, 1985 and Radi, et al 1991). Peroxynitrite anion can protonate to peroxynitrous acid (ONOOH), an unstable species, which decomposes spontaneously. Peroxynitrous acid can undergo an intramolecular rearrangement to yield nitrate (NOs") or homolytically decompose to yield *OH and NO 2.
Peroxynitrite initiates lipid peroxidation and reacts directly with sulfhydryl groups at a thousand fold greater rate than hydrogen peroxide at pH 7.4 (Radi, et al 1990). In addition, peroxynitrite reacts with transition metals to form a powerful nitrating agent with reactivity of the nitronium ion (NO 2+). Many pathophysiological processes including reperfusion of ischaemic tissue, acute inflammation and sepsis might initiate events that stimulate the simultaneous production of NO and superoxide.
How could NO be involved in ischaemia reperfusion injury as the result of the formation of peroxynitrite? Ischaemia will result in calcium entry into endothelial cytoplasm due to failure of ionic pumps and opening of ion channels. This together with accumulation of L-arginine and NADPH will favour NO synthesis when, during Chapter 1 82 reperfusion, oxygen again becomes available. Ischaemia also results in intracellular superoxide production by xanthine oxidase, mitochondria, and other sources (Lynch, and Fridorich, 1978). Superoxide anions are also produced in the vascular lumen by activated neutrophils and by circulating xanthine oxidase released from liver (Yokogama, et al 1990) which produces oxygen radical by oxidising purines released from tissue into the circulation. The reaction of NO with superoxide anions leads to the formation of peroxynitrite (Blough and Zafirions, 1985).
3.1.2 Impairment of Mitochondrial Function by NO
The cytotoxic properties of NO were demonstrated by the treatment of tumour cells with the inducible form of nitric oxide. Toxicity was also mediated as a result of binding to the iron sulphur cluster at the catalytic site of certain mitochondrial enzymes {Drapier, 1988), leading to intracellular iron loss, and damaging surrounding tissues.
3.1.3 Experimental Evidence for the Toxic Role of Nitric Oxide 3.1.3.1 During Ischaemia and Reperfusion
Experiments have suggested that inhibition of NO synthesis reduces ischaemia- reperfusion injury in the heart and liver. Matheis and colleagues (1992) reported, using an in vivo piglet model of myocardial ischaemia, that inhibition of NO synthase using N-nitro-L-arginine methyl ester (L-NAME) prior to hypoxia followed by reoxygenation during cardiopulmonary bypass showed significant protection against myocardial reoxygenation injury. This was associated with improved contractility, reduced myocardial injury and increased lipid peroxidation. Treatment of rat hearts with nitro-L- arginine (NA), significantly reduced arrhythmias, myocardial necrosis and also preserved myocardial contractility following global ischaemia and reperfusion (Naseem Chapter 1 83
, et al 1992). Similarly in the liver, there is evidence that nitric oxide and its reactive products with superoxide (eg peroxynitrite) may be toxic mediators in hepatic reperfusion injury (Jaeschke, et al 1992).
Gaily et al ( 1990) have shown that neuronal nitric oxide may regulate local cerebral blood flow, and also plays an important role in the neurotoxic cascade leading to neuronal damage after local cerebral ischaemia in rats (Buisson, et al 1992). Similarly in mice, pretreatment with NG-nitro-L-arginine protects the cortex from ischaemic insult induced by irreversible focal ischaemia by Nowicld et al (1991).
Furthermore, immune complex mediated injury of rat lungs is greatly attenuated by the presence of the L-arginine analogue NG-monomethyl-L-arginine (L-NMMA) (Mulligan, et al 1991) and N-iminoethyl -L-omithine (NIO) which is protective against immune complex induced vascular injury by activated macrophages (McCall, et al 1991).
3.1.3.2 Treatment with L-arginine
Further evidence for a deleterious action of NO in ischaemia and reperfusion comes from experiments in which NO concentrations were augmented. Pretreatment with L- arginine potentiates ischaemia and reperfusion injury in the isolated perfused rabbit heart (Takeuchi, et al 1993). Further evidence for toxicity of L-arginine comes from in vitro studies examining mechanisms of toxicity and the effects of nitric oxide synthesis inhibitors in cultured cells (Dawson, et al 1991 and Nowicki, et al 1991) and from an in vivo study examining the effects of NG-nitro-L-arginine on stroke outcome in a mouse model of cerebral ischaemia (Nowicki, et al 1991).These harmful effects of L- arginine may be related to nitric oxide production although other mechanisms may also be involved. Chapter 1 84
3.1.3.3 Nitric Oxide Donors
High doses of glyceryl trinitrate 3.1.4 Experimental Evidence Showing Protection with Nitric Oxide Nitric oxide (NO) released by the endothelium is one of the most important factors controlling the vascular tone of resistance vessels and large arteries such as the coronary arteries (Warren, et al 1994). When the endothelium is damaged by atherosclerotic, toxic or mechanical injury of the vessel wall, endothelium-dependent vasodilatation is lost. Endothelial dysfunction occurs early in myocardial ischaemia, with loss of loss of endothelium mediated relaxation due to inactivation of NO by oxygen derived free radicals produced by adherent leucocytes and injured endothelial cells (Lefer and Aoki, 1990, Ma X-L, et al 1992). The loss of NO coupled with the release of naturally occurring vasoconstricting substances such as endothelin promotes vasoconstriction (Brady, et al 1992 and Tsao, et al 1990a,b), and may contribute to reperfusion injury. This contention is supported by several studies which have shown that augmentation of NO levels during ischaemia and reperfusion preserves endothelial function (Nakanishi, et al 1992 and Morikawa, et al 1992). Chapter 1 85 3.1.4.1 Exogenous Nitric Oxide or L-arginine Furchgott and Zawadzki (1980) have reported that intact endothelium maintains proper vascular tone, and because endothelial dysfunction occurs early in repeifusion, resulting in diminished NO release from the endothelial cells. Efforts have been made to supplement EDRF during myocardial ischaemia-reperfusion. Exogenous nitric oxide has been shown to be cardioprotective in ischaemia-reperfusion injury £Ma X-I, et al 1993 and Siegfried, et al 1992). Also, Johnson et al (1991) reported that infusion of authentic NO significantly reduces myocardial necrosis and neutrophil accumulation in ischaemic tissues. Such protection has been shown with acidified sodium nitrite (Johnson et al 1991) and organic NO donors such as SIN-1 or C87-3754 (Siegfried, et al 1992). Weyrich and colleagues (1992) reported that intracoronary administration of L-arginine which is the physiological nitric oxide precursor attenuated post ischaemic damage (Nakanishi, et al 1992 and Morikawa, et al 1992). Further studies both in vivo and in vitro have also demonstrated that L-arginine can augment vasodilatation under certain conditions (Aisaka, et al 1989, Gold M E et al 1989, Rosenblum, et al 1992 and Schini, et al 1991). Under normal conditions, NO synthase may well be saturated with substrate (Baydoun, et al 1990 and Buhler and Luscher 1991). However, when L-arginine becomes depleted or is in greater demand, or when endothelium is damaged (Aisaka, 1989, Gold, et al 1989, Rosenblum, et al 1992 and Schini, et al 1991b), administering the L-amino acid enhances the vasodilatation presumably through a mechanism involving NO synthesis. It has been reported that L-arginine reduces infarct volume in two models of focal cerebral ischaemia (Morikawa, et al 1992b). It was suggested that L-arginine augments NO production and increases regional cerebral blood flow above the ischaemic threshold (Morikawa, et al 1992b). The protection obtained by NO donors or L-arginine as shown may be due to reduced neutrophil accumulation and infarct size and the infusion preserved endothelial Chapter 1 86 function, possibly by increasing NO release or production by the endothelium (Nakanishi, et al 1992). L-arginine does also have effects which are independent of its property as the substrate for NO synthase, for example increased release of insulin, which may itself cause vasodilatation. However, Girerd et al (1990) have demonstrated that both L and D-arginine infusion increased serum insulin to comparable levels in rats. Whereas the protective effects of arginine are restricted to the L isomer only. 3.2 Conclusion There is evidence that NO may mitigate against tissue injury in a variety of experimental models of different species. The fine balance between toxicity and protection in several systems may depend on the relative production of nitrogen monoxide, nitrosonium ion, or the reduced form of nitric oxide (Lipton, et al 1993). The findings that nitric oxide is cytotoxic, that this cytotoxicity is selective, and that its production can be modified in both directions suggests possible targets for ischaemia/reperfusion injury, immunological injury, antimicrobial therapy. Chapter 1 87 Section 4.0: Objectives Of Research Were: 1) In view of this biphasic role for nitric oxide, we have investigated the effects of inhibition and augmentation of endothelium-derived nitric oxide production during regional myocardial ischaemia and reperfusion in the in situ and in vitro rabbit hearts; a) using infarct size as the endpoint b) by investigation of biochemical markers of cellular function c) by examining the role of other endogenous protective mediators in the control of vascular tone (such as adenosine), following inhibition of nitric oxide synthesis. 2) To confirm that the preconditioning phenomenon can protect the myocardium in both the in vivo and in vitro rabbit hearts, we have investigated; a) the role of nitric oxide in the ischaemic preconditioned hearts. b) the role of adenosine in ischaemic preconditioning. 3) Adenosine has a cardioprotective effect during ischaemia-reperfusion injury when administered both prior to ischaemia and during reperfusion. Adenosine has also been implicated in the mechanism of ischaemic preconditioning. The aim of this study was to investigate whether there was a concentration-response between the administration of adenosine prior to ischaemia-reperfusion and reduction in subsequent infarct size in the in vitro rabbit hearts. 4) High levels of nitric oxide impair the function of mitochondria and other FeS- containing enzymes. This is mediated through binding to an iron atom, the iron- sulphur cluster at the catalytic site of certain mitochondrial enzymes (Drapier, 1988 ). This is of great interest during reperfusion where there is an increase in nitric oxide synthesis (during reperfusion hyperaemia) which may further result in reperfusion injury. Therefore, the aim of this study was to measure mitochondrial function following ischaemia and reperfusion; Chapter 1 88 a) in normal control hearts b) ischaemic preconditioned hearts c) hearts treated with a nitric oxide inhibitor. This thesis is presented as nine chapters. Chapter one is the major introduction. Chuter two includes the methods employed, chapters three to eight present experimental results and discussion. The final chapter (nine) is a summary of the results. Chapter 2 89 Chapter 2: Experimental Models and Methods Section 1.0: In Vivo and In Vitro Models 1.0 Species Used New Zealand white male rabbits were used in all the studies to be described. The rabbit was used because there is minimal coronary collateralisation and the hearts are large enough to allow for the necessary surgical interventions. In addition it is economical. The rabbits were housed in the animal unit at University College London, and had free access to a commercial animal diet. Rabbits were not starved prior to operative procedures. All procedures described complied with United Kingdom Home Office guidelines for the care and use of laboratory animals (Project Licence No. 70/01759). 2.0 Anaesthesia 2.1 Introduction to Anaesthesia Used 2.1.1 Administration of Anaesthetic Prior to the administration of the anaesthetic, the rabbit was wrapped firmly in a sheet in order to both restrain any movements and enable effective and fast administration of the anaesthetic. The anaesthetic used was sodium pentobarbital (60mg in 1ml). Fur covering the vein was removed and lignocaine gel (2%) applied to the skin. The vein was cannulated with a butterfly needle (23 int) through which the general anaesthetic was administered. This provided a short length of flexible catheter between the needle and the syringe so that movements of the rabbit during injection was less likely to result in the needle sliding out from the vein. As soon as the rabbit was lightly Chapter 2 90 anaesthetised, the ears drooped and the rabbit became unresponsive. Thereafter, additional anaesthetic (0.2mls of sodium pentobarbital at a concentration of 60mg in 1ml) was given as required to maintain adequate anaesthesia throughout the experiment. 2.1.2 Assessing and Maintaining Anaesthesia After successful anaesthesia, the following procedures were carried out regularly to ensure that an adequate depth of anaesthesia had been achieved: a) Pedal withdraw reflex: this was performed by extending one of the animals limb and pinching the web of the skin between the toes firmly. If the rabbit withdraws the limb, and muscles twitch, then the depth of anaesthesia is inadequate. b) Eye reflex: this is lost as the depth of the anaesthesia increased. However, this is of a limited value in the rabbit as the palpebral reflex is not lost until dangerously deep levels of anaesthesia have been attained. 3.0 General Surgical Preparation 3.1 Calibration of Transducers and Amplifiers Prior to the surgical preparation all pressure transducers and the amplifiers used were balanced daily and calibrated using a mercury sphygmomanometer: Calibration procedure: 1) Open transducer to air (pressure is zero), 2) Then turn pre-amp attenuator switch to off, 3) Set the pen to base line with zero control, 4) Rotate pre-amp attenuator switch from 50 to 500 scale, 5) Moving pen back to baseline using balance control, 6) Then calibrate the transducer to 100 mmHg, using a manometer: and Chapter 2 91 7) Move pen + to 100 mmHg using calibration control. 3.2 Tracheostomy The rabbit was shaved in the midline cervical region, and 3mis of 1% lignocaine solution was injected under the skin over the trachea using a 22G needle. An incision was made from the jugular notch to the laryngeal prominence, cutting through the various layers of skin, subcutaneous fat, striated muscle and pretracheal fascia to expose the trachea. A small incision was made in the trachea between the rings of cartilage. The endotracheal tube was inserted and secured in position with silk thread (2/0). The tube was connected to a MDI ventilator (MD Industries, Mobile, Ala.) set to provide continuous mechanical ventilation with 100% oxygen. The ventilation rate was set between 30 and 35 breaths per minute, with a tidal volume of approximately 15ml. Aorterial oxygen and CO 2 concentrations were determined at regular time points during the experimental procedures to ensure adequate ventilation was maintained (ABL2, Radiometer, Copenhagen, Denmark). The left common carotid artery was exposed, and cannulated with a short rigid polythene cannula connected to a pressure transducer (Gould Electronics, Valley view, Ohio USA), for continuous monitoring of both arterial blood pressure and heart rate. The arterial line was also used for sampling for blood gas analysis as required. In some experiments the left jugular vein was also cannulated to permit infusion of drugs. 3.3 Thoracotomy The left thoracic region from the tip of the scapula laterally around the rib cage was shaved. A left lateral thoracotomy was performed with a diathermy being used to simultaneously cut through tissue and achieve haemostasis. The chest was opened through the fourth intercostal space, and a retractor inserted to expose the heart and lungs. The lungs were gently pushed aside using a saline soaked swab to expose the Chapter 2 92 heart, and the pericardium was stripped 3.4 Ligation of the Left Circumflex Artery Regional myocardial ischaemia was achieved by ligation of the left circumflex artery. This epicardial artery was located on the ventricular surface and a curved taper needle with silk thread (2/0) passed underneath. The two ends of the ligature were passed through a plastic tube (10mm in length and 1.5mm in diameter) to create a snare. As artery and vein are sometimes in close proximity to one another in the rabbit heart it was sometimes necessary to ensnare both vessels together. 3.4.1 Induction of Ischaemia and Reperfusion Regional ischaemia was achieved in both in vivo and in vitro hearts by occlusion of the vessel, by firmly pulling the ligature through the tubing. Thus squeezing the vessel against the tip of the tube and clamping the snare in position using arterial forceps. Reperfusion was achieved by unclamping the tubing to release the snare. In order to prevent coronary thrombosis 1000 units of heparin was administered at the start of each experiment, 2 minutes prior to the end of the ischaemic period and before the removal of the heart at the conclusion of the experiment Ischaemia was confirmed in the in vivo model by regional cyanosis, loss of normal wall motion on the myocardial surface, and ECG changes (ECG amplifier and series 3000 recorder, Gould Inc, Ohio, USA). Reperfusion was confirmed by surface blushing of the previously cyanotic region, restoration of normal wall motion and resolution of ECG abnormalities. Chapter 2 93 3.5 Temperature Control It was necessary to maintain the physiological temperature of the rabbit throughout the experiment at 38±5°C using a heated electric under blanket The body temperature was continuously monitored using a rectal thermocouple (Type K thermocouple and Digital thermometer, Radiospares, Northants, England). 3.6 Haemodynamic Monitoring Heart rate and blood pressure were monitored to determine the effects of treatment with various drugs and were also used as an index of the viability of the rabbits during the experimental period. If any of the hearts developed ventricular fibrillation (VF), indicated by a sudden drop in blood pressure, a reduction in heart rate and severe disruption of the ECG trace, then mechanical cardioversion was attempted. This was achieved by gently flicking the apex of the heart with the index finger. If sinus rhythm was restored within two minutes the experiment was allowed to continue but if it took >2 minutes then the experiment was terminated. 3.7 Assessment of Infarct and Risk Size At the conclusion of the experimental protocol the heart was processed to determine the risk and infarct volumes. 3.7.1 Risk Area: Staining with Fluorescent Dye At the completion of the in vivo experiment, the heart was excised rapidly by cutting the great vessels and transferred directly into a beaker of saline at room temperature. The heart was mounted on a Langendorff apparatus and was retrogradely perfused with 0.9% saline at room temperature via the aorta for 2 minutes in order to wash out Chapter 2 94 residual blood from the coronary vasculature. In the in vitro model, the heart was already mounted on the Langendorff apparatus, and at the completion of the experiment the pacing electrode and ventricular balloon were removed and buffer perfusion stopped. In all experiments, the coronary artery was reoccluded and the heart was perfused with 5ml of 0.5% suspension of fluorescent particles (Zinc cadmium sulphide 1-lOpm particles, Duke Scientific, California, USA). When the heart was examined under ultra violet (UV) light the borders of the myocardium supplied by the occluded artery were easily visualised, as the risk area exhibited no fluorescence whereas the non-ischaemic area had been perfused with fluorescent particles, and could clearly be visualised (figure 2.1 and 2.2). 3.7.2 Infarct Area a) Staining of hearts following in vitro VR protocol: The ligature was released and the heart perfused with Krebs Henseleit buffer (KHB) to wash out excess fluorescent particles. The heart was then perfused for 2 minutes with Triphenyl- tetrazolium chloride (TTC, Sigma Chemicals, Dorset) at 370C, at a concentration of lOmg/ml in phosphate buffer at pH 7.4. This stained the viable myocardium brick red whereas the infarcted myocardium did not take up the stain due to lack of enzymes and co-factors and therefore remained pale. The heart was then frozen at -18°C overnight. b) Staining of hearts following in vivo VR protocol: The heart was frozen at -18°C overnight, and then sectioned as described below. The slices were incubated in TTC for 20 minutes at 370C. 3.7.3 Sectioning After 24 hour at -18°C, tissue from both the in vivo and in vitro experiments were partially thawed for 2 minutes before sectioning. All hearts were sliced into sequential Chapter 2 95 2mm sections, perpendicular to the intraventricular septum. The slices were washed in saline, then left to thaw completely. Stained slices (see above) were fixed in 1% formaldehyde solution for 24 hours before being analysed for infarct and risk areas. This enhanced the delineation of risk and infarct areas (figure 2.1 and 2.2). Determination of Infarct and Risk Zone Size n D)ET w 1 forve,! A , 6). Figure 2.1: Shows slices from a frozen heart that had been perfused with fluorescent particles and triphenyl tétrazolium to determine infarct and risk size. The slices viewed under ultraviolet light are shown in panel A: Under ultraviolet light (366nm) the area at risk appears dark whilst the area of perfusion fluoresces brightly. Panel B: shows the same slices viewed under white light. Under white light the slices contain red areas and pale sections. The red areas are the normal tissues that have been stained with triphenyl tétrazolium and the pale areas represents infarction.. Chapter 2 97 A) Schematic diagram showing how the infarct and risk areas are analysed A schematic representation of the coronary vascular anatomy in the rabbit. The left circumflex artery has been occlud^ by a ligature. At the end of the experimental protocol the heart was processed for infarct area and risk area determination.The ligature was reoccluded and the heart perfused with fluorescent particles which delineated the risk area. The shaded (non-fluorescent) area represents the risk area for the development of infarction. The heart was then sectioned into 2mm slices from the apex to the aorta and determined for infarction. B) Determination of infarction and risk area from a sectioned heart slice area perfused with particles right ventrid^.cC^ ^ ^TTC stained area papillary muscle left ventri isk area When the heart was examined under infarct area Ultra violet (UV) light the borders of the dependent myocardium supplied Under normal light the infarcted area by the occluded artery (shaded area) appears pale yellow (in vitro hearts) can be visualised, as this area is or grey (in vivo hearts). While the unperfused with the fluorescent viable tissue takes up the TTC particles. While the normal area was staining appearing deep red. perfused with fluorescent particles, this can be clearly separated from the risk area. Figure 2.2 Shows a schematic diagram of the coronary vascular anatomy in the rabbit. A) showing a branch of the coronary artery occluded and how the heart was determined for risk and infarct areas. B) The lower diagram represents a section from the heart at right angles to its long axis, this illustrates how infarct and risk ratios were analysised. Chapter 2 98 3.7.4 Analysing Infarct and Risk Areas Slices of heart were placed caudal surface upwards and compressed between the two perspex sheets separated by 2mm spacers. A clear piece of acetate film was clipped in position and using super fine coloured pens the outline of each slice was traced onto the film. Under UV light the Zinc Cadmium Sulphide fluorescent particles could be seen marking the distinct division between the fluorescent perfused area and the non perfused (ie risk) area (fig 2.1). Then the infarct area (TTC-ve) was drawn. Using a photocopier, these drawings were enlarged by a factor of 1.41 and the infarct and risk areas were planimetered using dedicated software (Summa Graphics, Summa Sketch n, Connecticut, USA) based on Simpson’s Rule. 4.0 The Isolated Langendorff Perfused Heart 4.1 The Principles of Retrograde Perfusion A schematic diagram of the apparatus is shown in figure 2.3 and a photograph of the rabbit heart perfused on the Langendorff apparatus is shown on figure 2.4. The principal mechanism for the working of the isolated heart, is to force the perfusate (or blood), towards the heart through a cannula inserted in the ascending aorta. Retrograde perfusion closes the aortic valves, as in a normal heart during diastole, and the perfusate passes through the coronary circulation exiting via the coronary sinus and the opened right atrium. Thus the cardiac cavities remain empty throughout the experiment. Changes in coronary vascular resistance can be measured by perfusing the heart in constant pressure system and measuring flow or perfusing in constant flow system and measuring pressure. The myocardium can remain viable and contract for many hours (Kreb H A and Henseleit H A 1932), as long as the perfusate contains the essential substrates and oxygen, and the flow rate is sufficient to supply these substrates and remove metabolites. THE LANGENDORFF APPARATUS n=r Perfusion reservoir(perfusate is D: ■ S bubbled with O 2 95%, and CO2 5%) n> Bubble trap to Peristaltic pump Pressure Transducer, for coronary perfusion pressure In Line Filter: exclud particles >20^im Three-way tap Connected to amplifier synnge to mcrease balloon volume Aortic Cannula: sidearm for measuring coronary pressure and sampling perfusate. Heart paced through To amplifier the right atrium Left ventricular balloon Water jacketed camber Pressure Transducer, for left ventricular pressure. ow rate can be determined by measuring Cannular: connected to the balloon flow of perfusate per minute Figure 2.3. Schematic diagram of the Langendorff perfusion apparatus. The apparatus was surrounded by a water jacket to maintain the perfusate at 3?0C in the right ventricle. An in line filter exclude large particulate contamination. The perfusate is drawn up from the perfusion reservoir through the peristaltic pump and into the bubble trap. Chapter 2 100 Heart perfused on a Langendorff Apparatus Figure 2.4. Shows a heart perfused with Krebs buffer on the Langendorff apparatus. The heart was electrically paced at 180 beats per minutes, both coronary perfusion pressure and left ventricular developed pressure determined, and a temperature probe inserted into the right atrium. Chapter 2 101 4.2 The Perfusion Medium The perfusion medium used in all the experiments was Krebs Henseleit buffer (KHB), containing: (in mM) 118 NaCl, 4.7 KCi, 1.2 MgS04.2H20, 1.8 CaCl2.2H20, 14.6 NaH2P04, 1.2 KH2PO4, 11.0 Glucose. This perfusate was equilibrated with oxygen and carbon dioxide (95% O 2, 5%C02) in an open oxygenator reservoir. The perfusate was drawn up by a variable rate peristaltic pump (Watson Marlow 5025/R, Com well England), passed through a 5pm in line filter (Waterman, Sigma) and passed through a bubble trap. The bubble trap was sealed by a rubber plug with a metal cannula in the centre. The cannula had four sidearms, one of which was used for the measurement of coronary perfusion pressure using a pressure transducer and amplifier (Gould Electronics, USA). The remaining sidearms were sealed. The peristaltic pump generates a constant perfusion flow with the pressure determined by the coronary resistance. Increased coronary vasomotor tone in this setting of constant flow will lead to a rise in perfusion pressure. Additional cyclic pressure fluctuations may develop fiom the motion of the roller pump. These do not correspond to the cardiac rhythm and may cause functional disturbances in the heart. The perfusion medium was temperature controlled by the oxygenator, as well as the thermocontrolled glass vessels, so that a small circulation thermostat was sufficient. 4.3 Surgical Preparation L ' Surgical preparation of rabbits were as described before. 4.4 Preparation of the Langendorff Perfused Heart The heart was quickly excised by cutting the great vessels and placed in a pre-weighed beaker tilled with isotonic saline at room temperature. The beaker was re-weighed and then the heart was mounted onto the Langendorff apparatus. The aorta was tied onto the Chapter 2 102 perfusion cannula under a meniscus of saline to prevent air entering from the coronary vessels. The time between removal of the heart and perfusion on the Langendorff apparatus was less than one minute; speed was necessary to prevent any significant ischaemia of the heart prior to subsequent experimentation. Once the heart was mounted onto the Langendorff apparatus the pulmonary artery was opened to allow drainage of the perfusate. The left appendage was excised to allow insertion the left ventricular balloon. 4.4.1 Electrical Stimulation of the Heart In order to reduce variability it was necessary to keep the heart rate constant by electrical pacing via the right atrium set at 180 beats per minute. The heart was paced by an isolated stimulator (Digitiser DS2, Hertfordshire, England) and triggered by a pulse generator (Digitiser DG2, Hertfordshire, England). The square wave pulse amplitude was set at twice threshold with a pulse duration of 2mS. 4.4.2 Temperature Control The temperature was continuously measured from the right ventricle using a K type thermocouple (Radiospares, Northants, England) connected to a digital thermometer (Digitron, Northants, England). The temperature of the heart was maintained at 3 7 +O.50C throughout the experimental procedure. This was achieved by circulating warm water within the sleeve surroimding the perfusion reservoir, heat exchanger and bubble trap (Techne C-Circulator, Cambridge UK).The heart was suspended in KHB in a double-walled vessel containing an overflow of perfusate, which was thermostatically controlled to maintain myocardial temperature. Chapter 2 103 4.4.3 Flow Rate All hearts were perfused at a constant flow rate with variable coronary perfusion pressure. In order to assess the rate at which the heart was being perfused, it was necessary to measure coronary flow but in these studies perfusate was collected in measuring cylinder and flow measured in ml/min.This procedure for the assessment of flow per minute is accurate and reproducible. 4.4.4 Left Ventricular Developed Pressure (LVDP) Left ventricular pressure was measured using a balloon, this was made from a piece of cling film of 4-5cm2 size, which was tied around the tip of the catheter with a thin silk thread (3/0). These balloons were not spherical in shape, but adjusted to the conical interior of the ventricle when inflated with KHB (2.5ml). The catheter to which the balloon was attached was 10cm in length and was connected to a three-way tap, which itself was attached to a pressure transducer and amplifier (Gould Electronics, USA). The pressure transducer was positioned at the same level as the balloon in the left ventricle, to minimise differences in hydrostatic pressure. Once the connection was made, the balloon was inserted into the left ventricle through the left atrium. It was important that the tip of the catheter did not press on the tissue of the heart’s apex, which might block the catheter opening, making a proper pressure measurement impossible. In addition the cardiac tissue would also be damaged by this pressure. The catheter was properly positioned by gently elevating the apex of the heart with the index finger and positioning the balloon. Once the balloon was correctly positioned it was secured in place, by taping the catheter to the aortic cannula. Then the balloon was inflated with KHB via a syringe attached to a three-way tap. The balloon volume was increased until the end diastolic pressure reading was 10 mmHg. The insertion and filling of the balloon does not injure the myocardium and coronary flow is not impeded, even when the balloon is inflated at higher pressures. Chapter 2 104 The beating heart exerts a rhythmic force on the balloon, which was recorded as a contraction curve (diastolic and systolic pressures see results section). The advantages of using a ventricular balloon were that the force and preload developed can be measured reproducibly in mmHg. In addition the ventricular contraction curve can be used for further calculations without limitation and a continuous heart rate recording obtained. 4.4.5 Coronary Perfusion Pressure (CPP) A pressure transducer and amplifier (transducer amplifier, Gould, USA) was connected to the sidearm on the aortic cannula, and was used to record CPP continuously throughout the experiment. The CPP at the start of the experiment was in the range of 40-60 mmHg. 4.4.6 Reduction of Tissue Oedema Perfusion of the isolated heart with a protein-free medium (albumin free perfusate), causes the exclusion of fluid from the vascular bed, due to osmotic pressure causing interstitial oedema to develop with the passage of time. However, tissue oedema can be reduced significantly when the heart is immersed into a chamber filled with perfusate (Doring and Dehnert 1961) which elevates tissue pressure, and considerably reduces transcapillary fluid leakage. 4.4.7 Measurement of Oxygen, Carbon Dioxide and pH Perfusate was collected from the sidearm of the aortic perfusion cannula, and analysed for pH, P02 and PCO2 using an automated blood gas analyser (ABL2 Radiometer Copenhagen, Copenhagen, Denmark). Chapter 2 105 4.4.8 Arrhythmias During the experiment the development of ventricular fibrillation was associated with a rapid increase in CPP and loss of the developed pressure trace. Under such conditions cardioversion was attempted by gently flicking the apex of the heart with the index finger. If sinus rhythm was restored within two minutes the experiment was allowed to continue but if it took > 2 minutes then the experiment was terminated. 4.4.9 Confirming Ischaemia and Reperfusion on the Isolated Hearts For the isolated heart model, ischaemia was determined by loss of normal wall motion, an increase in coronary perfusion pressure (CPP) and a rise in left ventricular diastolic pressure. Reperfusion was occasionally difficult to determine, however, the signs were a decrease in CPP, decrease in diastolic pressure and restoration of normal wall motion of myocardium. At the end of the experimental protocol all hearts were determined for risk and infarct volume. Chapter 2 106 Section 2.0: Biochemical Assays 3.1 Experimental Procedures Myocardial tissue was obtained from both in vivo and in vitro experiments. a) For the in vivo model: Myocardial tissue samples were obtained at various time points from the left ventricle. Using fine biopsy forceps, pre-cooled in liquid nitrogen. b) For the in vitro model: The whole heart mounted on the Langendorff apparatus. Tissue samples were obtained using large aluminium tongs pre-cooled in liquid nitrogen. The whole heart was clamped and immediately stored in liquid nitrogen. 3.2 Deproteinisation of Tissue Samples Handling small tissue samples can be very difficult, especially when measuring labile metabolites. Tissue samples were therefore freeze-dried over night and stored at -TO^C until analysed. Freeze-dried tissue samples were weighed in duplicates. The tissue was homogenised in an Eppendorff tube. This was carried out manually using a hand held pestle. Once the tissue was broken up, 350pl of 6% perchloric acid (PCA) solution was added and the sample was immediately homogenised using a motorised device (Yellon et. al., 1981). The sample, with the pestle in the Eppendorff tube was then spun in a micro centrifuge for 10 seconds. During centrifugation the tissue settled to the bottom of the tube with no loss of tissue with removal of the pestle. The sample was vortex mixed and stabilised on ice for 10 minutes, and then recentrifiiged in a micro centrifuge Chapter 2 107 for a further minute. From the Eppendorff tube a 200|il aliquot of the supernatant was transferred to a second Eppendorff tube. The sample was then neutralised to pH 7.4, by adding lOpl Universal Indicator and then the appropriate amount of neutralising solution. The neutralised sample was incubated for 30 minutes on ice, after which the sample was vortex mixed and micro centrifuged for 1 minute. The sample was deproteinised and stored for analysis. 3.3 Adenosine Triphosphate Determination (ATP) The ATP assay was carried out enzymatically using a standard spectrophotometric method (Yellon et.al., 1981). The reaction is based on the principle that ATP phosphorylates glucose to glucose-6-phosphate (G-6-P) in the presence of hexokinase (HK) and Mg2+. Glucose-6-phosphate dehydrogenase (G6P-DH) catalyses the dehydrogenation of G-6-P with NADP to give 6 phosphonoglucono lactone and NADP‘*'H'*". Each mole of ATP forms 1 mole of NADPH. The conversion of ATP to ADP by hexokinase was determined by measurement of its extinction at 340 nm. Mg2+ ATP + Glucose > G-6-P + ADP G-6-P + NADP'*" ^ 6-phosphonoglucono-lactone + NADPH + H"*" 3.2.1 Experimental Procedures for ATP Assay The reaction was carried out in a cuvette at 25^0 by adding 50pl of neutralised heart extract (or standard or blank) to 1.9 ml of assay medium containing 0.2M TRIS, 1% NADP,1M MgCl2, lOOmM glucose, 2pl of 2.4M G6P-DH suspension and finally, 1.8|il of hexokinase suspension, pH 7.4. The small changes in extinction that occurred Chapter 2 108 on adding the enzyme were determined once and the change in extinction was completed 15 minutes after the addition of hexokinase suspension and standards and blanks were determined with every assay. When the change in extinction was established, 0.02ml of lOOmM ADP was added to provide excess ADP for the subsequent assay of creatine-phosphate and any further change in extinction was noted. The result was expressed in pmol /g/dry weight. 3.4 Tissue Creatine Phosphate Determination (CP) The enzyme creatine phosphokinase (CPK) catalyses the transfer of phosphate from creatine phosphate and adenosine diphosphate (ADP) (Lamprecht and Trautschold 1974). In the presences of hexokinase (HK), the ATP formed phosphorylates glucose to glucose-6-phosphate (G-6-P) with the regeneration of ADP. Glucose-6-phosphate dehydrogenase (G-6P-DH) catalyses the oxidation of G-6-P with NADP+. In the overall reaction, 1 mole of NADPH is formed for each mole of creatine phosphate. The increase in optical density at 340nm due to the formation of NADPH+ H+. Mg2+ Creatine Phosphates ADP------> Creatine + ATP Mg2+ Glucose + ATP------> Glucose-6-Phosphate + ADP Glucose-6-Phosphate+NADP^ ^6 Phosphonoglucono lactone + NADPH+ H"*" 3.4.1 Experimental Procedures for CP Assay The reaction was initiated by adding 306pl of creatine kinase solution and reading the changes in extinction which followed, this was due to the formation of NADP. The change in extinction was completed in 20 minutes and standards and blanks were Chapter 2 109 determined with every assay. Creatine kinase was made up fresh each day, the result was calculated in jiMole /g/dry weight 3.5 Tissue Lactate Determination Lactate dehydrogenase (LDH) catalyses the oxidation of L-lactate by NAD (Hohorst, 1959): Lactate + NAD > Pyruvate + NADP + H+ The reaction products must be removed from the mixture to obtain quantitative oxidation of L-Lactate. The protons are bound by use of alkaline reaction medium and pyruvate is trapped as the hydrazone. The basic equation for the spectrophotometric assay of L-lactate is: ; L(+)Lactate + NAD + Hydrazine — > Pyruvate Hydrazone + NADH + HgO^ The equilibrium constant for the reaction is Kc~ 7x102 at pH 9.5. The high concentration of NAD and LDH are necessary to obtain a quantitative and sufficiently fast reaction. 3.5.1 Experimental Procedures for Lactate Assay Tissue samples were assayed from both in vivo and in vitro hearts for the determination of lactate. Samples were obtained and de proteinised (as described above). The experimental cuvette contains 1.95ml assay buffer which was 0.4M Hydrazine buffer at pH 9.5, 5x1Q-2M NAD and distilled H2O. To the assay buffer 50pl of neutralised tissue extract was added, mixed and the optical density (ODi) was read at 340 nm. Then lOjil of lactate dehydrogenase was added to the cuvette and mixed, this was allowed to stand at room temperature for 30 minutes prior to the measurement of optical Chapter 2 110 density (OD 2). The result was expressed in jxmol /g/dry weight. 3.6 Adenosine Measurement in Coronary Effluent 3.6.1 The Principles of High Performance Liquid Chromatography High performance liquid chromatography (HPLC) has its origins in classical column chromatography although in both theory and practice it is similar to gas chromatography. The sample is introduced into a liquid mobile phase which is pun^jed though the column at pressures up to about 300 psi and flow rates of 1 to 5 cm3 min-t This can be achieved through 10-25 cm columns packed with particles as small as 3pm in diameter. 3.6.2 Mobile Phase Preparation The low-strength effluent consisted of 0.02mol/l of KH 2PO4 and the high-strength effluent was 60% methanol. Both the eluents were degassed prior to use. The low strength effluent was degassed by a water-aspirator vacuum and the methanol solution was degassed using a stream of helium gas (60ml/min) for approximately 30 seconds. A discarded line-filter from a Waters M 6000 pump inlet serves as an efficient aspirator for the helium gas. 3.7 Sample Collection for the Adenosine Assay The heart was perfused with Krebs buffer, 5ml aliquot of coronary effluent was collected and quickly frozen in liquid nitrogen, then freeze-dried over night as previously described. Chapter 2 111 3.8 Measurements of Adenosine Concentration Coronary effluent samples were assayed by reverse phase high-performance liquid chromatography (HPLC) according to the method employed by Jenkins and Bellardinelli (1988). The HPLC system included a Waters 710B injector (Milfor, Pennsylvania), an LKB2150 pump (Bromma, Sweden), and a Beckman 160 absorbance detector (Palo Alto, California). The freeze-dried samples were reconstituted with de-ionised water to a volume of 0.5ml. A sample volume of 40pl was passed through a Waters 8NVC18 (4pm) column (Milford, Pennsylvania). The gradient slope was 0.69%/min (0-60 methanol in 87 min ), linear and the adenosine sample were assayed under ambient temperature. The flow rate of the effluent was at a rate of 1.5ml/min, the adenosine peak was detected at 254nm and the retention time was 20 minutes. The peak was recorded on a BBC Goerz metrawatt chart recorder, integrated using planimetry and referenced to a standard curve. Adenosine concentration in the perfusate was expressed in pM/lOOg heart tissue. Chapter 2 112 Section 4.0: Isolated Mitochondrial Function 4.1 Purity and Functional Characterisation of Intact Mitochondria The mitochondrial electron transport chain conserves the energy of electron transfer in the form of a proton gradient which is then used to drive the synthesis of ATP via ATP synthetase. This function is critically dependent on the inner membrane being impermeable to protons. In mitochondria with an intact inner membrane electron transfer can only proceed if the proton gradient is continuously dissipated by the flow back into the mitochondrion via the ATP synthetase. This phenomenon is described as ‘coupling’.The consumption of oxygen in the presence of substrate and ADP (state 3 respiration) is measured and compared with the rate of respiration after the ADP has been consumed (state 4 respiration). Mitochondria which show no difference in state 3 and state 4 respiration are ‘uncoupled*, a state which can also be induced by some reagents (e.g. dinitrophenol) appropriately termed uncouplers. 4.2 General Method for the Isolation of Mitochondria The first step involves rupture of the cell membrane while maintaining the structural integrity of the mitochondria. After breaking open the cells, differential centrifugation was used to separate the mitochondria from other organelles and cell debris. However, the yield of mitochondria and the ease with which pure preparations may be obtained depends very much on the type of tissue and the amount of tissue available. Chapter 2 113 4.3 The Oxygen Electrode Mitochondrial respiration is most conveniently and quickly measured using a Clarke oxygen electrode (figure 2.5). This consists of a silver/silver chloride reference anode surrounding a platinum cathode. These electrodes are immersed in saturated KCl solution and separated from the reaction vessel by a thin Teflon membrane that is permeable to oxygen‘but which prevents electrode poisoning. The electrodes are polarised at a voltage of 0.6 V. At the platinum cathode the electrons reduce oxygen molecules to water. This results in a net transfer of electrons from the cathode to the anode causing a current to flow between the two electrodes which can be measured in an external circuit. The current is proportional to the partial pressure of oxygen in the sample and, as the response is linear, only two calibration points are necessary. The current generated is very temperature dependent and it is therefore important to operate the electrode at constant temperature. The oxygen is consumed by the electrode, and the solution close to the electrode becomes progressively anaerobic. It is therefore necessary to stir the solution vigorously to ensure that the bulk solution under study is continuously brought into contact with the electrode. The electrodes can conveniently be enclosed in a small chamber of approximately 3 ml volume. This chamber is isolated from contact with the atmosphere by a close-fitting cap. Solutions can be introduced into the chamber and their oxygen concentrations measured directly once the apparatus has been calibrated. 4.4 Surgical Preparation For the studies in which mitochondrial function was assessed using the Clarke Oxygen Electrode, rabbits were prepared as described below. Chapter 2 14 Diagram of the Clarke Oxygen Electrode Samples are introduced into the vessel through a hole in the stopper Stopper Temperature control Rubber ring 'water jacket Reaction vessel Water Magnetic stirrer Water Rubber ring KCL solution Teflon membrane +ve Annular silver anode Leads to polarising unit -ve Platinum cathode Magnetic stirrer base Figure 2.5. Schematic diagram of the Oxygen electrode. This is used for the measurement of mitochondrial function. The reaction vessel can hold a total volume of 3ml, samples are introduced from the hole in the stopper. Once all the mitochondrial substrates are in the chamber they are mixed continuously using a small magnetic stirrer. The vessel is surrounded by a water jacket to maintain the temperature of the reaction chamber at 370C. Chapter 2 115 4.4.1 Isolation of Mitochondria from the Rabbit Heart At the end of the experimental protocol the heart was quickly removed and placed in ice cold saline (9%). The heart was washed in saline to remove excess perfusate or blood. The aorta, both atria and the ring of fat around the ventricles were removed. The ventricles were placed on a pre-cooled petri dish and finely chopped. The pieces were transferred into a homogenising tube containing 50ml of medium A; this was then homogenised using an Ultra Turrex homogeniser (XKA-Ultra-Turrax T25, Janke and Kunkel, Labortechnik, Staufen) at a speed setting of SOOOrpm for 5 seconds (figure 2.6) Medium A contained 180mM KCl, 10 mM ethylenediaminotetracetic acid (EDTA) and 0.5% free fatty acid bovine serum albumin (BSA), and was adjusted to a pH of 7.2 with TRIS [hydroxymethyljamino methane (TRIS). The homogenate was centrifuged at 30(X)rpm (Heraeus Sepatech Biofuge 22R, USA) for 10 minutes at 40C. The supernatant was filtered in a nylon mesh, this was recentrifuged for 10 minutes at 8500 rpm at 40C. The resulting crude mitochondrial pellet was resuspended in 6ml of medium B and centrifuged for 10 minutes at 6500 rpm at 40C. Medium B contained 180 mM KCl, 0.5% free fatty acid bovine serum albumin (BSA), and adjusted to pH 7.2. The mitochondrial pellet obtained from this centrifugation was washed again in 6 ml medium B and recentrifuged for 10 minutes at 6500 rpm at 40C. The final pellet was collected and resuspended in 0.5ml of medium B. Chapter 2 116 Isolation of Mitochondria from the Myocardium. Tissue mince; Homogenisation Buffer A Homogenate Centrifugation 6500 rpm x 10 minute ^Pellet (discard) Supernatant Filter Centrifugation 8500 rpm x 10 minute -►Supernatant (discard) Pellet Resuspend Buffer B Centrifugation 6500 rpm x 10 minute ►_ Supernatant (discard) Pellet Resuspend Buffer B Centrifugation 6500 rpm x 10 minute "►Supernatant (discard) Pellet mitochondrial enriched fraction Figure 2.6 Schematic representation for the different stages to isolate mitochondrial enriched fraction from the rabbit ventricular tissue. Chapter 2 117 4.4.2 Mitochondrial Protein Determination The mitochondrial protein concentration was determined by the method of Bradford (1978) using bovine serum albumin as a standard The assay was carried out using 3ml of a solution containing lOOmg coomassie brilliant blue, 50ml 95% ethanol and 100ml of othophosphoric acid in a total volume of 250ml distilled water, this was added to samples, mixed and allowed to stabilised for 10 minutes at room temperature. The measurement was made using a spectrophotometer (UVIKON 810, Kontron Instruments ltd, Watford, Herts) and the absorbance measured at 595nm. Blanks and bovine serum albumin standards were assayed at the same time. 4.4.3 Polarographic Determination of Mitochondrial Respiration A schematic diagram of the Clarke oxygen electrode is shown in figure 2.5. Mitochondrial respiration was measured polarographically at 370C using a Clarke oxygen electrode. The Clarke electrode assembly is enclosed in a glass vessel of 3ml capacity and surrounded by a water jacket which is continuously warmed by circulating water from a water bath adjusted to 37®C. The reaction medium was continuously mixed using a magnetic stirrer and the reagents were added through a small hole on the top of the glass vessel, using a Hamilton microsyringe. The electrode was enclosed in a small chamber of a approximately 3ml volume. The reaction media used for the activation of the mitochondrial respiration were as follows: Incubation medium (IM), 250mM sucrose, 3mM KH2P04 (Pi), 0.5mM EDTA (Na), 3mM glutamate pH 7.4 adjusted with Tris buffer (Peng et al 1977). For the mitochondria, to provide a final concentration of 1.25mg, mitochondrial protein/ml IM and the other substrates (these include glutamate. Pi and EDTA) were added to the reaction chamber and allowed to equilibrate and respire for 1 minute, after which state 3 of the respiration was initiated Chapter 2 118 by adding a concentration of 0.25mM ADP, pH 7.4 (Ferrari 1982). Mitochondrial function was assessed in terms of Q2, RCI and ADP/O ratio, where QO2 is n atoms of oxygen used/mg of mitochondrial protein/ml in response to the addition of ADP. RCI (respiratory control ratio) is the ratio of oxygen used in the presence of ADP to that used in the absence of ADP. ADP/O ratio is the nanomoles ADP used/n atoms of oxygen consumed. Figure 2.7 shows an example of how these indices can be directly calculated from a typical oxygen electrode tracing. After each mitochondrial extraction, duplicate mitochondrial respiration measurements were established, so that each estimation could be made in duplicate and the results averaged. 4.4.4 Assessment of Mitochondrial Function Figure 2.7 shows a schematic representation of a mitochondrial trace and how the indices can be directly calculated from a typical oxygen electrode tracing Equation 1 represents the atoms of O2 used for the mg of mitochondrial protein per minute in response to the addition of ADP. n=r ■S K> calculated from the trace. 1cm Substrate Mitochondria ADP ADP 10cm ADP Figure 2.7. Mitochondrial respiration was measured polarographically at 37^0 using a ClarkClark oxygenoxygen electrode.electrode. MitochondrialMitochondrial function was assessed in terms Q2 (n atoms of oxygen used/mg of mitochondrial protein/ml in response to the addition of ADP), RCR (respiratory control ratio, is the ratio of oxygen used in the presence of ADP to that used in the absence of ADP) and ADP/O ( ratio is the nanomoles ADP used/n atoms of oxygen consumed). Chapter 2 120 Equation (1) QO2/3 = 960 X (Al X 2 = (n atoms 0 2 / mg protein / min) (C) 1.25 Equation 2 represents the atoms of O2 used for the mg of mitochondrial protein per minute when all molecules of ADP have been converted to ATP. Equation (2) QO2 /4 = 960 X (B) X 2 = (n atoms 0 2 / mg protein / min) (C) X 10 1.25 Equation 3 represents the nanomoles of ADP used per n atoms of O 2 consumed. Equation (3) RCI = Amm= O2 used in the presence of ADP to Bmm O2 used in the absence Equation 4 represents the ratio of O2 used in the presence of ADP to that used in the absence of ADP. Equation (4) ADP/O = 250 X (Cl = (n moles of ADP used / n atoms of O 2 consumed) 960 X (Z) 5.0 Statistical Analysis of Data All results are expressed as mean ± standard error (SEM). Statistical analysis between groups was performed using AND VA followed by Fisher’s Protected Least Significance Difference FPLSD (Armitage and Berry 1987). Statistical comparisons were made with commercially available software (Stat View version 3.0, Apple Macintosh softwear run on a Cherwell Scientific Publishing, Oxford, England). The criterion of statistical significance was defined as p<0.05. Chapter 3 121 Chapter 3: Effects of Inhibition of Nitric Oxide in Both the In Vivo and In Vitro Hearts 1.0 Introduction The mechanism of myocardial injury following ischaemia and reperfusion is multi factorial and depends on the duration of ischaemia, the presence of a collateral circulation, the adhesion, activation and extravasation of neutrophils together with the generation of free radicals and progressive vasoconstriction of the microcirculation. The release of NO by the endothelium is widely perceived as having a protective role following ischaemia since it is a powerful endogenous vasodilator and also inhibits activation and aggregation of neutrophils and platelets. Consistent with this, the infusion of L-arginine or treatment with NO donors during ischaemia and reperfusion reduces myocardial injury [Weyrich, et al 1992, Johnson, et al 1990, Nakanishi, et al 1992, Morikawa, et al 1992 and Siegfried, et al 1992). Although some evidence does exist which suggests that NO may be cytotoxic when present in substantial excess (Beckman, et al 1990). This deleterious role of NO could potentially be relevant during the reactive hyperaemia which follows ischaemia and in the presence of activated leucocytes which express inducible NO synthase (Hibbs, et al 1988 and Moncada, et al 1991). 1.2 Aims To investigate the possibility of a detrimental role for NO during ischaemia and reperfusion, we have studied the effects of inhibition of the synthesis of endothelium- derived NO production during regional ischaemia and reperfusion in vivo rabbit hearts. Chapter 3 122 using infarct size as the end-point. To investigate a possible deleterious role for NO derived from circulating leucocytes, we have repeated these experiments using an in vitro model in which rabbit hearts were mounted on the Langendorff apparatus and perfused with Krebs Henseleit buffer (KHB). 2.0 Experimental Methods New Zealand White rabbits (mean weight 2.59± 0.04 kg, n=59) were anaesthetised using sodium pentobarbitone and surgically prepared as described previously. After surgical preparation, the rabbits were stabilised for 20 minutes before being sequentially assigned to the various experimental protocol (figure 3.1). Heparin (KXK) units) was administered intravenously at the start, at the end of sustained ischaemia and at the conclusion of each experiment. Experimental protocol for sustained regional ischaemia and reperfusion in the in vivo rabbit heart n=r TIME "S -ta> (minutes) U» stabilisation 1’ 10’ 30’ Ischaemia 120’ Reperfusion L-NAME or Saline determination of infarct and risk volume Stabilisation 50’ Ischaemia 180’ Reperfusion L-NAME/saline or determination of infarct Infusion of L-arginine and risk volume Figure 3.1. Experimental protocol used for the in vivo hearts. The rabbits were treated with saline or L-NAME, or a continuous infusion of L- arginine. The rabbits received 30 or 50 minutes of relouai ischaemia followed by 120 or 180 minutes reperfusion. At the conclusion of the experimental the hearts were determined for risk and infarct volume. Chapter 3 124 2.1 Exclusion Criteria Of the 59 rabbits used in this study, 10 animals in all were excluded from the analysis. Three rabbits (from groups 1, 3 and 5) developed intractable ventricular fibrillation and did not recover, two failed to manifest electrocardiographic changes with reperfusion (from groups 5 and 3); two rabbits failed to exhibit ischaemic changes (ST) on coronary artery occlusion (from groups 2 and 3). Two rabbits were excluded as a result of no change in blood pressure on treatment with L-NAME (from group 2 and 4). One rabbit died during surgical preparation (group 3). Data for 49 rabbits are presented below. 2.2 In vivo Experimental Groups Group 1: Control (n=10) Following an intravenous infusion of 5mls normal saline over 1 minute, rabbits were monitored for a further 10 minutes followed by 30 minutes of coronary branch occlusion and 120 minutes reperfusion. Group 2: Control+L-NAME (n=10) Following an intravenous infusion of (10 mg/kg) L-NAME over 1 minute, rabbits were monitored for 10 minutes followed by 30 minutes sustained ischaemia and 120 minutes reperfusion. Group 3: Control (n=7) Following stabilisation, 50 minutes of coronary branch occlusion was induced followed by 180 minutes reperfusion. Chapter 3 125 Group 4 : L-NAME (n=10) An intravenous infusion of (10 mg/kg/1 min) L-NAME over 1 minute was given 10 minutes prior to 50 minutes ischaemia and 180 minutes reperfusion. Group 5: L-arginine (n=8) An intravenous infusion of L-arginine (300mg/kg/90min) was started 10 minutes prior to 50 minutes ischaemia and continued for the first 30 minutes of reperfusion which lasted 180 minutes. Group 6: D-NAME (n=2) Following an 10 minute infusion of D-NAME (lOmg/kg/lmin), rabbits were monitored for 175 minutes. A sham thoracotomy was performed in these rabbits and haemodynamics were monitored only. Group 7: D-Arginine (n=2) Rabbits received a continuous infusion of D-arginine (300mg/kg/90min) for 175 minutes. A sham thoracotomy was also performed and haemodynamics were monitored. 3.0 Specific Methods for the In Vitro Model New Zealand White rabbits (mean weight of 2.59+0.04 kg, n=16), were anaesthetised and ventilated mechanically as described previously. The heart was exposed through a left thoracotomy and the left coronary artery selected for occlusion. The heart was then rapidly excised and mounted onto the Langendorff apparatus and perfused with Krebs buffer. All hearts were stabilised for 30 minutes followed by the experimental protocol. 3.1 Exclusions For these experiments 16 rabbits were used from which one was excluded. This rabbit Chapter 3 126 was from group 1, and developed intractable ventricular fibrillation during reperfusion. A total of 15 rabbits contributed to the data presented below. 3.2 In Vitro Experimental Groups There were two experimental groups with rabbits being assigned sequentially to each . Group 1: Control (n=7) All heart received 45 minutes regional ischaemia and 180 minutes reperfusion. Group 2: L-NAME (n=8) The heart was perfused with L-NAME 10 minutes prior to 45 minutes ischaemia and continuing into the first 15 minutes of reperfusion. 4.0 In Vivo Results 4.1 Infarct and Risk Determination Inhibition of NO synthesis by L-NAME prior to ischaemia and reperfusion significantly reduced the ratio of infarct to risk volume following 30 minutes of coronary branch occlusion and 120 minutes of reperfusion, from 40.6± 4.3% (Control) to 18.39+7.7% (L-NAME), p<0.01 (see table 3.1 and figure 3.2). Following the longer ischaemic insult of 50 minutes and 180 minutes reperfusion, the protection seen with L-NAME was still present with a significant reduction in infarct/risk volume from 60.7+5.9% (Control) to 39.9+4.3% (L-NAME), p<0.02. Pretreatment with L-arginine numerically reduced the ratio of infarct and risk volume to 47.3±4.3% (L-arginine, p<0.08) but this failed to meet the criterion of significance (see figure 3.2 ). The volume of myocardial tissue at risk following ischaemia and reperfusion were not significantly different between groups (figure 3.3). Infarct size and haemodynamic data for the in vivo hearts Group Control L-NAME Control L-NAME L-Aiginine Ischaemic Time (min) 30 30 50 50 50 n 10 10 7 10 8 Body weight (kg) 2.59±.02 2.58±0.1 2.64±0.03 2.49±0.O2 2.63±0.06 Heart weight (g) 6.8±0.1 7.Q±Q.05 6.5±0.5 7.2±0.2 6.4±0.04 Risk (cm3) 0.637±0.18 0.524±Q.16 0.704±0.07 0.837±0.07 1.035±Q.14 Infarct (cm3) 0.259±0.04 0.096±0.03* 0.426±0.05 0.332±0.04 0.49±0.66 Infarction (%) 40.7±4.29 18.3±7.74* 6 0 .^5.96 39.7±4.34* 47.3±4.32 T=0 min RPP 23793±2035 22755±1940 26983±1103 24352+1092 23332±1362 T= 10 min BP -9±2 21±3* -1±3 23±5* -Q±4 T=10 min HR -9±4 -19±3* -1±6 -27±6* -1±9 T= 10 min RPP 21899±1560 21973±1543 27085±1064 26982±1636 23297±1473 T= 120 min RPP 1688Q±958 16885±2540 18296±824 16973±1398 15502±2256 The table 3.1 .Shows experimental data, and infarct volume for the in vivo hearts. Infarct volume is expressed as a percentage of the risk volume for each of the experimental groiq)s. l^emodynamic data shown at start (T= 0 min), 10 min after saline, L-NAME or L-aiginine treatment ( I - 10 min) and at conclusion of exprim ent (Rate Pressure Product; RPP, changes in blood pressure BP mmHg; and change in Heart Rate, HR (bpm). Values are meap±SEM. L-NAME (10 y mg/kg), L-arginine (300mmg/kg),*p<0.01 Chapter 3 128 Percentage infarction/risk for in vivo hearts p<0.08 80 < 0.02 60 p<0.01 r 40 ■ i Control L-NAME Control L-NAME L-arginine L j I______I 30’ischaemia/120’reperfusion 50’ischaemia/180’ reperftision igure 3.2. Infarct volume is expressed as a percentage of the risk volume and the relationship etween infarct volumes for each of the experimental groups. Chapter 3 129 Determination of infarct and risk volume in the in vivo hearts 1.5 p = \S p 0 .5 0.0 (A)iitrol L-NA.VIH Control p_\L-argininc ControlI L-NAMl: Control L-NAMH 1 .-arginine I ■ ■ ■ ■ ■ i ■ ■ 301 120’R 501 180’R 301 120’R 501 180’R Risk Zone Infarct Zone Figure 3.3. Risk volume (R) and infarct volume (I) expressed in cm^ The area at risk was delineated with fluorescent micro spheres and the infarct volume by triphenyltetrazolium. Chapter 3 130 4.2 Haemodynamîc Data An example of the physiological traces obtained during the experimental protocol is shown in figure 3.4. The haemodynamic data is represented graphically in figures 3.5 and 3.6. The heart rate and arterial blood pressure did not differ significantly among the groups at the start of the experiments. The infusion of 10 mg/kg/1 min L-NAME led to a rapid rise in mean blood pressure from a basal value of 90.6±4.4mmHg to 132+2.7mmHg which was significantly greater than control group (95+1.8, p<0.05) (figure 3.5). This hypertensive response seen on treatment with L-NAME was accompanied by a reflex bradycardia (fig 3.6) and were present thoughtout ischaemia until the early part of reperfusion. However, there was no differences in rate pressure product prior to coronary occlusion, during treatment or reperfusion, implying that myocardial work and therefore oxygen demand was likely to be similar between groups (see table 3.1). The infusions of L-arginine (300mg/kg/90min), D-NAME or D-arginine had no effect on blood pressure or heart rate. Table 3.3 includes acid/base balance data for all the groups, during stabilisation and at the conclusion of the experiments. There were no significant differences between groups with regard to acid/base balance. The pH prior to ischaemia or end of reperfusion showed no significant changes. Arterial blood gas measurements were taken every 30 minutes during the experimental protocol, however, only two time point recordings are shown on the data table 3.3. Chapter 3 131 Trace of heart rate and blood pressure in the in vivo experiments ICO figure 3.4. Represents a trace obtained during an experiment in the in vivo rabbit heart. The rabbit was given a bolus of L-NAME (lOmg/kg) for 5 minutes prior to stabilisation. From the experimental trace both heart rate and blood pressure was monitored. As shown above , L-NAME led to a agnificant increase in blood pressure. Blood pressure in the in vivo rabbit hearts. n sa=r “S ft) ------. jÿ ■■■■■■ -1 Stabii I Ischaei»!» Reperfusion UJ + 4 0 - Control (30’) L-NAME (30’) c/5 L-arginine (50’) I Control (50’) + 20- L-NAME (50’) B .S & § 6 -20-^ Time (minutes) Figure 3.5. Represents the change in mean blood pressure seen in the in vivo hearts, for the Control groups, L-NAME groups and L-arginine treated group . Regional ischaemia lasted 30 or 50 minutes and followed by 120/180 minutes of reperfusion. *p<0.01. U) K) Heart rate in the in vivo model ■ Control(30’) n:r #L-NAME(30’) D: "S O L-Arginine(50’) -tn> ^ Control(50’) ® L-NAME(50’) 250 I s pti t: I200 I Reperfusion Ischaemia# Stabii 150 0 60 Time (minutes) 120 180 Figure 3.6. Shows the heart rate for the in vivo hearts . For the Control groups, L-NAME groups and L-arginine treated group. Regional ischaemia lasted 30 or 50 minutes followed by 120 or 180 minutes of reperfusion. Treatment with L-NAjte shows a significant decrease in heart *p<0.01 U) Measurement of blood gases for the in vivo rabbit hearts IU) Group Control L-NAME Control L-NAME L-Arginine Ischaemic Time (min) 30 30 50 50 50 Stabilisation pH 7.40±0.G2 7.4Qt0.01 7.38±0.04 7.46±0.03 7.45±0.02 pC02 4.82±0.44 4.27±0.26 4.29±0.49 3.38±0.54 4.15±0.38 p02 56.38±8.7 46.77±6.0 43.27±8.3 43.03±1.2 50.04±4.9 End of repafusim pH 7.38±q.02 7.4Q±0.01 7.42±0.02 7.42±0.03 7.46±0.02 pC02 4.52±0.50 4.60±0.40 3.61±0.40 5.1±0.4 3.91±0.38 p02 63.92+8.3 55.8Q±3.7 51.14±3.9 52.04±4.6 42.63±7.3 Table 3.3. Shows data obtained from blood gas analysis for pH ,pC02 and p02 (kpa) .Blood samples were taken at Stabilisation and aftarl20 minutes reperfusion frxxn the in vivo rabbit heart p=NS. U) 4^ Chapter 3 135 5.0 In vitro Results 5.1 Infarct and Risk Determination Inhibition of NO synthesis by L-NAME prior to ischaemia reperfusion significantly reduced the ratios of infarct to risk volume following 45 minutes coronary branch occlusion and 180 minutes reperfusion from 64.4+4% (Control) to 41.4+5% (L- NAME), p<0.05. (see table 3.2 and figure 3.7). The volume of myocardial tissue at risk following ischaemia and reperfusion were not significantly different between groups (see table 3.2 and figure 3.7). 5.2 Haemodynamic Data Haemodynamic data relating to ventricular performance for all experiments are presented in table 3.2. There were no differences between groups in coronary perfusion pressure (CPP) measured at the start and the conclusion of the experiments. However, CPP was significantly increased 10 minutes after the administration of L-NAME in all treated hearts. After 30 minutes of reperfusion, CPP was significantly higher than control in all hearts, (see table 3.2 and figure 3.8). There were no differences between groups in the haemodynamic variables measured following stabilisation. In all groups the left ventricular systolic pressure decreased and diastolic pressure increased over the duration of the experiments (table 3.2). At the end of reperfusion, the left ventricular systolic pressure was significantly higher in the L-NAME treated group in comparison to control p<0.05 (figure 3.9). The left ventricular diastolic pressure in the L-NAME treated group was significantly lower than control p<0.05. (figure 3.10) IUI Infarct and haemodynamic data from the isolated perfused rabbit hearts. Group Control L-NAME n 7 8 Weight (kg) 2.47+0.21 2.20±Q.14 Heart weight (g) 5.45+0.1 5.09±0.10 Risk (cm^) 0.685±0.1 0.609+0.1 Infarct(cm^) 0.463±0.09 0.24±0.03* Infarct/Risk% 64.6±4.28 41.4±5.06* Time- Omin (at end of stabilisation) CPP 42+3 47+2 Systolic 89±6 96±6 Diastolic 7±1 8±1 Time- 15min (prior to ischaemia) CPP 45±3 83±8* Systolic 89±6 96±5 Diastolic 7±1 8±1 Time-30min reperfusion CPP 64±6 99+6* Systolic 69+7 62+7 Diastolic 17+3 14+3 Times 180min reperfusion CPP 104±9 111±11 Systolic 43±3 63±3* Diastolic 33±4 14±3* Table 3.2. Shows experimental data form the isolated perfused rabbit hearts. Infarct volume, risk volume and a percentage U) infarction in all experimental groups. Haemodynamic measurements for coronary pafusion pressure mmHg (CPP), left ventricular G\ pressures (systolic and diastolic) are recorded at Time=0 (stabilisation), T=15 ( post treatment with L-NAME prior to ischaemia), T=30 (30 minutes into reperfusion) and T=180 (conclusion of the experiment). Values are mean±SEM. *p<0.05 vs. control. n :t ■a -Ire U» Infarct area and risk area in the in vitro Langendorff perfused rabbit hearts 1.2 - 1.0 - 100 00 0 . 8 - 80 0 .6 - 60 £ •o 0.4H - 40 20 0 .0 - - 0 Control IGNAME Control L-NAME Control IGNAME J Risk Area cm^ Infarct Area cm^ l/R% Figure 3.7. Representation of the area at risk, the infarct area and the percentage I/R for the Langendorff perfused rabbit hearts. Risk areas were determined with fluorescent particles and risk areas determined with TTC. * U) -4 n Coronary perfusion pressure recorded at time points for the in vitro hearts =r "S 180 — -In UJ 120- Q S e 100 I £ 8 0 - I 6 0 - I # L-NAME c O Control I 40 — 2 0 - stab Ischaemia Reperfusion 0 _ 0 35 Time (minutes) 80 260 Figure 3.8. The mean coronary perfusion pressure mmHg, over the duration of the experiment. Control (n=7), L-NAME- treated (n=8). * 100 o Control # L-Name 90 - 80 -j 70 60 50 40 30 20 Ischaemia Reperfusion 10 0 ^ 20 Time (minutes) U) Figure 3.9 Shows the left ventricular systolic pressure trace for the in vitro groups. The pressure was continuously sO measured through a balloon in the left ventricle for the isolated Langendorff perfused rabbit hearts. Hearts were perfused with krebs for control groups or perfused with L-NAME prior to ischaemia and reperfusion. *p<0.05. n =r Left ventricular diastolic pressure trace for the in vitro Langendorff perfused -a 100 “I hearts o 90 Ischaemia Reperfusion 80 O Control 70 - # L-NAME 60 - 50 - 40 - 30 - 20 10 0 _ -10 _ -20 _ 0 20 24565 Time (minutes) Figure 3.10. The left ventricular diastolic pressure trace for the isolated perfused rabbit hearts.The hearts were perfused with krebs only for the control group or perfused with L-NAME in the treated hearts prior to ischaemia and reperfusion in the in vitro hearts. *p<0.05 Chapter 3 141 6.0 Discussion In this study, pretreatment with L-NAME reduced the ratio of infarctAisk area after both 30 and 50 minutes of regional myocardial ischaemia and reperftision in the in situ rabbit heart. NO has been implicated in tissue injury, firstly, as a result of its capacity to interact with oxygen-derived free radicals to produce toxic intermediates (such as the peroxynitrite anion) (Radi, et al 1991). This can diffuse or cross cell membranes through anion channels (Gruetter, et al 1979, Assoian, et al 1986 and Garg, et al 1989), for several micrometers before decomposing to form the powerful and cytotoxic oxidants hydroxyl radical and nitrogen dioxide. This potent oxidant is capable of initiating fatty acid oxidation and nitrosylation of aromatic amino acids. Supporting evidence for a nitrogen radical comes from a report which has detected the nitrogen- centered radical during reperftision of the ischaemic isolated rat heart model (Zweier, et al 1987). Secondly, nitric oxide induces biochemical changes by avidly binding to the iron-sulphur-centred enzymes resulting in the inhibition of mitochondrial respiration (inhibition of NADP: ubiquinone oxido-reductase and succinate; ubiquinone oxido- reductase of the mitochondrial electron transport chain) (Drapier and Hibbs 1988, Nathan, 1991and Geng, 1992), inhibition of the citric acid cycle enzyme aconitase, and inhibition of DNA synthesis (Hibbs, et al 1988, Lepoivre, et al 1992). Coronary artery reperfusion following a period of occlusion is associated with the rapid adhesion and extravasation of activated neutrophils (Tanaka, et al 1990 and Dreyer, et al 1990 and Harlan, 1987). Neutrophil recruitment to sites of tissue injury is mediated by multiple adhesion molecules, which are sequentially required for the attachment of the cells to the blood vascular endothelium and their subsequent extravasation into the surrounding tissue (Smith, Anderson, et al 1991 and Lasky, 1991). NO may be implicated in this process, Johnson et al (1990) demonstrated a reduction in both leukocyte accumulation and tissue injury in post ischaemic myocardium of animals given exogenous NO. Others have also reported that inhibition Chapter 3 142 of NO exacerbates tissue injury associated with various inflammatory conditions (Hutcheson, et al 1990 and Kubes, 1993). Uncontrolled release of cytotoxic quantities of NO produced by the inducible enzymes may contribute to PMN-mediated damage (Moncada, et al 1991, reviews). In fact at least two studies have demonstrated that inhibition of NO synthesis reduced tissue injury caused by deposition of immune complex (Mulligan, et al 1991), and myocardial reoxygenation injury (Matheis, et al 1992). The dichotomy between results may lay in the enzymatic source of NO. In this study the protection following pretreatment with L-NAME was not likely to have been mediated by an effect on neutrophil accumulation because inhibition of NO increases neutrophil adhesion to endothelial cells and this is associated with increased myocardial injury (Ma X-L, et al 1993). In addition, L-NAME would not have inhibited formation of neutrophil-derived NO because it is not an effective inhibitor of the inducible NO synthase expressed by activated neutrophils (McCall, et al 1991). Therefore the observed protection following NO inhibition may have resulted from another mechanism which is likely to have involved the inhibition of endothelium- derived NO which is known to be released in substantially greater quantities during reactive hyperaemia (Kostic and Schrader 1992 and Yamabe et al 1992). Paradoxically, pretreatment with L-arginine also reduced myocardial injury following 50 minutes of regional ischaemia and reperfusion in the in situ rabbit heart although this was not significantly different from control. An earlier study reported that pretreatment with L-arginine protected the in situ feline heart from regional ischaemia (Wayrich, et al 1992). The mechanism for L-arginine protection, and for the protection conferred by low concentrations of NO donors (Johnson, et al 1990, Siegfried, et al 1992), seems to involve the preservation of physiological endothelial cell layer integrity and enhancement of NO release leading to a reduction in the accumulation of neutrophils in the ischaemic zone. Although L-arginine has been shown to have cardioprotective effects (Weyrich, et al 1992, Morikawa, et al 1992 and Katsuhiko, et al 1992), L-arginine also exerts several Chapter 3 143 biological effects other than the generation of NO (Barbul, 1986) These include the increased release of insulin and glucagon from pancreatic cells (Palmer and Walter, et al 1975) as well as the secretion of growth hormone and prolactin from the pituitary (Rakoff, 1973). Under physiological conditions, addition of L-arginine does not cause vasodilation because adequate L-arginine is present to ensure saturation of NO synthase (Rossitch, et al 1991). However, when L-arginine is deficient, or when the endothelium is damaged (Aisaka, et al, 1989, Gold, et al 1989b, Rosenblum, et al 1992 and Schini and Vanhoutte, 1991b), administering the L-amino acid reduces vascular tone presumably through a mechanism involving NO synthesis. This protective effect of L-NAME was also demonstrated in the isolated perfused hearts. These results supports the in situ study in which we observed a similar cardioprotective effect after NO inhibition in the rabbit heart. Because the isolated, perfused heart is free from circulating leukocytes, we can now confidently conclude that the observed protection does not result from NO released by activated neutrophils. There was a progressive loss of myocardial function with falling left ventricular systolic pressure and increasing ventricular diastolic pressure. This is a feature of the isolated perfused heart preparation. However, the decreases in left ventricular systolic pressures were more marked in control hearts which reflects the larger infarcts sustained by the hearts in this group in comparison to L-NAME treated. Similarly, increase in diastolic pressure were more marked in hearts which had sustained larger infarcts (i.e. control) in contrast to the smaller infarct groups (i.e. L-NAME). Inhibition of NO synthesis by L-NAME increased CPP showing that NO is a determinant of coronary artery tone in the unstressed isolated perfused heart (Ameczua, et al 1989, Kelm and Schrader, 1990, Rees, et al 1989a,b). Following 45 minutes of regional ischaemia, CPP was significantly higher in the L-NAME treated hearts compared with control (figure 3.7) which supports earlier suggestions that the release of NO is important in the regulation of coronary tone during reperfusion. D-NAME, the biologically inactive enantiomer did Chapter 3 144 not affect blood pressure or heart rate, supporting the contentions that the protection seen with L-NAME was due to inhibition of NO synthesis. NO-induced injury is likely to be important in many pathophysiological conditions including reperfusion of ischaemic tissue, during reactive hyperaemia (Kostc and Schrader, 1992 and Yamabe, 1992), acute inflammation, and sepsis, all of which might initiate events that stimulate excess NO production. In the chronic condition when the latent inducible form of NO synthase begins to produce cytotoxic amounts of NO, what was initially beneficial could actually become detrimental. This increased release of NO may also occur in the presence of activated neutrophils and macrophages (Moncada, et al 1991 and McCall, et al 1991). This was exemplified by Miller et al (Miller and Sadowska, al 1993), who reported detrimental effects associated with NO inhibition (L-NAME) in normal animals but observed a beneficial effect of L-NAME in a chronic model of inflammation of the intestine. Other studies supporting a noxious role for endothelium derived nitric oxide comes from numerous experimental models of arrhythmias (Naseem, et al 1993, Bams, et al 1993), myocardial necrosis following hypoxia (Matheis and Sherman, et al 1992), post-ischaemic cerebral reperftision injury (Buisson and Plotkine, et al 1992), neutrophil-mediated tissue injury (Mulligan, et al 1992), and harmful effects of L-arginine (Takeuchi, et al 1993) and NO donors (Moncada, 1993). 7.0 Conclusion I These results show that the protection against myocardial injury following ischaemia and reperfusion can be derived from manipulation of the nitric oxide pathway. The data from the isolated perfused hearts provides evidence that it was not likely to have been mediated by an effect on neutrophil accumulation because inhibition of NO increases neutrophil adhesion to endothelial cells and this is associated with increased myocardial injury (Ma X-L, et al 1993). In addition, L-NAME would not have inhibited formation of neutrophil-derived NO because it is not an effective inhibitor of the inducible NO Chapter 3 145 synthase expressed by activated neutrophils (McCall, et al 1991). Therefore the observed protection following NO inhibition may have resulted from another mechanism which is likely to have involved the inhibition of endothelium-derived NO which is known to be released in substantially greater quantities during reactive hyperaemia (Kostic and Schrader, 1992). To explain the observed protection against myocardial ischaemia-reperfusion injury following inhibition of endothelium-derived nitric oxide we have proposed a novel hypothesis and have designed experiments to support this; see the next chapter. Chapter 4 146 Chapter 4: Biochemical Parameters Measured In Vivo and In Vitro 1.0 Introduction In the preceding chapter, contrary to much of the published work, inhibition of NO synthase was shown to protect the myocardium against ischaemia and reperfusion injury in both the in vivo and in vitro rabbit heart. There are however reports supporting a toxic role for NO during reperfusion hyperaemia as well as in experimental models of NO release is excess. However, even if this is the case manipulation of the NO system may also affect myocardial protection due to compensatory release of the other vasoactive factors from the endothelium. Constitute with this, Kostic and Schrader (1992) have reported that inhibition of NO synthesis results in the increased level of adenosine which is recognised to have cardioprotective properties (Thornton et al 1992a,b and L i, et al et al 1990). 1.2 Aims Therefore this study was designed to determine the mechanism and biochemical changes that may occur following inhibition of NO synthesis by L-NAME, in both the in vivo and in vitro rabbit heart models. 2.0 Experimental Model for the In Vivo Study New 2^aland white rabbits (mean at 2.54+0.5kg) were prepared as described previously. The rabbits were treated with normal saline (Control), L-NAME or Chapter 4 147 phenylephrine. Phenylephrine was used to increase blood pressure to the same degree as that obtained with L-NAME, to determine whether the hypertensive response alone was responsible for myocardial protection. Once the rabbits were instrumented, they were randomly assigned. All hearts were stabilised for 20 minute prior to an infusion of either L-NAME (lOmg/kg/lmin) for the L-NAME treated group, phenylephrine (SOOug/kg/lmin) for the phenylephrine treated group or saline for the control hearts, and monitored for a further 10 minutes. Then a biopsy from the apical myocardium was taken using pre-cooled (-180C) tissue forceps with the samples being stored in liquid nitrogen prior to being freeze dried before analysis. Tissue extraction was performed using 350^1 of ice cold perchloric acid (6%). The samples were immediately homogenised and neutralised to pH 7.4. The samples were then analysed for Lactate, ATP and CP. 3.0 Specific Experimental Models for the In Vitro Study New Zealand white rabbit (mean weight of 2.44+1kg) were used. The experimental procedures were the same as that described in the method section. For these experiments, coronary effluent samples were assayed for adenosine release by HPLC, for the Control group, the L-NAME group and the Global ischaemic group. At the end of treatment the hearts were freeze clamped and tissue lactate concentration was determined (figure 4.1). 3.1 Experimental Protocol Coronary effluent was collected in 5ml aliquots following stabilisation of the isolated perfused rabbit heart, further aliquots were collected at 1 minute, 5 minutes, 7 minutes and 10 minutes. The experimental groups studied were as follows; 1) Control, continuous perfusion (n=5) Chapter 4 148 2) L-NAME (addition of 30uM L-NAME to the perfusate, n=5) 3) Ischaemia ( 5 minutes global ischaemia followed by 10 minutes reperfusion, n= 5) 4.0 In Vivo Results 4.1 Lactate L-NAME significantly increased myocardial lactate concentration from 5.35+0.9nMol/g dry weight (control, n=4) to 16.36±2.4nMol/g dry weight (L-NAME, n=4), p<0.03 (figure 4.2). Although, there was an increase in lactate concentration in hearts treated with phenylephrine, this was not significantly different from control (p t t 1 11 L-NAME 15’ 10 ’ I------ L-NAME 1 1 111 Ischaemia/reperfusion 10’ 5’ 10 ’ t 1 111 I I Hearts perfused with Krebs buffer H Hearts perfused with Krebs buffer + 30|iM L-NAME 5 minutes of global ischaemia Coronary effluent collected at stabilisation, 1 minute, 5 minutes, 1 7 minutes and 10 minutes. Hearts were freeze clamped for measurement of tissue lactate Figure 4.1.Experimental protocol for isolated Langendorff perfused rabbit hearts, using a model of global ischaemia or perfusion with L-NAME.At time points coronary effluent was collected and assayed for adenosine, and at the conclusion of the experiment the hearts were fteeze clamped and tissue lactate concentration was determined. Chapter 4 150 Myocardial lactate concentration for in vivo hearts 20 n •fl5 I 5- 0- stabilisation lO'perfusion stabilisation lO’perfusion stabilisation lO’p^usion Control L-NAME Phenylephrine Figure 4.2. Lactate concentrations were measured in biopsies taken from rabbit hearts in the in vivo groups. Samples were analysai during stabilisation in all the groups and following treatment The groups studied were Control group which received an infusion of Saline, L-NAME group and Phenylephrine group. *pc0.03 Chapter 4 151 Parameters measured for the in vivo lactate study Group Control L-NAME Phenylephrine n 5 5 4 Body weight (kg) 2.59+0.2 2.48+0.6 2.54+0.83 Stabilisation (T=0 minute) T= 0 min BP 81+3.1 79+2.9 77+3.4 T=0 min HR 270+2.7 266+4.0 265+5.9 pH 7.4+0.07 7.4+0.08 7.4+0.03 p02 63.92+8.3 46.77+6.0 56.38+8.7 pC02 4.52+0.50 4.27+0.26 3.38+0.54 Treatment (T= 10 minutes) T= 10 min BP 81+1.9 119+4.8* 120+5.3* T=10 min HR 272+2.6 218+7.3* 212+8.5* pH 7.4+0.08 7.4+0.05 7.0+0.32* p02 55.80+3.7 43.27+8.3 39.80+3.7 pC02 4.60+0.40 3.91+0.38 7.3+1.8 Table 4.1. Shows data table for haemdynamic measurements from the in vivo study. Measurements were taken following stabilisation (T=0) and 10 minutes following treatment (T=10). blood pressure (BP), heart rate(HR), all measured in mmHg and blood gases measured in kpa *p<0.01 Chapter 4 152 4.2 ATP and CP Results There was no significant increase in tissue ATP or CP concentration in all the groups during stabilisation or following 10 minutes treatment with either L-NAME or phenylephrine, see table 4.2. 4.3 Haemodynamic Data In both L-NAME and phenylephrine treated groups there was a significant increase in blood pressure from an average of 79+2.6mmHg at stabilisation to 119.5+3.3 (p<0.01, see table 4.1 and figure 4.3). This increase in blood pressure was accompanied by a decrease in heart rate. On intravenous infusion of L-NAME there was no significant change in blood gases, but hearts treated with phenylephrine showed significant metabolic acidosis compared to both control and L-NAME treated groups, p<0.05 (table4.1). 5.0 In vitro Results 5.1 Results for Adenosine Release The concentration of adenosine released form the coronary effluent samples were referenced to a standard curve (figure 4.4). Mean adenosine concentration in the coronary effluent was the same in each group prior to treatment and did not increase after 10 minutes of perfusion in the control group (3.4+0.7nmol/L per lOOg heart weight versus 4.8±0.4^imol/L per lOOg heart, n=4. p NS). However the addition of 30^iM L-NAME to the coronary perfusate increased the adenosine concentration progressively to a peak of 24.0+3.7^imollL per lOOg of heart (n=4) after 10 minutes, p<0.06 versus control. Following 5 minutes of global ischaemia, the adenosine concentration in the perfusate peak at 1 minute of reperfusion and 139.()±17.9^mol/L per l(X)g of heart,(n=4) and remained significantly higher than control hearts even at 10 Chapter 4 153 minutes (83.1±7.2^imol/L per lOOg of heart. n=4), p<0.001 versus control (see table 4.3 and figure 4.5). 5.2 Results for Lactate Concentration Tissue lactate concentration in the control group was 4.58+0.5 p.Mol/g/dry weight (n=5). Treatment with L-NAME (30^M) showed no increase in lactate concentration at 10 minutes perfusion, 5.55±1.7^iMol/g/dry weight (n=5), p 5.3 Haemodynamic Data Perfusion with L-NAME lead a significant increase in CPF compared to controls hearts (table 4.3). There were no significant differences in left ventricular systolic or diastolic pressure in all groups at stabilisation. However, 5 minutes global ischaemia and 10 minutes reperfusion resulted in a significant decrease in left ventricular systolic pressure compared to the other groups p<0.05 ( table 4.3) and left ventricular diastolic pressure in the global ischaemic group showed a significant increase compared to the other groups p<0.05 (table 4.3). Chapter 4 154 Lactate, ATP and CP measurements for the in vivo hearts Group Control L-NAME Hienylephrine n 5 5 4 Stabilisation (T=0 minute) Lactate 5.4+1.2 5.35±0.9 5.S+0.4 ATP 20.0±0.92 19.5±0.6 21.3±0.9 CP 26.2±0.7 2 8 .^ .4 30.6±0.6 Treatment (T= 10 minutes) Lactate 5.6±0.9 16.3^.0* 10.3±1.6 ATP 22.5±0.86 24.3±0.6 24.2±0.3 CP 28.9±0.5 30.2±0.7 32.3±0.9 ible 4.2 Shows data from the in vivo rabbit hearts. Measurements were taken following ibilisation (T=0) and at the end of 10 minutes treatment in the following groups Control, L- (\ME and Phenylephrine (T=10). All results are measured in pMol/g/chy weight. *p<0.05. n 140 - Blood pressure graph =r rt 120 100 - 80 2 40 ■“ 20 - 0 Control L-NAME Phenylephrine Control L-NAME Phenylephrine I______I stabilisation 10 minutes treatment Figure 4.3. Blood pressure data during stabilisation and 10 minutes following treatment with L-NAME or Phenylephrine. The groups studied were Control group which received an infusion of saline, L-NAME treated group and Phenylephrine treated group. *p<0.01. Chapter 4 156 5 Adenosine standard curve 4 3 CM E CO 2 2 < 1 0 0 20 40 60 80 100 120 Adenosine concentration(p.Mol) Figure 4.4. A standard curve for the adenosine concentration obtained in the coronary effluent samples, measured using HPLC. The peak area was calculated using planimetry. Chapter 4 157 Adenosine concentration 160 n 140 - O Control # L-NAME □ IschaemiaAeperfusion S 120- ■s, 8 10 0 - 80 - 60 - 40 - 20 - 0-J T T T T 1 0 10 Time (minutes) Figure 4.5. Adenosine concentration measured at time points in the coronary effluent collected during the experimental protocol. The experimental groups studied were Control group, L-NAME treated group and Ischaemia reperfusion group. *p<0.01 Chapter 4 158 Lactate measurements from the in vitro hearts 35 30 - f 25 Î20 - 10 - 0 - stabilisation lO’perfusion stabilisation lO’perfusion stabilisation 5’ ischaemia Control L-NAME 5’Ischaemia lO’Repeifusion Figure 4.6. Represents tissue lactate measurements from the Langendorff perfused rabbit hearts. Tissue samples were taken following stabilisation and 10 minutes perfusion (with normal Krebs buffer for control group or Krebs buffer-1- L-NAME for the L-NAME groups) and for the ischaemic group samples were taken following 5 minutes global ischaemia and 10 minutes reperfusion. * p<0.001 Chapter 4 159 Parameters measured for the in vitro rabbit hearts Group Control L-NAME Ischaemia n 5 5 5 Body weight (kg) 2.5±0.3 2.38+1.3 2.44±1.5 Stabilisation (T=0 minute) 55±4.4 CPP 52±2.9 50±1.9 Systolic pressure 98±2.2 95±3.0 96±4.5 11±1.4 Diastolic pressure 12±0.6 1Q±1.5 Adenosine 3.4±0.7 4.8±0.4 3.8±0.6 Lactate 4.58±0.5 5.0±0.9 4.5±1.5 Repafusion (T= 10 minutes) 50+1.9 75±4.5** 68±4.5** CPP 95±2.6 93±5.3 75+3.0* Systolic pressure 11±1.5 Diastolic pre^ure 13+1.0 20±2.1* Adenosine 4.3±0.5 24.0t3.7 83.1±7.2 T 4.50+0.5 5.55+1.7 27.14±3.4 Table 4.3 Shows data from the in vitro rabbit hearts. Measurements were taken following stabilisation (T=0) and at the end of 10 minutes reperfiision for the Control, L-NAME and the ischaemic groups (T=10). Coronary perfusion pressure (CPP), left ventricular systolic and diastolic pressure mmHg, adenosine pmol/L per lOOg of heart and lactate pMol/g/dry weight *p<0.05, **p<0.001 Chapter 4 160 6.0 Discussion From this study, pretreatment with L-NAME in the in vivo hearts significantly increased myocardial lactate levels which may indicate the development of myocardial ischaemia. However, tissue ATP and CP levels were not significantly different. In this situation, the raised myocardial lactate concentration presumably reflected a combination of myocardial hypoperfusion due to coronary vasoconstriction and increased ventricular work due to increased systemic vascular resistance. Interestingly from the isolated perfused rabbit heart studies there were no differences in lactate concentration between the L-NAME treated and Control group, whereas for the global ischaemic group (IP) there was a significant increase. It is possible that we failed to detect localised ischaemia using this whole-heart assay or that the assay was inadequately sensitive to detect mild ischaemia. Our results are consistent with a recent report from Pohl et al. (1994) who measured lactate concentration in the coronary perfusate of the isolated rabbit hearts. They showed that inhibition of NO synthesis causes myocardial ischaemia only if perfusion is pressure controlled but not if the coronary flow rate is maintained in the face of increased coronary perfusion pressure. For the isolated heart study all hearts were perfused at 35ml/min constant flow rate, the increase in adenosine concentration in the coronary perfusate was not associated with increased myocardial lactate levels which suggests that these hearts were not ischaemic. Reports by Pohl, et al (1994) have shown basal release of ATP from the isolated perfused heart. The release of adenine nucleotides, principally from endothelial cells but also from cardiac myocytes and purinergic nerves, increases substantially during ischaemia (Pohl, et al 1994). ATP activates PzY receptors on the endothelium, leading to the release of NO (Ralevic and Bumstock, 1991), and therefore it is conceivable that inhibition of NO might affect endothelial release of ATP. However, we believe that the linkage between the release of ATP and NO from the endothelium is haemodynamic rather than metabolic. Therefore, Chapter 4 161 inhibition of NO under conditions of constant flow results in a significant increase in pressure resulting in coronary vasoconstriction. Due to the vasoconstriction the coronary flow increases luminal shear stress on the endothelium leading to ATP release which can be enhanced by increased shear stress due, for example, to adrenergic vasoconstriction (Paddle and Bumstock 1974, Vial, et al 1987). The inhibition of NO synthesis led to vasocontriction and under conditions of constant flow increased luminal shear stress and resulted in increased release of ATP, which was subsequently degraded to adenosine. Adenosine preserves myocardial blood flow during reperfusion (Zhao, et al 1993) and inhibits neutrophil accumulation (Cronstein, et al 1986). Furthermore the release of adenosine from the endothelium shows the importances of the control of vascular tone and autoregulation when the release of NO is impaired under certain diseases, such as atherosclerosis, diabetes mellitus and hyperlipidemia. Thus it is possible that in hearts affected by these diseases, the concentration of adenosine in the coronary perfusate is higher than in normal hearts. Therefore, one would hypothesise that the increased adenosine release may explain a recent observation that streptoztocin non-insulin-dependent diabetes mellitus protects the rabbit heart from ischaemia and reperfusion (Liu Y and Downey, 1993), although this observation was not repeated in the diabetic dog (Forrat, et al 1993). To explain our findings following the now well-established observation that exposure of the myocardium to a brief episode of ischaemia followed by reperfusion has been shown to minimise or delay the ischaemic damage associated with a subsequent period of prolonged ischaemia (Reimer, et al 1986); the phenomenon known as ‘ischaemic preconditioning’ (Murry, et al 1986, Li, et al 1990). The mechanism for ischaemic preconditioning is not fully understood. However, there is substaintial evidence that ischaemic preconditioning is mediated by the release of adenosine during the brief ischaemic period from the ischaemic cells which fail to cycle ATP (Thornton, et al 1992a, Tsuchida, et al 1992, Toombs, et al 1992a). During the brief period of reperfusion and prior to the sustained ischaemia, the increased ambient concentration of adenosine stimulate cardiac myocytes (via Ai/As receptors) which protects them against the subsequent ischaemic challenge. Chapter 4 162 In our studies reperfusion following 5 minutes global ischaemia resulted in a substantially greater amount of adenosine being washed out into the coronary perfusate than treatment of the heart with L-NAME alone. (Coronary artery branch occlusion which affects a smaller portion of the myocardium than global ischaemia would obviously lead to the washout of less adenosine into the perfusate although the affected myocardium would have been exposed to a similar interstitial concentration). In view of this observation, we suggest that a relationship may exist between the amount of adenosine washed out into the coronary effluent and the extent of cardioprotection. Unfortunately our data does not permit us to comment as to whether it is the peak concentration of adenosine achieved or the total released (i.e. either the peak or the area under the curve on figure 4.4) which determines the extent of protection. 7.0 Conclusion From these results it can be shown that pretreatment with L-NAME resulted in an increase in adenosine concentration, which may be responsible for the cardioprotection which we have previously noted. We have shown in these studies that the release of adenosine was not due to tissue ischaemia that may have resulted from combination of myocardial hypoperfusion and increased ventricular work following treatment with L- NAME. As was shown from the in vitro experiments, there was no increase in lactate concentration but we did show an increase in adenosine release following perfusion with L-NAME. The protective role of adenosine appears to be similar to that implicated in the ischaemic preconditioning mechanism. However, it must be noted that in this case the stimulus for the adenosine release appears to be as a direct effect of the inhibition of nitric oxide synthesis and not ischaemia. Chapter 5 163 Chapter 5: Investigating the Role of Adenosine Release Following Treatment With L-NAME in Protection of the In Vitro Ischaemic Heart 5.0 Aims The objective for this study was to investigate the role of adenosine release following treatment with L-NAME and to determine whether the protection seen following treatment with L-NAME is dependent on the release of adenosine prior to the ischaemic insult. 5.1 Specific Experimental Model New Zealand White rabbits (mean weight 2.35+0.12, n=14) were anaesthetised and ventilated mechanically (see methods chapter). The heart was exposed through a left thoracotomy and the left coronary artery selected for occlusion. The heart was then rapidly excised and mounted onto the Langendorff apparatus and perfused with Krebs Henseleit Buffer (KHB) as described in the methods chapter. The experimental protocol used following 20 minutes stabilisation was 45 minutes regional ischaemia and 180 minutes reperfusion (see figure 5.1). Both L-NAME (30pM) the inhibitor of NO synthase and 8-p-sulfophenyl theophylline (SPT; 75.5pM), an adenosine receptor antagonist were used. There were two treatment groups: 8-p-sulfophenyl theophylline treated hearts (SPT) and 8-p-sulfophenyl theophylline plus L-NAME treated hearts (SPT plus L-NAME) see section 5.1.2. Data for the control hearts were presented in results chapter 3. However, all Chapter 5 164 experimental data from chapters 3, 5 and 6 were randomly performed. Only for simplicity have these groups been q)lit into relevant chapters. At the conclusion of the experiments all hearts were determined for infarct size and risk volume. 5.1.1 Exclusions For these experiments 14 rabbits were used from which two were excluded. One rabbit was excluded from groups 1, this developed intractable ventricular fibrillation during reperfusion and did not recover. One rabbit died during the surgical preparation. A final total of 12 rabbits contributed to the data presented below. Rabbits were assigned sequentially to each group. 5.1.2 Experimental Groups Experimental protocol used for this study are shown in figure 5.1. Group 1: SPT+L-NAME (n=6) The heart was perfused for 5 minutes with SPT and then with perfusate containing both SPT+L-NAME until after the start of 45 minutes ischaemia. The L-NAME continued through and until 15 minutes into the reperfusion period. Group 2: SPT (n=6) The heart was perfused with SPT 15 minutes prior to the 45 minutes of regional ischaemia and continuing into the first minute of ischaemia. Chapter 5 165 5.2 Results 5.2.1 Infarct and Risk Volume determination Inhibition of NO synthesis with L-NAME prior to ischaemia and reperfusion significantly reduced the ratios of infarct to risk volume following 45 minutes coronary branch occlusion and 180 minutes reperfusion from 64.4.3+4.2% (Control n=7) to 41.4+5.1% (L-NAME, n=8), p<0.05 (as was shown from results presented in chapter 3). This protection was prevented in groups pretreated with SPT 58.09+7.9% (L- NAME+SPT n=7), although SPT on its own had no effect on the infarct to risk volume, 63.17±6.5% (SPT n=6), see table 5.1 and figure 5.2. The mean risk volumes were not significantly different between groups (see table 5.1 and figure 5.2). n tü=r ■Hq LA Experimental Protocol for the isolated Langendorfî Perfused Rabbit Heart Time (minute) stabilisation 5’ 10’ 45’ 180’ I 2 0' ir ir \ Ischaemia reperfusion SPT L-NAME Figure 5.1. Protocol studied for the isolated Langendorff perfused rabbit heart: control groups were stabilised for 20 minutes followed by 45 minutes coronary artery occlusion and 180 minutes of reperfiision. For the treated groups, hearts were perfused with SPT and /or L-NAME plus Krebs Henseleit buffer. Perfusion with SPT was started 15 minutes prior to ischaemia and continued into 1 minute of ischaemia, while treatment with L-NAME was started 10 minutes prior to ischaemia and continued into the early part of reperfusion. » Infarct and haemodynamic data from the isolated perfused rabbit hearts. s Group Control L-NAME SPT+L-NAME SPT ILA 8 6 Weight (kg) 2.47±0.21 2.20±0.14 2.33±0.12 2.37±0.13 Heart weight (g) 0.685±0.10 0.609±0.10 0.735±0.13 0.680±q.ll Risk (cm^) 0.69±0.1 0.609±6.1 0.735±0.13 0.68 ±0.01 Infarct(cm3) 0.463±0.09 0.240±0.03* 0.516±0.09 0.469±0.09 Infarct/Risk% 64.64±4.28 41.44+5.06* 58.09±7.9 63.17+6.49 Time- Omin (at end of stabilisation) CPP 42+3 47±2 49±3 42±5 Systolic 89±6 96±6 80±5 76±5 Diastolic 7+1 8±1 6+1 7±1 rime- 5min (prior to ischaemia) CPP 45±3 83±8* 79±8* 6Q±8 76±5 Systolic 89+6 96±5 80±5 Diastolic 7+1 8+1 6±1 6±1 Time-30min reperfiision CPP 64+6 99±6* 100+ 11* 115+20* Systolic 69+7 62±7 61±6 56±4 Diastolic 17+3 14±3 11±3 16+4 Time- 180min reperfiision CPP 104+9 111±11 126±12 123±17 Systolic 43+3 63±3* 40±10 34+3 Diastnlic______33+4 14+3* 28+2 30±2 The table 5.1. Experimental data for the isolated perfused rabbit hearts treated with L-NAME, L-NAME+SPT and SPT only. Infarct volume is expressed as a percentage of the risk volume for each of the experimental groups. Coronary perfusion pressure (CPP), left ventriciüar systolic and diastolic pressures are measured as mmHg. Data was recorded throughout the experiment, however, only some of the time points are presented above: which were at stabilisation (Time-Omin), following 10 minutes perfusion with L-NAME, L-NAME+SPT or SPT only (Time- 5min), 30 minutes into reperfusion (Time-30min reperfiision) and at the conclusion of the 3 experiment (Time-180 min reperfiision). Values are expressed as mean+SEM. *p<0.05. n=r Ln Infarct and risk data for the isolated perfused hearts treated with 1.2-j L-NAME and SPT - m 1.0- 400 oB y i 0.8 - -80 q a o > 0.6- - j o . 1 0.4- -40 T) - 0.2- -20 5 0.0- _0 Control L-NAME L-NAME SPT Control L-NAME L-NAME SPT Control L-NAME L-NAME SPT +SPT I I +SPT I______+SPT I Risk volume cm^ Infarct volume cm^ I/R% Figure 5.2. Representation of the volume at risk, the infarct volume and the percentage VR for the Langendorff perfused rabbit hearts. Risk areas were determined with fluorescent particles and infarct areas determined with triphenyl tétrazolium chloride. All values are expressed in mean +SEM *p<0.05. Chapter 5 169 5.2.2 Haemodynamic Data There was no differences between the groups in coronary perfusion pressure (CPP) measured at the start and the conclusion of the experiment. However, CPP was significantly increased 10 minutes after the administration of L-NAME from 47+2.0 mmHg to 83+8.0mmHg for the L-NAME treated group and from 49±3.0mmHg to 79+8.0 mmHg for the SPT+L-NAME. However, SPT alone increased CPP but the increase was not as great as that seen with L-NAME treated hearts. After 30 minutes of reperfusion, CPP was the same in all the hearts (see table 5.1 and figure 5.3). Haemodynamic data relating to ventricular performance for all experiments are presented in table 5.1, figures 5.4 and 5.5. There were no differences between groups in the haemodynamic variables measured throughout the experiment. In all groups the left ventricular diastolic pressure increased (see table 5.1 and figure 5.4) and systolic pressure decreased (see table 5.1 and figure 5.5) over the duration of the experiments. n g" O) LA Coronary perfusion pressure recorded at time points in the in vitro experiments 180-, 120 - % (S 80 - a o 60 — Oa ^ 4 0 - O SPT+L-NAME O m SPT o 20 SPT M l U L-NAME Stab Ischaemia Reperfusion 0 J 0 35 Time (minutes) 80 260 Figure 5.3. The measurement of mean coronary perfusion pressure over the duration of the experiment for the isolated perfused rabbit hearts. SPT+L-NAME-treated (n=6) and SPT-treated (n=6) *p<0.05. n=r ■SO - t 1 /1 Left ventricular diastolic pressure trace for the Langendorff perfused hearts 100 Ischaemia Reperfusion H SPT . O L-NAME+SPT Oh 50 40 N 30 20 - 3 (J I 10 - 0 - -4-h œ-10 - 2 0 - 0 35 26080 -J Time (minutes) Figure 5.4. Represents the left ventricular diastolic pressure trace for the isolated perfused hearts treated with L-NAME+SPT or SPT alone prior to ischaemia and reperfusion. n=r "SfC Ui Left ventricular developed pressure trace for the L-NAME and SPT Langendorff perfused hearts 100 n U SPT O L-NAME+SPT CD 80 - i 70 - P h CJ B N 50 40 1 - 20 ^ Ischaemia Reperfusion œ J 10 0 - 0 I s 260 Time (minutes) Figure 5.5 . Shows the left ventricular systolic pressure trace for the isolated perfused rabbit hearts treated with SPT and L-NAME+SPT. The pressure was continuously measured through a balloon in the left ventricle on the isolated Langendorff perfused rabbit hearts. Chapter 5 173 5.3 Discussion In the preceding chapter, prior inhibition of NO synthesis by L-NAME was shown to lead to the release of adenosine from the isolated rabbit heart independent of myocardial acidosis. In this study, the protection induced by the addition of L-NAME can be abrogated by the adenosine receptor antagonist (SPT). These findings suggest that cardioprotection following nitric oxide synthesis inhibition may be dependent on increased adenosine release. This adenosine-dependent cardioprotection is similar to that first reported by Liu et al (1991) who observed that the reduction in infarct size which follows ischaemic preconditioning in the rabbit heart is dependent on adenosine release and Ai receptor activation and can be abolished by SPT. These observations may therefore support a common mechanism for the protection induced by pretreatment with L-NAME in our experiment and that seen with ischaemic preconditioning. SPT is a methylxanthine derivative with a sulfophenyl group added which prevents the molecule entering the cell and therefore intracellular phosphodiesterases are not affected. SPT is an unselective adenosine receptor antagonist with a Ki of 4.5pM and 6.3jiM for the Ai and A2 receptors respectively (Daly, et al 1985), therefore the concentration used in our study (75.5pM) will have been sufficient to block both sub- types of adenosine receptors. In view of the cardioprotective properties of adenosine, it is perhaps surprising that pretreatment with SPT (group 4) did not increase infarct size, however, this finding is consistent with other reports (Zhao, et al 1993). Interestingly, Zhao et al (1993) have recently shown that SPT can worsen myocardial injury either if additional doses administered during ischaemia and reperfusion or, alternatively, if SPT is administered just prior to reperfusion (Zhao, et al 1993). Thus it appears that the release of adenosine during and after any ischaemic period will mitigate against the reperfusion injury which follows. Treatment with either L-NAME or SPT increased coronary perfusion pressure, although the increment reached statistical significance only after L-NAME administration (see figure 5.3). These data support an important role for NO but not Chapter 5 174 adenosine as a determinant of coronary artery tone in the unstressed isolated perfused heart (Ameczua, et al 1989 and Kelm and Schrader 1990). However, during the reperfusion period that followed 45 minutes of regional ischaemia, CPP was higher in both L-NAME and SPT treated groups compared with control (see figure 5.3), which indicates that both NO and adenosine contribute to vasomotor tone during reperfusion. Therefore, the concentration of adenosine present in the coronary perfusate during reperfusion may have been increased in L-NAME treated hearts, which could have further contributed to myocardial protection (Zhao, et al 1993). 5.4 Conclusion This study supports the hypothesis from the preceding chapter that cardioprotection following the inhibition of nitric oxide synthesis is largely dependent on the compensatory release of adenosine. However, these results do not exclude the possibility that some myocardial protection results from inhibition of NO release during early reperfusion, however, we conclude that most of the benefit is derived from the release of adenosine. Chapter 6 175 Chapter 6: Ischaemic Preconditioning in the In Vivo and In Vitro Rahbit Heart. And Does Nitric Oxide Play a Role? 6.0 Introduction During ischaemia, the release of adenine nucleotides from endothelial cells, cardiac myocytes and purinergic nerves increases substantially (Ralevic, et al 1991). Nucleotides are rapidly degraded by extracellular ectophosphatases to adenosine which accumulates in ischaemic tissue and is progressively washed out during reperfusion. Interest in the role of adenosine during ischaemia and reperfusion has increased following the observation that a brief period of ischaemia-reperfusion can increase tolerance against subsequent sustained ischaemia-reperfusion (Murry, et al 1986). This phenomenon, termed ‘ischaemic preconditioning’, is associated with infarct size limitation as well as preservation of function and an anti-arrhythmic action, using both in vivo and in vitro models of myocardial ischaemia (Walker and Yellon, et al 1992 review). We have reported previously in our results chapter 3, 4 and 5 that prior inhibition of nitric oxide synthesis by L-NAME protects the myocardium of both the in situ and the isolated rabbit hearts by an adenosine-dependent mechanism, this mechanism of protection was similar to that described following ischaemic preconditioning (Liu, Thornton, Van Winkle, et al 1991). Our evidence, for this association is that, in our previous studies (results chapter 3, 4 and 5), we have shown that the protection induced by L-NAME depends on the release of adenosine because it can be abrogated by SPT, an adenosine receptor antagonist. Secondly, L-NAME and brief global ischaemia both give rise to an increase in the adenosine concentration in the coronary Chapter 6 176 effluent, although the increment was less marked after L-NAME than after ischaemia. It may be possible that prior inhibition of nitric oxide synthesis by L-NAME ‘preconditions’ the rabbit hearts and this may be the mechanism for protection against subsequent ischaemia and reperfusion injury. 6.1 Aim i) The objective of this study was to precondition the rabbit heart, using both the in vivo and in vitro models and to investigate the effects of inhibition and augmentation of endothelium-derived nitric oxide production during ischaemic preconditioning. ii) The protective role of adenosine during ischaemic preconditioning was further investigated using both an adenosine antagonist and L-NAME. We used L-NAME (lOmg/kg for the in vivo rabbit experiments and 30pM for the in vitro rabbit experiments) to inhibit NO synthase and 8-p-sulfophenyl theophylline (SPT, 75.5pM), to inhibit the adenosine receptor. Infarct and risk volumes were determined at the end of the experimental protocol. 6.2 Experimental Model for the In Vivo Study New Zealand white rabbits (mean weight 2.59±0.04 kg, n=20) were anaesthetised and surgically prepared as described in chapter 2. All rabbits were randomly assigned to the experimental protocols described in figure 6.1. Heparin (KXX) units) was administered intravenously at the start, at the end of sustained ischaemia and at the conclusion of the experiment. 6.2.1 Exclusions Of the 24 rabbits used in this study, 4 animals in all were excluded from the analysis. Chapter 6 177 One rabbit (from group 3) developed intractable ventricular fibrillation and did not recover, one failed to manifest electrocardiographic changes with reperfusion (from group 3); one rabbit was excluded as a result of no change in blood pressure on treatment with L-NAME (from group 2); one rabbit died during the surgical preparation (group 3). The remaining 20 rabbits contributed to the data presented below. 6.2.2 Ischaemic Preconditioning Protocol For the in vivo experiments all rabbits were stabilised for 20 minutes followed by an infusion over one minute of normal saline (control groups ) or L-NAME (10 mg/kg in the treated groups) and the animals were then monitored for a further 10 minutes. This was followed by the ischaemic preconditioning protocol (IP), which consisted of 5 minutes coronary branch occlusion followed by 10 minutes reperfusion prior to 30 minutes sustained occlusion and 120 minutes reperfiision (figure 6.1). n Ischaemic preconditioning protocol for both the in vivo and in vitro rabbit hearts CO "S In vivo protocol Time (minute) stabilisation 1’ 10’ 5’ 10’ 30’ ischaemia 120’ reperfusion I r I------II 11------I ■ k\\] L-NAME à In vitro protocol Time (minute) stabilisation 10’ 5’ 10’ 45’ ischaemia 180’ reperfusion [ I :s : L-NAME^ stabiUsation 5’ 10’ 5’ 10’ 45’ ischaemia 180’ reperfusion [ s p t 4 - L-NAME i □ Perfusion of heart with drugs prior to ischaemia ^ = regional ischaemia = Perfusion of hearts with SPT followed by reperfusion and/or L-NAME Figure 6.1. Protocol studied for both the in vivo hearts and the isolated rabbit hearts. All heart underwent the ischaemic preconditioning protocol. Hearts were treated with L-NAME (lOmg/kg for the in vivo model or 30pM for the in vitro hearts) and for the isolated hearts a third group was studied, this was the SPT (75.5pM) treated group were hearts were perfused with both SPT and L-NAME. At the conclusion of the experiment the hearts were determined for risk and infarct areas. 00 Chapter 6 179 6.2.3 Experimental Groups Group 2: Ischaemic preconditioning, IP (n=10) Following an intravenous infusion of 5mls normal saline over 1 minute, rabbits were monitored for a further 10 minutes followed by the IP protocol. This was followed by 30 minutes sustained ischaemia and 120 minutes reperfusion. Group 3: IP+L-NAME (n=10) Following an intravenous infusion of (lOmg/kg) L-NAME over 1 minute, rabbits were monitored for a further 10 minutes followed by the IP protocol than 30 minutes of ischaemia and 120 minutes of reperfiision. 6.3 Experimental Model for the In Vitro Study For the in vitro experiments rabbits (mean weight 2.4+Q.05 kg, n=17) were surgically prepared as described in methods chapter 2. The heart was exposed through a left thoracotomy and the left coronary artery selected for occlusion. The heart was then rapidly excised and mounted on to the Langendorff apparatus and perfused with Krebs Henseleit buffer. All hearts were stabilised for 20 minutes followed by the ischaemic preconditioning protocol which consisted of 5 minutes regional ischaemia followed by 10 minutes reperfusion prior to 45 minutes sustained occlusion and 180 minutes reperfusion. Rabbits treated with L-NAME for the in vivo experiments showed a significant increase in blood pressure, this rise in pressure was present until the early part of reperfusion (see results chapter 3 figure 3.5 and 3.6). The protocol studied for the isolated heart model was designed such that when hearts were perfused with L-NAME plus KHB the increase in pressure was maintained throughout ischaemia and continued into the early part of reperfusion (see figure 6.1). For the L-NAME treated group, hearts were perfused with L-NAME plus Krebs Henseleit buffer for 10 minutes prior to the Chapter 6 180 ischaemic preconditioning protocol and continued into the early part of second reperfusion (see figure 6.1) 6.3.1 Exclusions For these experiments 20 rabbits were used from which 3 were excluded. One rabbit was from group 1, it developed intractable ventricular fibrillation during reperfusion and did not recover. One failed to manifest changes on reperfusion (group 2), and one rabbit was excluded because of technical problems (group 3). A total of 17 rabbits contributed to the data presented below. 6.3.2 Experimental Groups Group 1: Ischaemic Preconditioning (n=6) Followed the ischaemic preconditioning protocol, which consists of 5 minutes regional ischaemia followed by 10 minutes of reperfusion, prior to 45 minutes sustained ischaemia and 180 minutes reperfusion. Group 2: Ischaemic Preconditioning+L-NAME (n=6) The heart was perfused with L-NAME 10 minutes prior to the ischaemic preconditioning protocol and continued into the early part of second reperfusion. Group 3: Ischaemic Preconditioning+L-NAME+SPT (n=5) The heart was perfused with SPT plus KHB for 5 minutes, followed by perfusion with both SPT+L-NAME plus KHB for 10 minutes prior to the ischaemic preconditioning, and then hearts were perfused with L-NAME plus KHB alone until 30 minutes into the second part of reperfusion (see figure 6.1). Chapter 6 181 6.4 In Vivo Results 6.4.1 Infarct and Risk Volume Determination Following ischaemic preconditioning (IP) the infarct/risk ratio was reduced to 13.84+3.7% which was significantly less than control (40.75+ 4.3% as was reported in chapter 3) pcO.OOl. Furthermore the additional presence of L-NAME in the IP treated group conferred no additional benefit (11.26+2.9%) see figure 6.2. The volume of myocardial tissue at risk following ischaemia and reperfusion were not significantly different between groups (chapter 3 table 3.1 and table 6.1). Risk volume, infarct volume and percentage of risk and infarction are shown in table 6.1 and figure 6.2. 6.4.2 Haemodynamic Data The infusion of 10 mg/kg L-NAME led to a rapid rise in mean blood pressure from a basal value of 95+3mmHg to 118+4mmHg, this increase in pressure was still significantly higher than the control group at the start of the final reperfusion period (see table 6.1). The hypertensive response seen on treatment with L-NAME was accompanied by a reflex bradycardia. There was no differences in rate-pressure product prior to coronary occlusion, during treatment, IP or reperfusion, implying that myocardial work and therefore oxygen demand was likely to be similar between groups. All rate-pressure product data are shown in table 6.1 for all the groups. Arterial blood gas measurements taken at time points during the experiment showed no significant differences between groups (see table 6.1). n CD3" "O -In Infarct and risk volume for the in vivo hearts - 50 0.8 - J - 40 en 0.6 - S’ - 30 0.4 - - 20 2 0.2 - - 10 0.0 - - 0 Control IP IP+L-NAME Control IP IP+L-NAME Control IP IP+L-NAME I------1 I______I I------1 Risk area cm3 Infarct area cm3 %Infarction (I/R) Figure 6.2. Represents results for risk area and infarct area. Percentage infarction expressed as a percentage of risk volume for the in vivo model. Both ischaemic precondition (IP) and ischaentic precondition plus L-NAME (IP+L-NAME) have shown significant reduction in l/R.% vs Control hearts (Control data represented in results chapter 3). All values are expressed as the mean + SEM, *p< 0.01. Infarct and haemodynamic data from the in vivo rabbit hearts. Group Control IP IP+L-NAME ÎQ\ n 10 10 10 Weight (kg) 2.58±0.09 2.62±0.05 2.49±0.25 Heart weight (g) 6.82+0.09 7.21±0.18 6,92±0.13 Risk (cm^) 0.637±0.18 0.342±0.08 0.51±0.14 Infarct(cm^) 0.213+0.04 0.039±0.01* 0.036±0.01* Infarcl/Risk% 40.75±4.29 13.84±3.69* 11.26±2.90* Time- Omin (at end of stabilisation) RPP 23793±2035 26983±1103 23755±2040 pH 7.41±0.03 7.39±0.08 7.45±0.01 pC02 4.29±0.25 4.21±0.50 4.85+0.40 p02 46.80+5.0 56.45±7.5 44.27±8.5 Time- lOmin (After L-NAME) BP -9±2 -1±3 20±4* HR -9±4 -1±6 -18±2* RPP 218994-1560 28085±1060 22970+1580 pH 7.38±0.03 7.45±0.02 7.42±0.06 pÇ02 4.53+0.45 4.50±0.25 4.25±0.50 p02 48.80+3.0 51.75±1.5 47.57+5.5 Time-120min reperfusion RPP 16880±9580 1690Q±1368 16785±1940 pH j 7.48±0.02 7.40±0.08 7.45±0.01 pC02 4.4Q±0.50 4.65±0.40 4.58±0.30 p02i 43.80±6.0 55.60+5.0 48.3.0±5.8 The table 6.1 Shows experimental data for the in vivo rabbit hearts. Infarct volume is expressed as a percentage of the risk volume for each of the experimental groups. Groups studied were control, ischaemic preconditioning (IP) and ischaemic preconditioning plus L-NAME (IP+L-NAME). Haemodynamic data is shown at the start (T=Omin), 10 min after iirfusion of saline or L-NAME or after ischaemic preconditioning protocol (T=lQmin) and at the conclusion of the experiment (T=120min). Haemodynamic data represents; blood gas analysis for pH, pC02 and p02; rate pressure product mmHg(RPP); blood pressure mmHg (BP) and heart rate bpm (HR). Values are expressed as mean±SEM. *p<0.01 vs. control (experimental data represented in chapter 3) Chapter 6 184 6.5 In Vitro Results 6.5.1 Infarct and Risk Volume Determination Following ischaemic preconditioning (IP), the infarct/risk ratio was 21.4+1% (n=6) which was significantly less than control group, (64.4.3+4%, reported in results chapter 3) pcO.OOl, but the protection seen with the IP group was significantly greater than L-NAME (41.4+5%) treated hearts alone, p<0.05 (see chapter 3 table 3.2). The combination of L-NAME and IP (L-NAME +IP) conferred no additional benefit. However, pretreatment with SPT abolished the protection conferred by preconditioning and L-NAME (ie IP+L-NAME+SPT), see table 6.2 and figure 6.3. The volume of myocardial tissue at risk following the ischaemic preconditioning protocol were not significantly different between groups (see chapter 3 table 3.2, table 6.2 and figure 6.3). 6.5.2 Haemodynamic Data There was no differences between groups in coronary perfusion pressure (CPP) measured at the start and the conclusion of the experiment. However, CPP was significantly increased 10 minutes after the administration of L-NAME in all treated hearts. SPT alone increased CPP but this was not significantly greater than control. After 30 minutes of reperfusion, CPP was significantly high than control in all hearts except those assigned to ischaemic preconditioning alone (see results chapter 3 table 3.2). Infarct and haemodynamic data from the in vitro rabbit hearts. Group Control IP IP+L-NAME IP+L-NAME+SPT n 6 6 6 5 Weight (kg) 2.47±0.21 2.58±0.09 2.20±0.07 2.44±0.22 Heart weight (g) 5.54+0.1 6.01±0.05 5.68±0.14 6.21±0.14 Risk (cm^) 0.685±0.10 0.682±0.09 0.742±0.11 0.885±0.12 Infarct(cm^) 0.463±0.09 0.143±0.01* 0.147±0.02* 0.522±0.08 Infarct/Risk% 64.6+4.28 21.42+1.18* 2021±1.93* 58.46±2.39 Time- Omin (at end of stabilisation) CPP 42±3 39±1 44±3 48±5 Systolic 89±6 75±9 90±4 74±5 Diastolic 7±1 9±1 7±1 10±2 Time- 15min (prior to ischaemia) CPP 45+3 41±3 73±5* 94±13* Systolic 89±6 75±9 9Q±4 74±5 Diastolic 7+1 9+1 7±1 10±2 Time-30min reperfusion CPP 64+6 58±3 107±13* 108±8* Systolic 69±7 71±6 81±4 52+5 Diastolic 17+3 13±2 14±3 21+4 Time=i 180min reperfusion CPP 104±9 107±12 126±13* 122±11* Systolic 43±3 6Q±4* 71±5* 31+5 Diastolic 33+3 16±4* 18±2* 3Q±2 The table b-2 Shows experimental data for the isolated perfused rabbit heart Infarct volume is expressed as a percentage of the risk volume for each of the experimental groups. Experimental groups studied were control, ischaemic preconditioned (IP), ischaemic preconditioned plus L-NAME and ischaemic preconditioned plus L-NAME plus SPT (IP+L-NAME+SPT). All values are expressed as mean+SEM. *pc0.05 vs. control (experimental data for control group was represented in chapter 3) nrr ■s(D Risk and infarct volume for the in vitro rabbit hearts — 80 1.2 X en 1.0 - i - 60 a 0.8 - 0 § r? H 1 0.6 — o OJ 0.4 - * * a - 20 00 * r i 0.2 - DiC -r- vvvVvj 0.0 - L- 0 Control IP IP Control IP IP Control n>+ r: i IP+ +NAME +L-NAME +L-NAME L-NAME L-NAME+SPT +SPT L L I L J Risk area cm3 Infarct area cm^ % Infarction (I/R) Figure 6.3, Represents risk area, infarct area and percentage infarction expressed as a percentage of risk volume for the in vitro model. Groups that were ischaemically preconditioned (IP) or ischaemically preconditioned plus L-NAME (IP+L-NAME) both showed a significant reduction in I/R% vs Control (Control data represented in results chapter 3) and ischaemically preconditioned plus L-NAMEplus SPT (IP+L-NAME+SPT) treated groups. All values are expressed as mean + SEM, * p<0.01. Chapter 6 187 Haemodynamic data relating to ventricular performance for all experiments are presented in table 6.2. There were no differences between groups in the haemodynamic variables measured following stabilisation. In all groups the left ventricular systolic pressure decreased and diastolic pressure increased over the duration of the experiments. At the end of reperfusion, left ventricular systolic pressures were significantly higher in the L-NAME+ischaemic preconditioning treated and ischaemic preconditioning groups in comparison to SPT treated group, p<0.05. Also increases in diastolic pressure were more marked in the hearts which had sustained large infarcts (SPT+L-NAME+IP) in contrast to the smaller infarct groups (IP and L-NAME+IP). 6.6 Discussion The present study confirmed that 5 minutes of coronary occlusion, interrupted by 10 minutes of reperfusion, followed by sustained ischaemia (45 minutes) and reperfusion (180 minutes) showed a significant reduction in infarct size. It was therefore clearly possible to precondition both the in vivo and the isolated rabbit hearts. Pretreatment with L-NAME did not confer additional protection on hearts which were subsequently preconditioned. For the isolated hearts that were pretreated with SPT, all of the protection induced by the combination of L-NAME and ischaemic preconditioning was prevented. Therefore, these observations support a common mechanism for the protection induced by pretreatment with L-NAME and ischaemic preconditioning. However, in contrast to IP, we propose that the trigger for adenosine release after inhibition of NO synthesis is an alteration in coronary haemodynamic rather than myocardial ischaemia. Treatment with L-NAME or both L-NAME plus SPT increased CPP, although the increment reached statistical significance only after L-NAME (see table 6.2). These data support an important role for NO but not adenosine as a determinant of coronary artery tone in the unstressed isolated, perfused heart (Ameczua, et al 1989 and Kelm Schrader 1990). Our results contrast with those of Vegh et al (1992) who have demonstrated that NO generation contributes to the antiarrhymic effects of IP in the in situ canine heart. This Chapter 6 188 may reflect either a disparity between arrhythmogenesis and infarct size as measures of myocardial injury or that L-NAME may inhibit the opening of preformed collaterals in the canine coronary circulation (Yamamoto, et al 1984) that are absent in the rabbit heart. 6.7 Conclusion In conclusion, this study supports our previous findings (from results chapter 3,4 and 5) that the inhibition of nitric oxide synthase protects against ischaemia and reperfusion injury in the rabbit heart. The mechanism responsible for the cardioprotection was an increase in release of adenosine, this adenosine-mediated mechanism was similar to that implicated in ischaemic preconditioning. Current evidence suggests that a common adenosine dependent mechanism may be responsible for the cardioprotection observed following inhibition of nitric oxide synthase and ischaemic preconditioning, as was observed from our results (chapter 3,4 and 5). However, ischaemic preconditioning or ischaemic preconditioning plus L-NAME in both in vivo and in vitro gave greater protection than hearts treatment with L-NAME only, which may provide some evidence to support a dose response relationship between adenosine release and the degree of cardioprotection itself. Chapter 7 189 Chapter 7: Perfusion of Hearts With Adenosine Prior to Ischaemia and Reperfusion, Does This Confer Protection to the Myocardium 7.0 Introduction The release of adenosine from ischaemic cells and its subsequent washout during the preconditioning cycle has been implicated in the mechanism of ischaemic preconditioning (Liu, et al 1991). The effector pathway of adenosine-induced protection in preconditioning remains unclear but appears to involve Ai/As (and not A i) receptors on the cardiac myocytes (Liu, et al 1994b). In addition to the cardioprotective effect resulting from exposure to adenosine prior to sustained ischaemia-reperfusion as observed for preconditioning, the presence of increased concentrations of adenosine during reperfusion is also beneficial and further augmentation of adenosine levels during reperfusion offers protection against injury (Gruber, et al 1989 and Zhao, et al 1993). The mechanism of the cardioprotection offered by adenosine in this respect may involve A2-mediated coronary arteriolar vasodilatation (Ralevic, et al 1991), inhibition of local release of vasoconstrictors such as noradrenaline (Richard, et al 1987) and endothelin (Velasco, et al 1993) as well as the inhibition of activation and aggregation of circulating platelets and granulocytes (Gruber, et al 1989 and Cronstein, et al 1986). Thornton et al. (1992) has recently shown that brief intracoronary infusion of adenosine or intravenous infusion of the Ai adenosine receptor agonists, 2-Cloro-N6- Cyclophenyladenosine (CCPA) (Tsuchida A Lui G, Downey J 1993c) or Nti-phenyl- 2R-isopropyl adenosine (PIA) (Thornton et al 1992 and Van Winkle, et al 1994), was shown to mimic the reduction in infarct size achieved by a short period of ischaemia Chapter 7 190 (Li, et al 1990, Van Winkle, et al 1991a,b and Li, et al 1992b). We have also recently reported a relationship between elevated adenosine concentration in the coronary perfusate of the rabbit isolated heart and infarct size reduction by comparing two different experimental techniques which have adenosine-dependent mechanisms; see results chapter 4 and 5 (Woolfson, et al 1995). 7.1 Aims In the light of those findings, we have gone on to investigate whether there is a concentration dependent response between the administration of adenosine prior to ischaemia-reperfusion and subsequent infarct size. The experimental model used was the isolated perfused rabbit heart, perfused with a specific concentration of adenosine in Krebs Henseleit buffer (lOO^M), prior to regional ischaemia and reperfusion. In the studies by Lui et al (1991) using the isolated perfused rabbit heart model in which brief intracoronary infusions of adenosine or intravenous infusion of Ai adenosine receptor agonists were used, their experiments were performed using a model set at high coronary perfusion pressure (CPP) lOOmmHg and constant flow. The experimental model we used was of a mean CPP of 50 mmHg which is constant for all of our studies undertaken. i) Therefore in view of these differences in CPP used between our isolated heart studies and that of Liu et al., we decided to examine the effects of pretreatment with adenosine prior to regional ischaemia and reperfusion under conditions of both normal (50mmHg) and high (>100mmHg) CPP, using a constant flow rate model. We also perfused some hearts with 2-Cloro-N6-C!yclopentyladenosine (CCPA), being a potent long lasting specific Ai adenosine receptor agonist, unlike adenosine which is inactivated rapidly and has a very short half life of only seconds. We also collected coronary effluent from these hearts and later assayed for adenosine concentration, using HPLC (see methods chapter 2). Chapter 7 191 ii) The second part of this study was to determine whether there existed a concentration dependent relationship between the amount of adenosine released and myocardial protection obtained. As such, hearts were perfused with different concentration of adenosine prior to ischaemia and reperfusion and infarct size determined at the end of the experimental period. i) 7.1.1 Experimental Protocol for Different Coronary Perfusion Pressure Models New Zealand white rabbits (mean weight 2.54+0.3, n=30) were used. All rabbits were surgically prepared as described in the methods chapter 2. All hearts were perfused on the isolated Langendorff apparatus (see methods chapter) and randomly assigned to the experimental protocol. During stabilisation the heart was perfused with Krebs Henseleit buffer (KHB) alone. lOO^M adenosine was added to the KHB and the heart perfused for 5 minutes followed by 10 minutes of washout. This was followed by 45 minutes of regional ischaemia and 120 minutes of reperfusion. An ischaemic preconditioning group and a CCPA treated group were also studied with normal KHB (see experimental groups; section 7.1.3). At the end of the experimental protocol hearts were also analysed for infarct and risk volume. Coronary effluent was collected from the isolated perfused hearts and later assayed for adenosine concentration (methods chapter 2). The effluent was collected during stabilisation and 1 minute into reperfusion following 5 minutes treatment. Effluent was collected from both normal CPP studies and high CPP studies. Samples were collected and assayed from normal CPP; control hearts, adenosine treated hearts, CCPA treated and ischaemic preconditioned hearts. 7.1.2 Exclusions A total of 36 rabbits entered the study. 6 rabbits were however excluded from the Chapter? 192 analysis. Three rabbits were excluded (from groups 1; (1 at normal CPP and group 2 at high CPP) because of technical problems. Two rabbits failed to manifest changes on reperfusion (from group 1 and 4; normal CPP), and one rabbit was excluded because of inadequate staining (from group 1 high CPP). A total of 30 rabbits contributed to the data presented below. 7.1.3 Experimental Groups a) Normal Coronary Perfusion Pressure: All heart were perfused with KHB at a constant flow rate of 34+4ml/min, and CPP was set to 50mmHg. Group 1: Control (n=5) Following stabilisation all hearts were subjected to 45 minutes regional ischaemia and 120 minutes reperfusion. Group 2: Adenosine (n=5) Hearts were perfused with adenosine (lOOpM) plus KHB for 5 minutes and 10 minutes washout with KHB followed by 45 minutes regional ischaemia and 120 minutes reperfusion. Group 3 Ischaemic preconditioning (n=4) For the ischaemic preconditioning group hearts were subjected to 5 minutes of regional ischaemia and 10 minutes reperfusion, followed by 45 minutes sustained ischaemia and 120 minutes reperfusion. Group 4 CCPA (n=4) Hearts were perfused with CCPA (200^.1) plus KHB for only 5 minutes followed by 10 minutes washout with KHB, followed by 45 minutes of regional ischaemia and 120 minutes reperfusion. Chapter 7 193 b) Higher Coronary Perfusion Pressure: All hearts were perfused at a constant high flow (75±5 ml/min) and CPP of >100mmHg. Group 1 : Control (n=6) Hearts were subjected to 45 minutes regional ischaemia and 120 minutes reperfusion. Group 2: Adenosine (n=6) Hearts were perfused for 5 minutes with adenosine(lOOpM) plus KHB followed by 10 minutes washout with KHB and then 45 minutes regional ischaemia and 120 minutes reperfusion. 7.2 Results 7.2.1 Infarct and Risk Volume For the ischaemic preconditioning hearts, using normal CPP, the ratios of infarct to risk volume following 45 minutes coronary branch occlusion and 120 minutes reperfusion were significantly reduced from 57.84+.0.2% (Control n=5) to 17.37±0.7% (IP, n=4), p<0.(X)l. When hearts were perfused with CCPA 10 minutes prior to 45 minutes sustained ischaemia and 120 minutes reperfusion still showed a significant reduction in the ratios of infarct to risk volume (20.61+4.9%, n=4), p<0.(X)l. But for the hearts that were perfused with adenosine (lOO^iM) 10 minutes prior to 45 minutes sustained regional ischaemia and 120 minutes reperfusion, at normal CPP there was no reduction in infarct to risk ratios, 55.57+2.1% (n=5, p=NS). See table 7.1 and figure 7.1. The following infarct and risk results were from the study performed using high CPP. Following an increase in CPP and flow rate, pretreatment with adenosine (l(X)p.M) prior to 45 coronary branch occlusion and 120 minutes reperfusion, the infarct to risk volume was reduced to 23.87+2.9% (n=6, p<0.(X)l), which was significantly less than control hearts perfused at the high CPP and flow ie 56.68+6.3% (n=6, p<0.(X)l). Chapter 7 194 There were no differences in infarct to risk volume in both normal and high CPP control groups studied. See table 7.1 and figure 7.1. The determination of mean risk volumes were not significantly different between all the groups from both normal and high CPP studies (see table 7.1). 7.2.2 Coronary Perfusion Pressure (CPP) Coronary perfusion pressure traces for experiments performed at normal pressure are presented in figure 7.2. Data for the isolated perfused rabbit hearts . Hearts perfused with normal or high coronary perfusion pressures Normal CPP I-J HkhCPP ^ConïoT Adenosii^ Preconditioning CCPA r Control Adaiosine ■ Group No 2 4 5 6 n 5 5 4 4 6 6 Body weight (kg) 2.81±0.5 2.3±0.8 2.44±0.3 2.5±0.2 2.65±0.1 2.58±0.1 Heart weight (g) 8.8±0,1 6.4±0.1 6.8±0.5 7.2±0.4 6.8±0.3 7.Q±0.5 Risk (cm3) 3.944+0.1 1.10±0.4 0.986±0.8 1.066±0.3 1.035±0.1 1.066±0.5 Infarct (cm3) 0.547±0.1 0.613±0.1 0.173±0.1* 0.216±0.1* 0.584±0.1 0.25+0.1* Infarction (%) 57.84±0.2 55.57±2.1 17.37±0.8** 20.61±4.9** 56.68±6.3 23.87±2.9** Time-Omin (end of stabilisation) CPP 49±1.0 45.8±2.0 47.5±3.0 52±8.0 103±4.0 105±3.0 Systolic 92±2.5 92.8±^.6 91±7.0 102±2.0 143.5±6.6 149±5.0 Diastolic 9±1.0 8.4±1.3 7±1.0 7+3.0 7.3±3.7 8.6±1.7 Time-Smin (adenosine) oCPP. 50+2.0 43.2±1.0 61±1.0* 51.1±1.5 105.5±3.0 85±6.0* Systolic 92.5±2.0 91±4.2 94±6.0 101±1.0 138.3±7.2 142.5±3.3 Diastolic 10±2.0 8.4±1.9 8±0.5 6.5±3.5 9+2.0 9.1±1.8 Time-lOmin (perfusicm) 44.8±2.0 oCPP. 49±1.0 53±5.0 53±5.0 104.6±4.0 104.6±4.0 Systolic 95±5.0 93.6±4.4 90±8.0 10Q±2.0 14Q±5.7 143±4.8 Diastolic 10±2.0 9.4+1.8 9+1.0 7+3.0 9+2.0 9.1±1.9 Time-120min (reperfusion) CPP 82.5±3.0 95±6.0 90+5.0 183±8.0 151±6.0 Systolic 66±2.0 72±1.6 82±15* 85±9.0* 87.3±4.4 104.3±3.6* Diastolic 25±7.0 30±8.3 12.5±2.5 11±5.0 38±8.5 17±3.3 Table 7.1. Experimental data for the isolated perfused rabbit hearts. For groups 1-4, hearts were perfused at normal coronary perfusion pressure (CPP) for control, adenosine, ischaemic preconditioning and CCPA treated groups. Experimental groups 5 and 6 were perfused at the higher CPP (l(X)mmHg). Infarct volume is expressed as a percentage of the risk volume for each of the experimental groups. Haemodynamic data was recorded at stabilisation (0 min ), following 5 minutes perfusion with adenosine, CCPA or Sminutes region ischaemia for die preconditioning group (5 min adenosine). Following 10 minutes reperfusion prior to 45 minutes ischaemia (10 min pei&sion) and at the end of reperfusion (120 min reperfusion). All haemodynamic recording (CPP- coronary perfusion pressure, left ventricular systolic pressure and left ventricular diastolic pressure) were measured in rnmHg. All values are expressed in mean +SEM *p<0.01, **pcO.(X)l. n =r Bar graph represents infarction as a % of the risk area for the isolated "S hearts perfused at normal and high coronary perfusion pressures 60 — T 50 - _k: CO40 - be w M . X 20 - ■ 10 - 0 - I Control Adenosine Precondition CCPA I |_Control Adenosine Normal CPP High CPP Figure 7.1. Represents % infarct /risk. Isolated hearts were perfused with Krebs Henseleit buffer at a) normal coronary perfusion pressure (50mmHg) and b) at high coron^ perfusion pressure (lOOmmHg). Experimental groups studied were: control, adenosine, ischaemic preconditioning and CCPA treated hearts. * All experimental data are expressed ±SEM with p<0.001. Chapter 7 197 Coronary perfusion pressure trace (normal CPP) [mm a) Control heart. Stabilisation prior to ischaemia and reperfusion (lOOmmHg) M LxfOp 25mrn /sec b) Ischaemic preconditioned heart. Stabilisation prior to IP protocol ( lOOmmHg) I no feO ! c) CCPA treated heart. Heart treated with CCPA ( lOOmmHg^^ 7^5 /s«_c_ 2.5roim I ScjC CPP Lv d p d) Adenosine treated heart. Heart treated with adenosine prior to ischaemia and reperfusion ( 100mmHg). Figure 7.2. Represents experimental traces recorded throughout the experiment. All hearts were perfused at normal coronary perfusion pressure (mmHg). Traces a) Control heart, b) Ischaemic preconditioned heart, c) CCPA treated heart and d) Adenosine treated heart. The experimental protocol for all treated hearts was 5 minutes perfusion with adenosine+KHB, C(7PA+KHB or regional ischaemia followed by 10 minutes reperfusion with KHB prior to 45 minutes regional ischaemia and 120 minutes reperfusion Chapter 7 198 From the normal CPP data there were no differences between groups measured at the start of the experiments, with a mean of 48.66±2.6mmHg. When hearts were perfused with adenosine at normal CPP, there was no significant change (43.2±1.3mmHg), also perfusion with CCPA did not influence CPP (51.1±1.5mmHg). For the ischaemic preconditioning group there was a significant increase in CPP during 5 minutes coronary branch occlusion (61.0±1.0mmHg), as expected, due to the reduction of flow and an increase in pressure. At the end of the experiments there were no differences in CPP between control (82.5±3mmHg), CCPA (90±5mmHg) and adenosine treated groups (95.0±6.0mmHg). However, for the ischaemic preconditioning group there was a significant reduction in CPP (63.0+7.OmmHg) p<0.01. This we believe reflects the reduction in infarct volume. See table 7.1 and figure 7.3. Coronary perfusion pressure traces for experiments performed using the high pressures are presented in figure 7.4. Experiments performed at the higher CPP, showed no differences between group at the start of the experiments (mean of 101.22±2.5mmHg). Following 5 minutes perfusion with adenosine, hearts showed a significant decrease in CPP from 105+3.1 to 85+6.1 mmHg (p<0.05). But at the end of 10 minutes perfusion with KHB only (adenosine treated hearts) the CPP was back to a baseline value of 104.6+4.0mmHg. At the conclusion of the experiments there were no significant differences in CPP between control (183+8.OmmHg) and adenosine perfused hearts (151.16+6.0). See table 7.1 and figure 7.5. n Isolated rabbit hearts perfused at normal coronary perfusion pressure g* 100-1 rt □ Control 9 0 - V Precondition q c c P A . W) ♦Adenosine 8 0 - 2^ vap va 70J 2a Pu og 6 0 - va 5 0 - I o 4 0 - rfiision Y /////////^ m ^ ///////A reperfusion U stahilisatinn 5'ADO or 0 A + Krebs buffer or Ischaemia T" 1 1------1 0 ’ 20 ’ 25’ 35’ Time (minutes ) 80’ 200 ’ Figure 7.3. Represents coronaiy perfusion pressure data for the isolated perfused rabbit hearts at the normal pressure, mean of 48.5±3.5mmHg. Experimental groups studied were; control, adenosine treated, CCPA treated and ischaemic preconditioning. The experimental protocol for the treated groups were as follows; 5 minutes perfusion with Krebs+adenosine or Krebs +CCPA and for the ischaemic preconditioning group hearts werepreconditioned with regional ischaemia for 5 minutes followed by 10 minutes perfusion with Krebs. All hearts received 45 minutes regional ischaemia and 120 minutes reperfusion.* All experimental data are expressed ±SEM with p<0.01. . Chapter 7 200 Coronary perfusion pressure trace (High CPP) T .-T i r ir a) Control heart. Stabilisation prior to ischaemia and reperfusion (250mmHg) ITb ' ^ 6 lis 1^0 • >5 too b) Adenosine treated heart. Heart treated with adenosine prior to ischaemia and reperfusion (250mmHg) Figure 7.4. Represents experimental traces recorded for hearts perfused at high coronary perfusion pressure (mmHg). Traces a) Control heart, b) Adenosine treated heart. The experimental protocol for the adenosine group was 5 minutes perfusion with adenosine+KHB followed by 10 minutes reperfusion with KHB only, for the control group, hearts were perfused with KHB prior to 45 minutes regional ischaemia and 120 minutes reperfusion n=r "S -t(D Coronary perfusion pressure graph for the isolated perfused 170- rabbit hearts at high pressure toû X I 1^0- c e cüI I c/3 110- □ Control (high) 'C OAdenosine (high) oi U 70^ stabilisationlHnerfiLsio reperfusion 5^AD0 f 1 Time (minutes) 0 ’ 20’ 25’ 35’ 80’ 260’ Figure 7.5. Represents coronary perfusion pressure data for hearts perfused at high pressure o (lOOmmHg). Experimental groups studied were control and adenosine treated. All hearts received 45 minutes regional ischaemia followed by 180 minutes reperfusion. * All experimental data are expressed +SEM with p<0.01 Chapter 7 202 7.2.3 Left Ventricular Developed Pressures Left ventricular systolic and diastolic performance for all experiments are presented in figure 7.6, 7.7 and table 7.1. There were no differences between groups measured following stabilisation. For the hearts perfused at normal CPP; perfusion with adenosine appeared to show no effect on ventricular performance as judged by left ventricular systolic and diastolic pressures. At the end of the experiment the left ventricular systolic pressure for hearts treated with CCPA (85±9mmHg) and ischaemic preconditioning groups (82+2.5mmHg) were significantly higher than control (66±2mmHg) and adenosine (72±lmmHg) treated hearts (p<0.01). See figure 7.6 and table 7.1. For hearts perfused at high CPP there were no differences between groups measured following stabilisation (see table 7.1). At the conclusion of the experiment left ventricular systolic pressures were significantly higher in the hearts treated with adenosine, 104.3±3mmHg (lOO^M) in comparison to control group, 87.3±4mmHg (p<0.01). In all groups the left ventricular systolic pressure decreased and diastolic pressure increased over the duration of the experiments (see table 7.1). The significant decrease in systolic pressure was more marked in the hearts which had sustained large infarcts (for the normal perfused CPP; these were control hearts, adenosine perfused hearts and for the high CPP; control perfused hearts) in contrast to the smaller infarct groups (for the normal perfused CPP these were; ischaemic precondition hearts, CCPA treated hearts and for the high CPP; adenosine perfused hearts). n=r ID 1 0 0 - Left ventricular systolic pressure for isolated hearts perfused at normal - 1 coronary perfusion pressure W) X B B 90 B 3 V3V 2 (U o 2 8 0 - C/3 00 cd n~| Control S ^ Precondition ■c □ CCPA 7 0 - I Adenosine Ischaemia Reperfusion L iLj L iï 60— 5’ADO or CCPA+ Krebs buffer or ischaemia Time (minutes) 20’ 25’ 35’ 80’ 200 ’ Figure 7.6. Left ventricular systolic pressure for the isolated rabbit hearts perfused at normal coronary perfusion s pressure (mean GPP of 48.5±3.5mmHg). Groups studied were; Control, Adenosine treated, Ischaemic preconditioning and CCPA treated. * All valves are expressed as +SEM p<0.01. n 3“ "S -t 1 6 0 -, Left ventricular systolic pressure for isolated hearts perfused at high coronary perfusion pressure 1 4 0 - r~| Control 100- ► Adenosine 80 — 'TTV-r r r z / / / Stabilisation Perfusion Ischaemia Reperf usion 5’ ADO+Krebs buffer I------1---- 1------1------1------1 0 20’ 25’ 35’ Time (minutes) 80’ 260’ Figure 7.7. Left ventricular systolic pressure for isolated rabbit hearts perfused at high GPP (mmHg). Groups studied were: Control and Adenosine treated . * All valves are expressed as +SEM p < 0.01. Chapter 7 205 7.2.4 Adenosine Concentration in the Coronary Effluent Coronary effluent was collected and measured for adenosine concentration from hearts that were perfused at normal and high CPP. There were no differences in adenosine concentration during stabilisation between all the groups (see figure 7.8). When hearts were perfused with adenosine (lOOuM) at normal CPP, no differences were seen in adenosine release in the coronary effluent (4.2±0.2pmol/100g of heart, n=5) to the baseline value (4.8+0.3pmol/l(X)g of heart, n=5) p=NS. In addition perfusion with CCPA did not increase adenosine in the coronary effluent (from 4.8±1.0pmol/100g of heart to 3.8+0.7pmol/100g of heart, n=4) p=NS. For hearts that were ischaemically preconditioned, this showed a significant increase in adenosine release in the coronary effluent following 5 minutes regional ischaemia and 1 minute reperfusion (from 5.0±0.4pmol/100g of heart to 77.1±4pmol/100g of heart, n=4) p<0.001. The following adenosine results were measured from hearts that were perfused at high CPP. Perfusion of hearts for 5 minute with adenosine (IOOm-M), showed a significant increase in adenosine release in the coronary effluent from 5±0.3pmol/100g of heart (control, n=6) to 52±pmol/100g of heart (n=6, p<0.001). See figure 7.8. n=r Adenosine concentration measured in coronary effluent taken during ■o both normal and high coronar* perfusion pressures 8 0 - t:<73 o e 5 0 - o 4 0 - C 8 3 0 - C O o 20 - .1 c/3 0 10_ 1 < m m Pre Post Pre Post Post Pre Post Pre Post Control Adenosine CCPA Precondition | Control Adenosine i Normal CPP High CPP Figure 7.8. The bar graph shows the concentration of adenosine released in the coronary perfusate, measured by HPLC.All measurements were tScen during stabilisation and following 5 minutes post treatment. The groups studied were: a) Normal coronary perfusion pressure (CPP) groups; Control, Adenosine, CCPA and ischaemic preconditioning, b) Higher CPP groups; K) Control and Adenosine treated hearts. * All data are expressed as +SEM p<0.001. Chapter 7 207 7.3 Discussion It was observed from the above study that adenosine appears not to show any protection at the normal CPP used. However, at the higher CPP and flow rate, pretreatment with adenosine interestingly showed a significant limitation of infarct size. We cannot be totally sure why these differences exist, however they could occur as a result of the short half life of adenosine. As was shown from the adenosine concentration measured in the coronary effluent, that perfusion with adenosine at normal CPP showed no increase in adenosine release but showed a significant increase in adenosine release in hearts perfused at high CPP. During normal CPP adenosine may be rapidly broken down in the coronary vessels, before it can reach the target cell (myocytes). However, for the high CPP with the increased flow, a greater distribution of adenosine to target tissue may and could possibly account for the protection observed. From the CPP data it can be shown that pre treatment with adenosine showed a significant hypotensive response for the higher CPP group in comparison to the normal CPP group. However, it is still possible to show protection at normal CPP with the preconditioning group, because the ischaemic insult may be much more severe and results in a higher concentration of adenosine release, as well as activating many other mechanisms that may possibly be involved in myocardial protection. Finally pretreatment with CCPA also showed a reduction in infarct to risk volume at the normal CPP. This could possibly be due to the fact that CCPA is a very potent Ai receptor agonist with a longer half life. ii) 7.4 Adenosine Dose Response Study As reported in the above study the protection achieved with adenosine was only beneficial in the hearts that were perfused at the higher CPP, therefore a dose response study was performed at the higher CPP and constant flow rate to investigate this in Chapter 7 208 more detail. 7.4.1 Exclusions A total of 41 rabbits entered the study, of these 5 rabbits were excluded from the analysis. Two rabbits (from groups 1 and 6) developed intractable ventricular fibrillation and did not recover. One rabbit died during the surgical preparation (group 3). Two rabbits were excluded because of technical problems (from group 2 and 5). A total of 36 rabbits therefore contributed to the data below. 7.4.2 Experimental Protocol There were seven experimental groups with rabbits being assigned sequentially to each (mean weight 2.55+0.2, n=36). Group 1, the control group, was monitored for an additional 10 minutes before being subjected to ischaemia and reperfusion. The remainder of the hearts (Groups 2 to 7) were perfused with Krebs Henseleit buffer containing adenosine at a fixed concentration (3fiM, 6^M, lO^M, 20^M, 50p.M, or lOO^iM) for 5 minutes and then with KHB alone for 5 minutes prior to ischaemia and reperfiision. In each experiment, ischaemia lasted 45 minutes and was followed by 120 minutes of reperfusion. At the end of each experiment, the hearts were analysed for infarct and risk volume. 7.5 Results 7.5.1 Infarct and Risk Volume Hearts perfused with lOOpM adenosine prior to 45 minutes regional ischaemia and 120 minute reperfusion showed a significant reduction in infarct to risk volume from 58.8+2% (control n=7) to 23.1+0.6% (ADO-lOOpM, n=7), p<0.001. However, when hearts were perfused with adenosine at a low concentration of 3pM prior to 45 minutes Chapter 7 209 regional ischaemia and 120 minutes reperfusion, did not reduce infarct to risk volume 55.58±2.9% (n=4) p=NS. But as the concentration of adenosine was increased (6pM, lOpM, 20pM, 50|iM) there was a dose dependent reduction in infarct to risk volume from 44.1±2.02% (6pM), 33.3±1.9% (lOpM), 26.6±0.9% (20piM) and 21.6±3.5% (50pM) respectively, all valves were significantly reduced compared to control hearts, p<0.05. There were no significant differences in infarct to risk volume between 50pM and lOOpM adenosine perfused hearts (see table 7.2 and figure 7.9). Mean risk volumes were not significantly different between groups. See table 7.2. 7.5.2 Haemodynamic Data 7.5.3 Coronary Perfusion Pressure There was no significant differences in coronary perfusion pressure between groups during stabilisation and at the conclusion of the experiment (see table 7.2). However, 5 minutes perfusion with KHB plus adenosine (lOOuM, 50nM and 20^M, lO^M, p<0.05) lead to a dose-dependent reduction in coronary perfusion pressure which had returned to baseline when perfused with KHB only (5 minutes later). While lower concentration of adenosine (6^M and 3^M) used showed no differences. See table 7.2. 7.5.4 Left Ventricular Pressure Left ventricular performance for all experiments are presented in table 7.2. There were no differences between groups measured following stabilisation. Adenosine had no effect on ventricular performance as judged by left ventricular systolic and diastolic pressures. In all groups the left ventricular systolic pressure decreased and diastolic pressure increased over the duration of the experiments. At the end of reperfusion, left ventricular systolic pressures were significantly greater in the hearts treated with Chapter? 210 adenosine (IGOm-M, SG^M and 2Gp.M) in comparison to control group (p 45 g 40 (5 35 u 30 I25 20 - 15 - 10 - 5 - 0 - Control 3[iM 6|iM lO^M 20^M 50|,tM lOOpM Adenosine plus Krebs Henseleit Buffer Figure 7.9. Shows the % infarct/risk for isolated perfused rabbit hearts. All experiments were performed at a coronary perfusion pressure of^lOOmmHg and a flow rate of 70 ml/min. Hearts were perfused with different concentrations of adenosine plus Krebs Henseleit buffer (3, 6, 10, 20, 50 and lOO^iM) prior to 45 minutes regional ischaemia followed by 180 K) minutes reperfusion, at the conclusion of the experiments infarct/risk volume was determined. Results are expressed in w +SEM, * p<0.05 compared to Control and 3uM adenosine treated groups. Chapter 7 213 7.6 Discussion This study demonstrates that pretreatment with adenosine leads to a concentration- dependent reduction in infarct size in the rabbit isolated perfused heart. These results support the findings of an earlier study in which we had reported a correlation between adenosine concentration in the coronary perfusate and the degree of infarct size reduction; see results chapter 4 and 5 (Woolfson, et al 1995). Cardioprotection was noted following perfusion with adenosine concentrations of 6nM and the effect was maximal at a concentration of 50^M. Thus the graded cardioprotective response was present over a narrow dose range, only one order of magnitude. The degree of protection noted at higher doses was similar to that we have previously reported following ischaemic preconditioning in the rabbit isolated perfused heart and which could be abrogated by prior treatment with 8-p-sulfophenyl- theophylline (8-SPT), an adenosine receptor antagonist. Although there is one report of ‘dose-dependency’, of protection by ischaemic preconditioning against ischaemia-induced arrhythmias in the rat isolated heart, this has not to date been reported in other species (Lawson, et al 1993). Optimal protection against myocardial necrosis can be achieved in dogs and rabbits with a single preconditioning cycle of 5 minutes ischaemia and 5 minutes reperfusion and there appears to be no additional protection from further cycles (L i, et al 1990 and Van Winkle, et al 1991b). Although the detailed mechanisms which underlay the anti- arrhythmic and anti-ischaemic effects of ischaemic preconditioning may well be different, it remains possible that a graded response between preconditioning and myocardial necrosis could be demonstrated in the rabbit or dog if the preconditioning cycles were sufficiently brief. This conclusion is consistent with our observation that graded adenosine-dependent cardioprotection can be demonstrated over a relatively narrow range of concentrations of adenosine. Chapter 7 214 Treatment with adenosine leads to a brief fall in coronary perfusion pressure with no change in the indices of ventricular performance. No chronotropic effects of adenosine were identified because all hearts were paced electrically at 180 bpm. At the end of reperfusion, we note no differences in coronary perfusion pressure between the groups. 7.7 Conclusion In conclusion, this study demonstrated that pretreatment with adenosine leads to a concentration-dependent reduction in subsequent infarct size following ischaemia- reperfusion in the rabbit isolated perfused heart. The graded response to adenosine occurs over a narrow concentration range (one order of magnitude) and this may account for the supposition that ischaemic preconditioning has an ‘all-or-none*, action. Chapter 8 215 Chapter 8: Isolated Mitochondrial Function 8.0 Introduction (Mitochondrial Function) When nitric oxide is present in substantial excess it may be injurious. This may be relevant both during early reactive hyperaemia which is mediated by a substantial increase in nitric oxide release as well as in the presence of activated leucocytes and cardiac myocytes both of which can express inducible nitric oxide synthase. Two possible mechanisms have been suggested to account for nitric oxide-dependent toxicity: firstly, nitric oxide may interact with finee radicals to produce toxic nitric oxide- derived species, such as peroxynitrite anion (Beckman, et al 1990); and secondly, nitric oxide may inactivate iron-sulphur centred enzymes involved in essential cellular activity, such as mitochondrial respiration (Drapier, et al 1988). 8.1 Aim It was shown from our studies (see results chapter 3,4, 5, 6 and 7) that inhibition of nitric oxide synthesis prior to ischaemia and reperfusion appeared to be associated with myocardial protection. From those studies it was speculated that adenosine may play a major role in protecting the myocardium. Another theory is that nitric oxide itself can be very toxic. Therefore, it may be possible that the protection seen on inhibition of nitric oxide synthesis may also be due to preservation of mitochondrial function during reperfusion. As nitric oxide release increases during reperfusion hyperaemia, large amounts of nitric oxide binds to ion sulphur centred enzymes. Our hypothesis is that the protection seen with L-NAME was not only due to the Chapter 8 216 adenosine release but may also be involved in the reduction of nitric oxide released during reperfusion which may also be detrimental. It would be very interesting to be able to show that both forms (ischaemic preconditioning and L-NAME) of protection share a common pathway. So far both ischaemic preconditioning and treatment with L- NAME have shown an increase in adenosine concentration prior to sustained ischaemia and reperfusion. Although the protection seen may be adenosine dependent, as well as concentration dependent. It is a possibility that this increase in adenosine prior to ischaemia may reduce nitric oxide release during reperfusion. As a result of which there is a reduction in reperfusion injury, and an increase in cell survival (infarct size reduction). In this study mitochondrial function was determined as an index of ischaemia and reperfusion damage. Mitochondrial function was assessed at the start of reperfusion, studying the effects of reperfusion hyperaemia, as well as at the end of 120 minutes reperfusion. 8.1.1 Experimental Protocol Hearts were perfused with Krebs Henseleit Buffer (KHB) on the Langendorff apparatus as described in methods. Mitochondrial function was assessed at various time points (see figure 8.1). For the L-NAME treated group, hearts were perfused with 30m.M L-NAME 10 minutes prior to 30 minutes global ischaemia and 120 minutes reperfusion. At the end of reperfusion the hearts were quickly placed in cold saline and mitochondria isolated (see methods chapter). 8.1.2 Exclusions A total of 36 rabbits contributed to the following study. A total of 8 rabbits were excluded from the analysis. Four rabbits were excluded because of poor yield of mitochondria (from groups 3, 5, 6 and 8). Two rabbits were excluded because of Chapter 8 217 technical problems (from groups 2 and 8) and two rabbits had intractable ventricular fibrillation and did not recover ( from groups 4 and 6). 8.1.3 Experimental Groups Group 1: Sham (freshly excised) (n=4) Rabbits were anesthesized, the chest opened the heart rapidly excised and mitochondria isolated. Group 2: Aerobic Perfusion 60minutes (n=5) The heart was perfused on Langendorff apparatus at a constant flow rate for 60 minutes, at the end of which mitochondria were isolated Group 3: Ischaemia 30 minutes (n=4) Following 30 minutes stabilisation all hearts received 30 minutes of global ischaemia followed by 2 minutes reperfiision. At the end of this the mitochondria were isolated Group 4: Ischaemia and Reperfusion (n=5) The heart received 30 minutes global ischaemia followed by 120 minutes of reperfusion and at end of the reperfiision period the mitochondria were isolated. Group 5: L-NAME Perfusion (n=4) After 10 minutes perfusion with L-NAME+KHB, mitochondria were isolated Group 6: L-NAME Ischaemia (n=5) The heart was perfused with L-NAME 10 minutes prior to 30 minutes of global ischaemia and 2 minutes into reperfusion following which the mitochondria were isolated Group 7: L-NAME Ischaemia and Reperfiision (n=5) The heart received 5 minutes global ischaemia followed by 10 minutes reperfusion and Chapter 8 218 than followed by 30 minutes global ischaemia and 120 minutes reperfusion. At the end of the experiment mitochondria were isolated.. Group 8: Ischaemic Preconditioning (n=4) Following 5 minutes global ischaemia and 10 minutes reperfusion followed by 30 minutes global ischaemia and 120 minutes reperfiision the mitochondria were isolated. nsr -a Global Ischaemic Protocol for the Determination of Mitochondrial Function 00 Time (minute) O' 25’ 35’ 65’ 185’ n r ir - 1 I- ♦ ♦ i L-NAME i Ischaemic Preconditioning protocol 0’ 20’ 25* 35’ 65’ 185’ “ 1 r—II------II------11- Time points when mitochondrial function was 4 assessed t t L-NAME +KHB perfused, at 10 minutes prior to ischaemia and continuing into reperfusion Figure 8.1 : Isolated Langendorff perfused hearts, using global ischaemia model, heart were perfused with L-NAME or underwent the preconditioning protocol. At time points the hearts were processed for the isolation of mitochondria and mitochondrial function was assessed. Chapter 8 220 8.2 Results Tables 8.1 summarises the data obtained from mitochondria isolated by the extraction process as described in the methods section. A typical oxygen electrode tracing of mitochondrial respiration is also shown on figure 8.2 (a,b,c.) State 3 respiration represents the rate of oxygen consumption in the presence of ADP, while state 4 respiration is the rate of oxygen consumption in the absence of ADP and represents the energy consumed to maintain ion gradients in the face of ion leakage across the membrane. The mitochondria were isolated in a medium containing KCl, EDTA and albumin (medium 1) which has been proposed (Lindenmayer et al., 1968) to be ideal for the isolation of tightly coupled heart mitochondria. The addition of EDTA in the medium was used to to buffer all the Ca2+ during the extraction procedure. This result demonstrates that the isolation medium had an important effect on the degree of depression of mitochondrial respiration and phosphorylation, induced by ischaemia and reperfusion. Mitochondrial respiration was measured using glutamate as substrate and the reaction mixture described in section methods chapter. The yield of mitochondria isolated from the myocardium was not significantly different between groups. The results show that there was no significant difference between RCI, Q02/3, Q02/4 and ADP/0 values of mitochondria isolated from hearts after 60 minutes of constant perfusion with KHB compared to 10 minutes perfusion with L-NAME on the Langendorff apparatus. Also there was no significant difference between mitochondrial function from freshly excised(sham) non perfused hearts compared to hearts perfused on the Langendorff apparatus. î00 Data form the isolated perfused rabbit heart models for the determination of mitochondria function. Body Heart Ventricular Group No Group n weight (kg) weight (g) weight (g) 1 Sham 4 2.51+03 6.5+0.1 2.8+0.6 2 Aerobic Perfusion 5 2.4+0.2 6.0+0.09 2.5+0.4 3 Ischaemia (30’) 4 2.4+0.3 6.3+0.5 3.1+0.3 4 Ischaemia/Reperfusion 5 2.6+0.2 6.6+0.5 3.0+0.5 5 L-NAME Perfusion 4 2.6+0.3 6.3+0.5 2.7+0.6 6 L-NAME Ischaemia 5 2.7+0.09 8.8+0.3 3.2+0.8 7.2+0.4 2.5+0.7 7 L-NAME 5 2.68+0.2 Ischaemia/Reperfusion 6.6+0.5 3.1+0.6 8 Preconditioning 4 2.4+0.09 Table 8.1. Experimental data for the Isolated perfused rabbit hearts. Hearts were perfused on the Langendorff apparatus as indicated on the protocol. At time intervals mitochondria were isolated and function determined. The experimental groups studied were 1) Sham (freshly excised), 2) hearts perfused on the Langendorff apparatus for 60 minutes (Aerobic perfusion), 3) 30 minutes of global ischaemia following stabilisation (ischaemia), 4) following stabilisation hearts received 30 minutes global ischaemia followed by 120 minutes of reperfusion (ischaemia/reperfusion), 5) hearts were perfused with L-NAME for 10 minutes (L-NAME perfusion), 6) following 10 minutes perfusion with L-NAME hearts received 30 minutes of global ischaemia (L-NAME ischaemia), 7) hearts were perfused with L-NAME followed by 30 minutes of global ischaemia and 120 minutes of reperfusion (L-NAME ischaemia/reperfusion) and 8) for the precondition protocol hearts received 5 minutes global ischaemia followed by 10 minutes reperfusion and following 30 minutes global ischaemia and 120 minutes reperfusion. Chapter 8 222 Typical Oxygen Electrode Tracing of Mitochondrial Respiration From the Experimental Protocol a) Control mitochondrial trace: (O' m • LO; Spbstrocbe,. I 00 b) Ischaemic preconditioned mitochondrial trace: so: ADP Figure 8.2a/b: Typical traces obtained from mitochondrial respiration. Trace a) was from control heart, trace b) was from ischaemic preconditioned heart. From these traces the indices can be directly calculated (see methods for calculation). Chapter 8 223 Typical Oxygen Electrode Tracing of Mitochondrial Respiration From the Experimental Protocol c) Ischaemia and reperfusion mitochondrial trace: CD oicn Figure 8.2c: Typical traces obtained from mitochondrial respiration. Trace c) was from ischaemia and reperfused heart. From these traces the indices can be directly calculated (see methods for calculation). Chapter 8 224 8.2.1 Effects of Extraction of Mitochondria From Ischaemic and Reperfused Hearts The mitochondria isolated after 30 minutes of global ischaemia in both the control ischaemic group and the L-NAME treated group showed significantly reduced values for RCI, and QO2 . While the Q/O2 /4 values for these groups showed a significant increase (see Table 8.2). This indicated that the ischaemic mitochondria maintained the capacity to phosphorylate ADP even if at a reduced rate (indicated by the lower valves of RCI and QO2). The decrease in State 3 respiration and the increase in State 4 respiration rate is experienced in uncoupled mitochondria. Following 120 minute of reperfusion the Q02/3 and RCI values were still significantly low for both the control ischaemic reperfused group and the L-NAME treated group. However, these reperfusion values were numerically higher then the ischaemic values but not significantly different (see table 8.2).The QO 2 /4 values for both these groups were significantly higher than the control group. Finally for the Ischaemic preconditioning group there was a greater preservation of mitochondrial function compared to both ischaemia/ reperfused and L-NAME+ischaemia/reperfusion groups. Both RCI and Q02/3 were lower and the Q02/4 values higher for the ischaemic preconditioned group compared to control group but not significantly different. The ADP/O ratio, however, was not significantly different between all the groups. This clearly suggests that the mitochondria maintained the capacity to phosphorylate ADP at a lower rate (as suggested by the reduced valves of RCI and QO 2). I00 Mitochondrial Respiation in Mitochondria Isolated from Freshly Excised, Ischaemic or Reperfused Hearts Q02/4 Mitochondrial Group Experiments RCI QCn/Z ADP/O No n moles 0 2 /mg/protein n moles 02/mg/protem proteiir (mg/dL) 1 Freshly Excised 19.5+0.5 265+15 10.0+3.0 3.0+0.06 60.5+8.6 (sham) 2 Aerobic Perfusion 18.0+0.4 263+22 10.0+2.0 2.9+0.07 56.0+6.4 (60min) 3 Ischaemia (30*) 9.0+0.5* 190+20* 38.0+7.2 * 2.5+0.05 55.0+10.0 4 Ischaemia/ 12.0+0.8* 230+18* 24.0+6.5* 2.6+0.09 70.0+4.9 Reperfusipn 5 L-NAME Perfusion 19.0+0.7 270+10 9.0+0.8 2.8+0.03 63.5+10 6 L-NAME Ischaemia 14.1+0.3* 225+20* 18.5+7.1* 2.6+0.06 54.9+8.6 7 L-NAME Ischaemia/ 15.8+0.6 245+14 14.1+3.0 2.6+0.08 60.0+12.5 Reperfusion 8 Ischaemic 17.0+0.3 250+15 12.5+4.5 2.7+0.05 72.8+0.5 Preconditioning Table 8.2. Shows data obtained from the isolated mitochondria from 8 groups, 1) sham, 2) control perfusion, 3) control ischaemia, 4) control ischaemia reperfiision, 5) L-NAME perfusion, 6) L-NAME ischaemia, 7) L -N A ^ ischaemia reperfusion and 8) ischaemic precondition group. The mitochondria ^ere harvested following stabilisation, 30 minutes global ischaemia and following 120 minutes reperfusion. Q02/3 (response to the adition of ADP) or Q02/4 (conversion of ATP to ADP), RÇI (ratio of 02 used in the presence of ADP to that in its absence) and ADP/Q ( nanomoles ADP used per n atoms of 02 consumed). Values are expressed as mean+SEM. *p<0.05 vs. control. Chapter 8 226 8.2.2 Haemodynamic results There was no differences in all haemodynamic variables measured during stabilisation between all groups. At the end of 120 minutes reperfusion there was a significant increase in diastolic pressure in the control hearts and L-NAME treated hearts compared with preconditioned hearts. In addition there was a significant decrease in systolic pressure in both the control hearts and the L-NAME treated hearts compared to the preconditioned hearts. However, there was also a significant differences between the control hearts and the L-NAME treated hearts, showing a greater recovery of the myocardium during reperfusion in the nitric oxide treated hearts, but the recovery was not as great as that for the preconditioned hearts. 8.3 Discussion 8.3.1 Effects of Ischaemia, Reperfusion and Treatment With L-NAME on Mitochondrial Function In this study mitochondrial function was determined as an index of ischaemic and reperfusion damage. As reported by Drapier et al (1988), the inducible form of NO, when present in large amounts, binds to ion sulphur centred enzymes; this may cause further injury following ischaemia and reperfusion. Therefore, by inhibiting NO synthesis prior to ischaemia and reperfusion the protection seen may be due to preservation of aerobic respiration on reperfusion. Mitochondria isolated from control aerobic perfused hearts showed a respiration control ratio greater than 10 and ADP/O ratio close to the theoretical valve of 3 in all instances. These results are not different from those obtained for mitochondria from freshly excised non-perfused hearts and are in agreement with those of other laboratories (Lindenmayer, et al 1968, Jennings, et al 1978,1981, Lochner, et al 1976). Chapter 8 227 When calculated from the yield, the total mitochondrial content of control ventricular tissue was 23.7±0.4mg mitochondrial protein per g wet weight. This value was similar to that reported by Kleitke et al (1963), for left ventricular tissue and by Murfit et al (1978), for the anterior papillary muscle of the dog. Although we have not carried out quantitative studies, it has been reported by Jennings, et al (1969), using similar isolation procedures, that the majority of mitochondrial profiles were intact and of similar appearance. Most mitochondria appeared round in cross-section and closely packed (Jennings, et al 1969.). This indicates that the conventional technique we used, based on homogenisation and differential centrifugation in the absence of proteolytic enzymes, produces mitochondria qualitatively and quantitatively similar to those obtained by others using different techniques (Safer and Schwartz, 1967, Vercesi, et al 1978). In addition, our mitochondria oxidised NADH in the presence of ADP at low rates (QO 2 values 24% of that obtained after addition of glutairiate). Such low values are indicative of structural integrity of the isolated organelles. Many experiments carried out determining biochemical and structural integrity of mitochondria, isolated from ischaemic myocardium, have indicated considerable disagreement. Some laboratories (Lindenmayer, et al 1968, Schwartz, et al 1973 Peng, et al 1977) have reported depressed State 3 respiration using succinate, glutamate or pyruvate as substrate in mitochondria from ischaemic tissue, while others (Calva, et al 1966) have reported no changes. Respiratory control ratios of mitochondria isolated from ischaemic tissue have been reported to be depressed by most laboratories using a number of substrates, although one reported no significant changes (Jennings, et al 1969). They reported a decreased RCR after periods of ischaemia resulting from a decrease in State 3 respiration and not from an increase in State 4 respiration rate as is traditionally encountered in uncoupled mitochondria. In many studies the ADP/O ratio is unchanged, even after a longer period of ischaemia (Locher, et al 1975). Chapter 8 228 Our results show that the mitochondrial function, seen by the reduced value of RCI and Q02/3 were significantly reduced after 30 minutes global ischaemia. The ADP/0 ratio, however, was unchanged in all groups. This indicated that the ischaemic mitochondria maintained the capacity to phosphorylate ADP even if at a reduced rate (as suggested by the reduced valves of RCI and QO2). Restoration of coronary flow did not modify mitochondrial function when measured in vitro. Reperfusion after total ischaemia did not improve the function of isolated mitochondria in the control group. The most likely cause of a reduced function of isolated ischaemic mitochondria were reported to be loss of Krebs cycle intermediates, nicotinamide, adenine nucleotides and electron transport chain components (Calva, et al. 1966, Jennings, et al 1969, Schwartz, et al 1973 ) and a disorganisation of normal structure. 8.4 Conclusion As reperfusion results in further deterioration of mitochondrial function, there is no simple explanation for this effects seen in our studies. However from these results it can be shown that NO dependent toxicity dose not contribute to further injury as was hypothesised. Chapter 9 229 Chapter 9: Final Summary and Future Work 9.0 Summary of Results Nitric oxide, not only modulates the function of vascular smooth muscle, but also influences numerous other biochemical activities. The studies presented in this thesis have investigated the role of nitric oxide in the rabbit myocardium during ischaemia and reperfusion. Inhibition of nitric oxide synthesis has led to myocardial protection in both in situ heart and the retrogradely perfused isolated heart. It was shown from the in situ studies that inhibition of endothelium derived nitric oxide using L-NAME prior to ischaemia and reperfusion results in a significant reduction in infarct size. This protection was also seen in the isolated Langendorff perfused rabbit hearts and therefore appeared not to be mediated by an effect on neutrophil accumulation. Biochemical assays, performed in both the in situ and in vitro rabbit hearts, indicated that inhibition of NO synthesis resulted in an increase in adenosine concentration. It was therefore speculated that the increase in adenosine may be due to tissue ischaemia, which may have resulted from a combination of coronary vasoconstriction and increased ventricular work due to systemic vasoconstriction. This possibility was extended by measurements of lactate concentration in the isolated heart, which indicated that myocardial protection following NO inhibition could occur in the absence of myocardial acidosis. Release of adenosine following NO inhibition suggested the origin of the myocardial protection and administration of adenosine receptor antagonist (SPT) in vitro hearts abolished the protection with L-NAME. Thus the release of adenosine following NO inhibition seems likely to result from altered coronary haemodynamics with evidence Chapter 9 230 from the literature to support flow-mediated release of purines from endothelial cells. Ischaemic preconditioning or ischaemic preconditioning plus L-NAME in both in situ and in vitro gave greater protection than hearts treatment with L-NAME only. This may provide some evidence to support a dose response relationship. From a further study using the rabbit isolated perfused heart, prior to ischaemia and reperfusion, pretreatment with adenosine led to a concentration-dependent reduction in subsequent infarct size. It has been reported by Drapier et al (1988) that locally released excess NO may also mediate tissue injury during reperfusion, as a result of binding to the iron sulphur cluster at the catalytic site of certain mitochondrial enzymes damaging tissue. However from the last study described in this thesis in which isolated mitochondrial function was studied there was no evidence showing that NO itself may be damaging. Interpretation of this study was complex but the changes occurring in the mitochondrial cells following inhibition of NO showed no protection or preservation of mitochondrial function following ischaemia and reperfusion. Evidence from other physiological models suggest that adenosine is capable of protecting the rabbit myocardium from injury, and also plays an important role as a trigger for ischaemic preconditioning. The results presented in this thesis are certainly compatible with the adenosine hypothesis, but do not identify the mechanism which associates the release of adenosine and nitric oxide. It may be possible that the increase in adenosine release on inhibition of NO may be acting to maintain vascular tone. Chapter 9 231 9.1 The Design of Future Studies Control of Vascular Tone 1) Phenylephrine administration in these studies produced a rise in blood pressure. It would be very interesting to extend this study by measuring infarct size in both in vivo and in vitro models. Adenosine release in the coronary effluent could be measured. From such studies it may be possible to determine whether; a) the protection is present with phenylephrine and whether an increase in the level of adenosine is present, then it would be possible to conclude that there is a compensatory relationship between control of vascular tone and adenosine release. b) if there is no protection, then the protection observed with NO inhibition may be unique. 2) It would be interesting to look at the effects of NO donors on infarct size. Measure the Production of Nitrite and Nitrate Studies reported in this thesis where we have looked at the inhibition of NO synthase, it may be appropriate to measure NO in both L-NAME treated and in control groups. The method used to measure NO is chemiluminescence, in which production of nitrite and nitrate (is assessed). Such a study would also provide information about the effect of reactive hyperaemia on NO release. 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Some results in this thesis have already been presented elsewhere; a list of publications with results in common with this thesis together with copies of manuscripts can be found below and on the following pages. Robin G. Woolfson, Vanlata C Patel, Guy H. Neild and Derek M. Yellon. Supplement I Circulation 1992. Vol 86, No 4, p 1-829, 3298. Richard H. Heads, Vanlata C Patel, David S Latchman and Derek M Yellon. Supplement I Circulation 1992. Vol 86, No 4, p 1-556, 2213. Vanlata C Patel, Robin G Woolfson, Karen J Singh, Guy H Neild and Derek M Yellon. Supplement I J Mol Cell Cardiol 1992,Vol 24, pS.152, P-01-46. Richard J Heads, Vanlata C Patel, David S Latchman and Derek M Yellon. Supplement I J Mol Cell Cardiol 1992. vol 24, P S.94, 0-18-8. Vanlata C Patel, Robin G Woolfson, Guy H Neild and Derek M Yellon. Proceedings Supplement B J Phamacol January 1993. Vol 108, 124P. Vanlata C Patel, R G Woolfson, M Singer, G H Neild and D M Yellon. Supplement Clinical Science November 1992. Gary F Baxter, Michael S Marber, Vanlata C Patel and Derek M Yellon. Supplement to Circulation 1993. Vol 88, No 4, Part 2, p 1-101, 0534. Vanlata C Patel, Robin Woolfson, Guy H Neild and Derek M Yellon. Supplement to Circulation 1993. Vol 88, No 4, Part 2, p 1-330, 1767. Vanlata C Patel, Robin G Woolfson, Guy H Neild, Derek M Yellon. Proceedings Supplement B J Parmacol January 1994. Vol 112, 385P. , j(, 1 94, No. ^ . 1993 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Vol. 194. No. 1. 1993 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS July 15. 1993 Pages 234-238 INHIBITION OF NITRIC OXIDE LIMITS INFARCT SIZE IN THE IN SITU RABBIT HEART VC Patel. DM Yellon. KJ Singh, GH Neild", and RG Woolfson"'+ The Hatter Institute for Cardiovascular Studies, Department of Academic Cardiology, and ’Department of Nephrology, Institute of Urology and Nephrology, University College London Medical School. Gower Street, London WCIE 6AU, UK R eceived May 24 , 1993 Recent data has suggested a dual role for nitric oxide (NO) so that it can both attenuate myocardial injury during ischaemia and reperfusion as well as mediate reperfusion injury. In this study in the in situ rabbit heart, we have shown that p re treatment with intravenous N^- nitro-L-arginine metliyl ester (L-NAME, an inhibitor of NO synthesis) significantly reduced infarct size following sustained coronary artery occlusion and reperfusion. L-NAME was also noted to increase myocardial lactate concentration. This study provides further evidence that protection against ischaemia-reperfusion injury can be derived from manipulation of the microcirculation. o 1993 Acadwle Trm%m, Inc. . «'-%. ‘ ' -V The mechanism of myocardial injury following ischaemia and reperfusion is multi- factorial and depends on the duration of ischaemia, the presence of a collateral circulation, the adhesion, activation and extravasation of neutrophils together with the generation of free radicals. The basal release of low levels of NO by the endothelium maintains the coronary micro vasculature in active vasodilatation and minor augmentation of NO levels during ischaemia and reperfusion, which are insufficient to affect blood pressure have been shown to mitigate against myocardial injury (1-3). Conversely, it has been suggested that NO may be cytotoxic when present in substantif excess (4). This deleterious role of NO may be important firstly, during early reactive hyperaemia which is in part a response to increased NO release (5,6) and, secondly, in the presence of activated leucocytes which express inducible NO synthase (7,8). Evidence to support a noxious role for NO comes from experimental models of myocardial injury following hypoxaemia (9% post-ischaemic cerebral reperfusion injury (10) and neutrophil-mediated tissue injury (11). To whom correspondence should be addressed at The Hatter Institute for Cardiovascular Studies, Department of Academic Cardiology, University College London Medical School, Gower Street, London WCIE 6AU, UK. 0006-291X/93 S4.00 \ Copyright O 1993 by Academic Press. Inc. All rights of reproduction in any form reserved. 234 ;|. 194, No. 1, 1993 biochemical AND BIOPHYSICAL RESEARCH COMMUNICATIONS In view of ihis biphasic role for NO. we have invesiigaied the effects of inhibition and augmentation of endothelium-derived NO production during regional myocardial ischaemia and reperfusion in the in situ rabbit heart, using infarct size as the endpoint. MATERIALS AND METHODS Male New Zealand White rabbits (mean weight 2.59 ± 0.04 kg, n=54) were anaesthetised with pentobarbital (40 mg/kg i.v.), ventilated mechanically and the left carotid artery cannulated. Following a left thoracotomy, the heart was exposed and a snare permitting intermittent occlusion placed proximally around the left circumflex artery. Myocardial ischaemia was confirmed by regional cyanosis and akinesis, with reperfusion being confirmed by blushing of the previously cyanotic myocardium. Heparin (I(X)0 units) was administered intravenously at the start and conclusion of each experiment. L-NAME (lOmg/kg), L-arginine (300mg/kg) or normal saline was infused over 1 min and the animals then monitored for 10 min. Sustained ischaemia lasted either 30 or 50 min and was followed by either 2 or 3 h reperfusion. GROUP 1: Control - 30 min ischaemia and 2h reperfusion. GROUP 2: L-NAME - 30 min ischaemia and 2h reperfusion. GROUP 3: Control - 50 min ischaemia and 3h reperfusion. GROUP 4: L-NAME - 50 min ischaemia and 3h reperfusion. GROUP 5: L-arginine - 50 min ischaemia and 3h reperfusion. At the conclusion of the experiment, the heart was excised, mounted on a Langendorff apparatus and perfused with saline for 1 min. The snare was occluded and the heart perfused with 1-10 pm diameter fluorescent particles to define the borders of the dependent myocardium supplied by the occluded artery (i.e. risk area). The heart was frozen, sliced into 2 mm thick sections, thawed and incubated in 1% tiiphenyl tétrazolium chloride (TTC) at 37*C for 20 minutes to define infarcted tissue. Infarct fTTC-negative) and risk (lacking UV fluorescence) areas were planiraetered for the entire heart with infarct area being expressed as a percentage of risk area. This experimental method is well established (12). In a second series of experiments, in s itu biopsy of apical myocardium was performed 10 minutes after the intravenous injection of either L-NAME (lOmg/kg) or saline. Biopsies were taken with pre-cooled (-180°C) tissue forceps with the samples being stored in liquid nitrogen prior to being freeze dried before analysis. Tissue extraction was performed using 350pl of ice cold perchloric acid (6%) followed by neutralisation to pH 5.5-6.0. Quantitative lactate Mtimation was performed using the enzymatic reaction of lactate dehydrogenase linked to nicotinamide adenine dinucleotide in glycine-hydrazine buffer medium (13). Statistics: Results are expressed as mean ± standard error. Statistical analysis between groups was performed using ANOVA followed by Scheffe’s F-tesL RESULTS L-NAME significantly reduced the ratios of infarct/risk area following either 30 min (Groups 1 vs. 2. p<0.05) or 50 rain (Groups 3 vs. 4. p^.02 ) of ischaemia and reperfusion 235 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Vol. 194, N o. 1, 1993 3 0 m in 5 0 m in is c h a e m ia is c h a e m ia { X 0 . G 2 Control L-NMMA Coalrol L>NMMA L-ARC • FTailRE 1. Infarct area cjçrcssed as percentage of risk area following either 30 or 50 min of coronary artery occlusion and repcrfusion in the in situ rabbit heart (see Figure 1 and Table 1).'Pre-treatment vdth L^arginine reduced the ratio of infarct/risk area (Group 3 vs. 5. p<0.08) but this failed to meet our criterion of significance. Mean risk areas - were not significantly different between comparable groups (see Table 1). L-NAME but not L-arginine lead to à rapid hypertensive response together with a j,;? I reflex bradycardia. However, there was no sigiilficant differences between rate pressure products of comparable groups at any time. See Table 1. V L-NAME significantly increased myocardial lactate concentration from 5.35 ± 0.85 praol/g dry weight (control. n=4) to 16.36 ± 2.41 praol/g dry weight (L-NAME, n=4), p<0.03. TABLE 1 Hacmodynaxnic data are shown at start (T=0 min), 10 min after saline, L-NAME or L-argininc (L-ARG) infusion (T=10 min) and at conclusion of cxperimenL (RPP, rate pressure product; ABP. change in blood pressure from time = 0 min, mmHg; AHR, change in heart rate from time c 0 min, bpm.) . CROUPS 1 2 3 4 5 B to 10 7 8 Ischaemic period (min) 30 30 50 ".IS 50 CONTROL L-NAME CONTROL •^N A M E L-ARG InfarcVRisk (*) 40.6 ± 4.4 18.7 * 4.6 60.7 * 5.9 39.9 * 43 473 * 43 Risk area (cm^ 5.2 ± 05 5.7 * 0.7 3.5 * 0.4 4.2 * 0.4 5.1 * 0.7 T B 0 min RPP 23793 ± 2035 22755 * 1940 26983 * 1103 24352 * 1092 23332 * 1362 T B 10 min ABP -9*2 21 *3 • 1*3 23*5 0 * 4 . AHR -9*4 - 19 *3 -1*6 -27 *6 -1*9 RPP 21899 * 1560 21973 * 1543 27085 * 1064 26982 * 1636 23297 * 1473 T B conclusion of experiment RPP 16S80 ± 9SS 16885 * 2540 18296 * 824 16973 * 1398 15502 t 2256 236 Vol 194. No. 1. 1993 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS DISCUSSION In this study, pretreatment with L-NAME reduced the ratio of infarct/risk area after both 30 and 50 minutes of regional myocardial ischaem.ia.and repeifusion in the in situ rabbit heart- NO has been implicated in tissue injury, firstly, as a result of its capacity to interact with oxygen-derived free radicals to produce toxic intermediates (such as the peroxynitrite anion) (4) and, secondly, because NO avidly binds and thereby inactivates iron-sulphur- centred enzymes which are required for essential cellular metabolic activity including mitochondrial respiration (14). NO-induced injury is likely to be important in conditions of relative NO excess, for example in the presence of activated neutrophils (8,15) or during reactive hyperaemia (5.6). In this study the protection following pretreatment with L-NAME was not likely to have been mediated by an effect on neutrophil accumulation because inhibition of NO increases neutrophil adhesion to endothelial cells and this is associated with increased myocardial injury (16). In addition, L-NAME would not have inhibited formation of neutrophil-derived NO because it is not an effective inhibitor of the inducible NO synthase expressed by activated neutrophils (15). Therefore the observed protection following NO inhibition may have resulted from another mechanism which is likely to have involved the inhibition of endothelium-derived NO which is known to be released in substantially greater quantities during reactive hyperaemia (5.6). Paradoxically, pre-treatment with L-arginine also reduced myocardial injury following 50 minutes of regional ischaemia and reperfusion in the in situ rabbit heart although this was not significantly different from control. An earlier study reported that pretreatment with L- arginine protected the in situ feline heart from regional ischaemia (3) and therefore we consider our findings to demonstrate a Type 2 statistical error which reflects inadequate group size. The mechanism for L-arginine protection, and for the protection conferred by low concentrations of NO donors (1,2), seems to involve the preservation of physiological endothelial cell layer integrity and enhancement of NO release leading to a reduction in the accumulation of neutrophils in the ischaemic zone. An alternative and novel mechanism to explain our findings follows the now well- established observation that a brief period of ischaemia and reperfusion protects the myocardium against subsequent sustained ischaemia and reperfusion (17). This phenomenon, termed "ischaemic preconditioning", is mediated by the release of adenosine during the brief ischaemic period from ischaemic cells which fail to cycle ATP (12). During the brief period Of reperfusion and prior to the sustained ischaemia, the Increased ambient concentrations of adenosine stimulate cardiac myocytes (via A, receptors) which protects them against the subsequent ischaemic challenge by an unidentified mechanism (ref. 18 for review). In our 237 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATION; [- Vol. 194, No. 1. 1993 Study, pre-treatment with L-NAME significantly increased myocardial lactate levels which may indicate mild tissue ischaemia. This evidence that L-NAME causes myocardial ischaemia is consistent with a previous study which noted that L-NAME increases the concentration of adenosine in the coronary perfusate (5). Thus the% observations give rise to the novel possibility that L-NAME caused myocardial ischaemia with a resultant increase in adenosine release; this increase in ambient adenosine levels effectively "preconditioned" the myocardium against the subsequent sustained ischaemic insulL In summary, our study provides further evidence that protection against myocardial injury following ischaemia and reperfusion can be derived from manipulation of the NO system. Furthermore, we have provided preliminary evidence of a novel mechanism to explain the observed protection against myocardial ischaemia-reperfusion injury following inhibition of endothelium-derived NO. ACKNOWLEDGMENTS: This work was supported by grants from the British Heart Foundation and the Medical Research Council. REFERENCES * * Johnson. G., Tsao, P.S., MuUoy, D., and Lefer, A.M. (1990) J. Pharm. Exp. Ther. 252:35-41. 2. Siegfried, M.R., Erhardt, J., Rider, T.. Ma, X-L., and Lefer, A.M. ( 1992) J. Pharm.Exp. Ther. 260: 668-675. 3 Weyrich, A.S., Ma, X-L., and Lefer, A.M. (1992) Circulation 86: 279-288. 4 Beckman, J.S., Beckman, T.W., Chen, J., Marshall, P.A., and Freeman, B.A. (1990) • Proc. Natl. Acad. Sci. (USA) 87: 1620-1624. 5 Kostic, M.M., and Schrader, J. (1992) Circ. Res. 70: 208-212. 6 . Yamabe, H., Okumura, K., Ishizaka, H., Tsuchiya, T., and Yasue, H. (1992) Am. J. Physiol. 263:H8-H14. 7 Hibbs, J.B., Taintor, R.R., Vavrin, Z., and Rachlin, E.M. (1988) Biochem. Biophys. Res. Commun. 157:87-94. & Moncada, S., Palmer, R.M.J., Higgs, E.A. (1991) Pharmacol. Reys. 43:109-142. if Matheis. G.. Sherman, M.P., Buckberg, G.D., Haybron, D.M., Young, H.H., and Ignarro, L.J. (1992) Am. J. Phvsîol. 262; H616-H620. 10 Buisson, A., Plotkine, M„ and Boulu, R.G. (1992) Br. J. Pharmacol. 106: 766-767. U ' Mulligan, M.S., Mondcada, S., and Ward, PA. (1992) Br. J. Pharmacol. 107:1159- 1162. 12. Liu, G.S., Thornton, J., Van Wrinkle, D.M., Stanley, A.W.H., Olsson, R.A., and Downey, J.M. (1991) Circulation 84: 350-356. 13. Hohorst, H-J. (1963) In Methods of Enzymatic Analysis (H. Bergmeyer, Ed ), pp. 266- 270. Academic Press, New York, NY. ^ Geng, Y-j., Hansson, G.K., and Holme, E. (1992) Circ. Res. 71:1268-1276. (15) McCall, T.B., Feelisch, M., Palmer, R.M.J., and Moncada, S. (1991) Br. J. Pharmacol. 102:234-238. ^16) Ma, X-L., Weyrich, A.S., Lefer, DJ., and Lefer, A.M. (1993) Circ. Res. 72:403-412. 17. Murry, C.E., Jennings, R.B., and Reimer, K.A. (1986) Circulation 74:1124-1136. 18. Walker, D.M., and Yellon, D.M. (1992) Cardiovasc. Res. 26: 734-739. 238 1545 Inhibition of Nitric Oxide Synthesis Reduces Infarct Size by an Adenosine-Dependent Mechanism Robin G. Woolfson, MD; Vanlata C. Patel, BSc; Guy H. Neild, FRCP; Derek M. Yellon, PhD, DSc, FACC, FRSC Background Nitric oxide (NO) is both a potent endogenous L-NAME was lost in hearts pretreated with SPT (I/R ratio, vasodilator with potential to attenuate ischemia-reperfusion 59%). In a separate set of experiments, adenosine concentra injury and a mediator of tissue injury. The aim of the present tion in the coronary perfusate and myocardial lactate concen study was to investigate the mechanism by which prior inhibi trations were measured. Treatment with L-NAME increased tion of NO synthesis can lessen ischemia-reperfusion injury in the average adenosine concentration in the perfusate from 5.7 the isolated rabbit heart. /Ltmol/L per 100 g of heart (control) to a peak of 24.0 p,mol/L Methods and Results We examined the effects of inhibition per 1 0 0 g of heart; however, there was no effect on average of NO synthesis on infarct size using a model of coronary artery myocardial lactate concentration (control, 4,6 ju,mol/g dry wt; ligation in isolated rabbit hearts perfused at a constant flow L-NAME, 5.5 jamol/g dry wt). In contrast, after 5 minutes of rate of 35 mL/min. Infarct size averaged 65% of the zone at risk global ischemia, the average adenosine concentration peaked after 45 minutes of ischemia and 180 minutes of reperfusion. at 139.0 )u,mol/L per 100 g of heart, and the average myocardial The addition of 30 p,mol/L N°-nitro-L-arginine methyl ester lactate concentration increased to 27.1 p.mol/g dry wt. (L-NAME), an inhibitor of NO synthesis, to the perfusate Conclusions Infarct size limitation after inhibition of NO reduced the infarct-to-risk (I/R) ratio to an average of 41% synthesis shares a common mechanism with that of ischemic (P<.05 versus control). This effect was abolished by pretreat preconditioning and is dependent on the release of adenosine. ment with 75.5 /xmol/L 8 -p-sulfophenyl theophylline (SPT), an However, in this model, adenosine release after inhibition of NO adenosine receptor antagonist (I/R ratio, 63%). Ischemic pre synthesis is not secondary to myocardial ischemia. The protection conditioning (5 minutes of ischemia and 10 minutes of reper of the heart against ischemic injury by adenosine appears to be fusion) before 45 minutes of ischemia and 3 hours of reperfu concentration dependent. {Circulation. 1995;91:1545-1551.) sion reduced the I/R ratio to an average of 21%, and this was not augmented by pretreatment with L-NAME (I/R ratio, Key Words nitric oxide • adenosine • reperfusion 20%). However, all protection due to preconditioning and ischemia oronary artery occlusion followed by reperfusion preservation of function and an antiarrhythmic action, leads to infarction of a proportion of the isch using both in vivo and in vitro models of myocardial emic area. The extent of injury depends on the ischemia (see Reference 2 for a review). The intracellu Cavailability of collateral blood flow and the duration of lar mechanism responsible for IP may involve activation ischemia as well as the accumulation of neutrophils and of ATP-dependent potassium channels or protein kinase the generation of free radicals during reperfusion. Much C. Cogent evidence appears to support an important of this injurious activity is mediated at the level of the primary role for adenosine released by ischemic cells endothelium. during the preconditioning period.^ The final effector The emphasis of recent work has been on devising pathway of adenosine-induced protection remains un strategies to mitigate against ischemia-reperfusion in clear, but it is mediated by A, (and not A^) receptors on jury. In 1986, Murry et aP showed that a brief period of the cardiac myocytes. This emphasis on adenosine is ischemia followed by reperfusion leads to increased supported by other studies that have identified a cardio tolerance to a subsequent and sustained ischemic insult protective role 4 for adenosine in ischemia-reperfusion as determined by infarct size. This phenomenon, termed injury.^'5 “ischemic preconditioning” (IP) has been demonstrated Nitric oxide (NO), synthesized by a constitutive en by many other groups and is associated with both zyme from L-arginine,^ is a highly reactive species that is released continuously by endothelial cells."^ NO main Received June 1, 1994; revision received August 17, 1994; tains the coronary microcirculation in a state of active . revision accepted October 27, 1994. From the Department of Nephrology (R.G.W., G.H.N.), Institute vasodilatation,*^-^ prevents platelet and leukocyte adher- hi Urology and Nephrology; and The Hatter Institute for Cardiovas ence,’°'" and preserves physiological vascular imperme cular Studies (V.C.P., D.M.Y.), Department of Academic Cardiology, ability.'^ Although other endothelium-derived auto- University College London Medical School, London, UK- coids, such as adenosine and prostacyclin, have similar Correspondence to Professor D.M. Yellon, The Hatter Institute p r o p ertie s,th ey are less important physiological de for Cardiovascular Studies, Department of Academic Cardiology, University College London Medical School, Gower St, London, terminants of coronary tone. Inhibition of NO synthesis UK WCIE 6 AU. in the coronary vasculature by L-arginine analogues, © 1995 American Heart Association, Inc. such as /V^-nitro-L-arginine methyl ester (L-NAME),'^ 1546 Circulation Vol 91, No 5 March 1, 1995 leads to vasoconstriction of the microcirculation®’^ and The perfusate was Krebs-Henseleit buffer equilibrated with can cause myocardial ischemia, In response, there is 95% 02-5% CO2 at 37°C that contained (in mmol/L) NaCl increased release of adenosine and prostacyclin, which 118.0, KCl 4.7, MgS04 • 7 H 2O 1.2, CaCb • 2 H 2O 1.8, NaH2P04 vasodilate and partially compensate for the myocardial 14.6, KH 2PO 4 1.2, and glucose 11.0, pH 7.4. All hearts were hypoperfusion that results from diminished NO electrically paced at 180 beats per minute via the right atrium. Left ventricular pressures were measured by means of a production. ^8-20 fluid-filled latex balloon, connected by polyethylene tubing to a Continuing controversy surrounds the involvement of pressure transducer, and inserted into the left ventricle via an NO in ischemia-reperfusion injury. Ischemia-reperfu incision in the left atrial appendage. The balloon volume was sion injury leads to the loss of endothelium-dependent adjusted to give an initial diastolic pressure of 10 mm Hg. NO-mediated relaxation due to inactivation of NO by Coronary perfusion pressure (GPP) was measured via a side oxygen-derived free radicals produced by adherent leu- arm in the aortic cannula using a pressure transducer. The kocytes2i-23 and injured endothelial cells.^^ Given its heart was allowed to stabilize for at least 30 minutes before the antiadhesion and vasodilatation properties, NO defi experiment was begun. ciency during reperfusion would be expected to contrib ute to reperfusion injury. This contention is supported Infarct Size Experiments by several studies that have shown that augmentation of Experimental Protocols NO levels during reperfusion preserves endothelial func There were seven experimental groups with rabbits assigned tion, reduces neutrophil accumulation, and decreases sequentially to each. In each experiment, ischemia lasted 45 markers of myocardial injuiy,24-“ However, there is minutes and was followed by 180 minutes of reperfusion. other evidence that supports an injurious role for NO if Group 1, the control group, was monitored for an additional it is present in substantial excess. This could be relevant 15 minutes before being subjected to ischemia-reperfusion. In during early reactive hyperemia that is mediated by a group 2, 30 p-mol/L L-NAME was added to the perfusate 10 substantial increase in NO release*®’^» or later when minutes before ischemia and continued for 15 minutes into the activated neutrophils capable of expressing inducible reperfusion period. In group 3, 75.5 jumol/L 8 -p-sulfophenyl theophylline (SPT) was added to the perfusate 5 minutes NO synthase^ have accumulated. NO-dependent toxicity before the addition of 30 jumol/L L-NAME; SPT was continued results from the formation of NO-derived free radical until after the start of ischemia and the L-NAME continued species, such as the peroxynitrite anion,^^ and the inac until 15 minutes into reperfusion. In group 4, 75.7 p.mol/L SPT tivation of iron-sulfur-centered enzymes involved in was present in the perfusate until after the start of ischemia. essential cellular activity, such as mitochondrial respira- Group 5, the preconditioned group, received 5 minutes of tion.2« Evidence to support a noxious role for NO when coronary branch occlusion followed by 1 0 minutes of reperfu overproduced comes from experimental models of myo sion before the 45-minute occlusion and 180 minutes of cardial injury after hypoxem ia,^’ postischemic cerebral reperfusion. Group 6 received 30 p,mol/L L-NAME 10 minutes reperfusion injury,^» and neutrophil-mediated tissue in- before IP. Group 7 received 75.5 p.mol/L SPT, followed by 30 jury.31 We have recently found that prior inhibition of p.mol/L L-NAME before IP. NO synthesis reduces infarct size after regional coronary Measurement of Infarct and Risk Area artery occlusion and reperfusion in the in situ rabbit heart. However, we did not determine whether the At the end of each experiment, the heart was flushed with room-temperature saline for 1 minute. The coronary branch mechanism of this protection depended on either inhi was then reoccluded, and 1 - to 1 0 -/im fluorescent zinc cad bition of physiological production of NO before isch mium sulfide particles were infused into the perfusate until,the emia or inhibition of overproduction of NO during risk area could be visualized with UV light. The ligature was reperfusion. The present study was designed to elucidate then released; 2 to 3 mL of 1% triphenyl tétrazolium chloride the mechanism by which inhibition of NO synthesis by (TTC) was injected through the side arm; and the heart was L-NAME protects against ischemia-reperfusion injury immersed in Krebs-Henseleit buffer at 37°C for 2 minutes. using a model of coronary artery occlusion in the (TTC stains viable tissue deep red.) The heart was weighed and isolated, buffer-perfused rabbit heart free of circulating frozen. While frozen, the heart was cut into 2-mm transverse joeutrophils. slices. Tracings were made of the risk zones (lacking fluores cence under UV light) arid the infarct zones (TTC-negative Methods tissue), and the areas were determined by planimetry of the Isolated Heart Model tracings. This experimental method is well established. Male New Zealand White rabbits (weight, 2.0 to 3.0 kg) were Biochemical Assays anesthetized with sodium pentobarbital (40 mg/kg IV) after Adenosine Assay cannulation of a marginal ear vein. A tracheostomy was performed with the rabbit under local anesthetic (3 mL 2% In a separate group of hearts, coronary effluent samples were lidocaine), and the rabbit was ventilated mechanically with assayed for adenosine by reverse-phase high-performance liq 100% oxygen. The chest was opened through a left thoracot uid chromatography (HPLC) according to the method used by omy, and the pericardium was incised to expose the heart. A Jenkins and Bellardinelli.^^ The HPLC system included a 3-0 silk suture on a small round-bodied needle was passed Waters 710B injector, an LKB2150 pump, and a Beckman 160 through the myocardium underneath a proximal branch of the absorbance detector. EflRuent was collected in 5-mL aliquots, left coronary artery, and the ends were threaded through a freeze-dried, and reconstituted with deionized water to a small vinyl tube to form a snare, which was used to occlude the volume of 0.5 mL. Samples were passed through a Waters artery as required. The rabbits were given 1000 U heparin IV; 8NVC18 (4-p,m) column containing 20 mmol/L KH 2PO 4 (pH then, the heart was rapidly excised by cutting the great vessels, 5.6) with 60% vol/vol methanol. The adenosine peaks were placed in room-temperature saline, and mounted on the Lan detected at 254 nm, recorded on a BBC Goerz metrawatt chart gendorff apparatus within 1 minute. recorder, integrated using planimetry, and referenced to a The hearts were perfused retrogradely at a constant flow rate standard curve. Adenosine concentration in the perfusate was of 35 mL/min in a nonrecirculating system using a roller pump. expressed in micromoles per liter per 1 0 0 g of heart tissue. Woolfson et al NO Inhibition Reduces Ischemia-Reperfusîon Injury 1547 30 /xmol/L L-NAME to the perfusate or 5 minutes of global p<0.001 ischemia. At the conclusion of the experiment, the hearts were freeze-clamped, and the myocardial lactate concentration was measured. Chemicals L-NAME was purchased from Sigma Chemicals Ltd, and SPT was purchased from Semats Technical Ltd. All studies were performed in accordance with the United Kingdom Home Office regulations for the care and use of laboratory animals (project license no. 70/01759). Contrai L-NAME L-NAME L-NAME L-NAME ♦ SPT Statistical Analysis Fig 1. Bar graph of ratio of infarct area to risk areas expressed All results are given as mean±SEM . Statistical analysis as percentages for the seven groups studied; control (n = 7); between groups was performed using ANOVA followed by L-NAME (n=8): L-NAME + SPT (n = 7); SPT (n=6); IP (n = 6); IP + Scheffe’s F test. L-NAME (n=6); and IP + L-NAME + SPT (n = 5). L-NAME in dicates /V'^-nitro-L-arginine methyl ester hydrochloride; SPT, Results 8-p-sulfophenyl theophylline; and IP, ischemic preconditioning. Infarct-to-Risk Ratios iMctate Assay Prior inhibition of NO synthesis by L-NAME signifi cantly reduced the ratios of infarct to risk area after 45 When coronary perfusate collection was complete, the same hearts were freeze-clamped with precooled (-180X) tissue minutes of branch coronary artery occlusion and 180 forceps and stored in liquid nitrogen to permit subsequent minutes of repcrfusion from 64.6±4.3% (control, n = 7) measurement of myocardial lactate concentration. Tissue ex to 41.4±5.1% (L-NAME, n = 8 ) (P<.05). This protection traction was performed using 350 /iL ice-cold perchloric acid was prevented by pretreatment with SPT (ie, L-NAME (6%) followed by neutralization to pH 5.5 to 6.0. Quantitative plus SPT), which alone had no effect on the infarct-to- lactate estimation was performed using the enzymatic reaction risk ratio (Fig 1 and Table). After IP, the infarct-to-risk of lactate dehydrogenase linked to nicotinamide adenine dinu ratio was 21.4±1.2% (n=6 ), which was significantly less cleotide in glycine-hydrazine buffer medium.Myocardial lac than both control (P<.001) and L-NAME-treated tate concentration was expressed in micromoles per gram of (P<.05) hearts. The combination of L-NAME and IP dry weight. (L-NAME plus IP) conferred no additional benefit. Experimental Protocol However, pretreatment with SPT abolished the protec For the adenosine assays, a 5-mL aliquot of coronaiy effluent tion conferred by preconditioning and L-NAME (ie, IP was collected after stabilization of the isolated, perfused rabbit plus L-NAME plus SPT) (see Fig 1 and Table). Mean heart. Additional aliquots were collected at 1, 2, 5, and 10 risk areas did not significantly differ between groups (see minutes after continuous perfusion (control) or the addition of Table). Hemodynamic Data and Infarct Sizes After 45 Minutes of Ischemia and 180 Minutes of Reperfusion for 45 Isolated, Perfused Rabbit Hearts IP -t- L-NAME IP + L-NAME Group Control L-NAME -I- SPT SPT IP L-NAME 4- SPT n 7 8 7 6 6 6 5 I/R, % 64.6±0.3 41.4±5.r 58.1 ±7.9 63.2 ±6.5 21.4±1.2t 20.2±1.9t 58.5±2.4 Risk area, cm^ 3 .4 ± 0 ,5 3.0±0.5 3.7±0.6 3.4±0.5 3.4±0.6 3.7±0.6 4.4±0.6 Tlme=0 min (at end of stabilization) CPP 42 ±3 47±2 49±3 42 ±5 39±1 44±3 48±5 LVDP 8 9 ± 6 96±6 80±5 76±5 75±9 90±4 74±5 DP 7±1 8±1 6±1 7±1 9±1 7±1 1 0 ± 2 Tlme=15 min (before Ischemia) CPP 45±3 83±8* 79 ±8* 60±8 41 ±3 73 ± 5 ' 94±13* LVDP 89 ±6 96±5 8G±5 76±5 75±9 90±4 7 4±5 DP 7±1 8±1 6 ± 1 6±1 9±1 7±1 1 0 ± 2 Tlme = 30-mln reperfuslon CPP 64 ±6 99 ±6- 100± i r 115±20* 58±3 1Q7±13* 108±8- LVDP 69 + 7 62±7 61 ±5 56±4 71 ±6 61 ±4 52±5 DP 17±3 14±3 11±3 16±4 13±2 14±3 21 ±4 Tlme = 180-mln reperfuslon CPP 104±9 111±11 126±12 123±17 107±12 126±13 122±11 LVDP 43 ±3 63 ±3" 40±10 34 ±3 60±4* 51 ±5* 31 ±5 DP 33 ±4 14±3* 28±2 30±2 16±4* 18±2* 30 ±2 I/R indicates Infarct area as a percentage of riskCPP, area; coronary perfusion pressure (mm Hg);LVDP, left ventricular developed pressure (mm Hg); and DP. diastolic pressure (mm Hg). ■P<.05, tP<.001 vs corresponding values from control hearts. Values are m eaniSEM. Seven groups were studied: control and combinations of L-NAME (W^-nltro-L-arglnine methyl ester hydrochloride), SPT (8-p-sulfophenyl theophylline), and IP (Ischemic preconditioning). 1548 Circulation Vol 9L No 5 March 1, 1995 30jiML-NAME □ 5 min Global Ischemia ISJS ^lM SPT • 30 nM L-NAME CONTROL 160 -, 1 8 0 - O C O N T R O L 120 - 120 - 100 8 0 80 - 6 0 40 - 4 0 20 - T3 0 0 - REPERFUSION 0 1 s 7 10 Reperfusion Time (minutes) Time (minutes) F ig 3. Plot of adenosine concentration (p.mol/L per 100 g of Fig 2. Plot of mean coronary perfusion pressure over the heart) in the coronary perfusate before (tim e-0 minutes) and at duration of the experiment for control (n = 7) and L-NAME- various times during 10 minutes of reperfusion (control, n=5) or treated (n=8) and SPT-treated (n=6) hearts. Treatment groups after the addition of 30 /xmol/L L-NAME to the coronary perfu were compared with control at each time point. *P<.05. L-NAME sate (n=5) or after 5 minutes of global ischemia (n = 6). Treatment indicates /V°-nitro-L-arginine methyl ester hydrochloride; SPT, groups were compared with control at each time point: *P<.06, 8-p-sulfophenyl theophylline. **P<.02, ***P<.001. L-NAME indicates A/'^-nitro-t-arginine methyl ester hydrochloride. Hemodynamic Data Hemodynamic data relating to ventricular performance of ischemia and 10 minutes of reperfusion, myocardial for all experiments are presented in the Table. There were lactate had risen to 27.1±3.4 /xmol/g dry wt, n= 6 ; P<.OI no differences between groups in the hemodynamic vari versus control; see Fig 4). ables measured after the period of stabilization. In all Discussion groups, the left ventricular developed pressure (LVDP) decreased and diastolic pressure increased over the dura The present study demonstrates clearly that inhibition tion of the experiments. At the end of reperfusion, LVDP of NO synthesis by L-NAME reduces infarct size after was significantly higher in L-NAME-treated and IP hearts coronary artery occlusion and reperfusion in the rabbit in comparison to control (P<.05). Diastolic pressures were isolated, perfused heart. This result supports an earlier lower in hearts treated with L-NAME, IP, or both (L- study in which we observed a similar cardioprotective NAME plus IP) in comparison to control (P<.05). There effect after NO inhibition in the in situ rabbit heart. were no differences between groups in coronary perfusion Because the isolated, perfused heart is free from circu pressure (CPP) measured at the start and the conclusion of lating leukocytes, we can now confidently conclude that the experiment. However, CPP was significantly increased the observed protection does not result from either 10 minutes after the administration of L-NAME in all altered neutrophil accumulation or overproduction of treated hearts. SPT alone increased CPP, but this was not NO by activated neutrophils. significantly greater than control. After 30 minutes of We propose that prior inhibition of NO synthesis by reperfusion, CPP was significantly higher than control in L-NAME protects the myocardium of the isolated rabbit every group of hearts except those assigned to IP (see heart by an adenosine-dependent mechanism that is Table and Fig 2). similar to the mechanism described in IP.^ Our evidence is that, first, in the present study, we have shown that the Biochemical Assays protection induced by L-NAME depends on the release Adenosine Assay Mean adenosine concentration in the coronary effluent p < 0 .0 0 1 was the same in each group before treatment and did not 3 0 - increase after 10 minutes of perfusion in the control group (3.4±0.7 versus 4.8±0.4 p.mol/L per 100 g of heart, n=5, P=NS). However, the addition of 30 /xmol/L L-NAME to the coronary perfusate increased the adenosine concentra tion progressively to a peak of 24.0 ±3.7 yamol/L per 100 g of heart (n=5) after 10 minutes (P<.06 versus control). p N S After 5 minutes of global ischemia, the adenosine concen tration in the perfusate peaked at 1 minute of reperfusion (I39.0±17.9 p.mol/L per 100 g of heart, n= 6 ) and re mained significantly higher than control hearts at 10 min utes (83.1 ±7.2 p,mol/L per 100 g of heart, n= 6 ; P<.001 versus control; see Fig 3). Control L-NAME Ischemia F ig 4. Bar graph of myocardial lactate concentration (/xmol/g Lactate Assay dry wt) measured after 10 minutes of continuous perfusion Myocardial lactate concentration was the same after (control, n = 5) or the addition of 30 /xmol/L L-NAME to the coronary perfusate for 10 minutes (L-NAME. n = 5) or after 5 10 minutes of perfusion with either 30 p.mol/L L-NAME minutes of global ischemia and 10 minutes of reperfusion (5.5±1.2 jamol/g dry wt, n=5) or control (4.6±0.5 (ischemia, n = 6). *P<.001. L-NAME indicates A/'^-nitro-i-arginine /amol/g dry wt, n=5; P=NS). In contrast, after 5 minutes methyl ester hydrochloride. Woolfson et al NO Inhibition Reduces Ischemia-Reperfusion Injury 1549 of adenosine because it can be abrogated by SPT, an that we failed to detect localized ischemia using this adenosine receptor antagonist. Second, L-NAME does whole-heart assay or that the assay was inadequately not confer additional protection on hearts that are sensitive to detect mild ischemia, our result is consistent subsequently preconditioned. Third, SPT prevents all of with a recent report from Pohl et al,‘^ who measured the protection induced by the combination of L-NAME lactate concentration in the coronary perfusate of iso and IP, and, fourth, L-NAME and brief global ischemia lated rabbit hearts. These reseachers showed that inhi both give rise to an increase in the adenosine concen bition of NO synthesis causes myocardial ischemia only tration in the coronary effluent, although the increment if perfusion is pressure controlled, not if the coronary was less marked after L-NAME than after ischemia. flow rate is maintained in the face of increased CPP. These observations support a common mechanism for Their report is consistent with our previous finding that the protection induced by pretreatment with L-NAME inhibition of NO synthesis in the in situ rabbit heart and IP. However, in contrast to IP, we propose that the causes myocardial ischemia"^; in that situation, raised trigger for adenosine release after inhibition of NO myocardial lactate concentration presumably reflected a synthesis is an alteration in coronary hemodynamics combination of myocardial hypoperfusion due to coro rather than myocardial ischemia. nary vasoconstriction and increased ventricular work Liu et aP have previously observed that the reduction due to increased systemic vascular resistance. in infarct size that follows IP in the rabbit heart is ATP activates P2Y receptors on the endothelium, dependent on adenosine release and A; receptor stimu leading to the release of NO^^^; therefore, it is conceiv lation, which can be abolished by SPT. SPT is a methyl- able that inhibition of NO might affect endothelial xanthine derivative with an added sulfophenyl group release of ATP. However, we believe that the linkage that prevents the molecule from entering the cell; there between release of ATP and NO from the endothelium fore, intracellular phosphodiesterases are not affected. is hemodynamic rather than metabolic. Coronary flow SPT is an unselective adenosine receptor antagonist with exerts shear stress on the endothelium, leading to ATP a Ki of 4.5 /i,mol/L and 6.3 ju,mol/L for the A% and Aj release, which can be enhanced by increased shear stress receptors, respectively^^; therefore the concentration due to, for example, adrenergic vasoconstriction.^^-^® We used in our study (75.5 ju.mol/L) would have been propose that inhibition of NO synthesis led to vasocon sufficient to block adenosine receptors. In view of the striction that under conditions of constant flow increased cardioprotective properties of adenosine,'*'^ it is perhaps luminal shear stress and resulted in increased release of surprising that pretreatment with SPT (group 4) did not ATP, which was subsequently degraded to adenosine. increase infarct size; however, this finding is consistent Under physiological conditions, NO is probably the with other reports. Interestingly, Zhao et aP recently major determinant of flow-mediated coronary vasodila showed that SPT can worsen myocardial injury either if tation and eclipses the contributions of other autocoids additional doses are administered during ischemia and such as ATP, adenosine, bradykinin, substance P, and early reperfusion or if SPT is administered just before reperfusion. Thus, it appears that the release of adeno prostaglandins.^"^ However, the contribution from these sine during and after any ischemic period will mitigate autocoids is likely to be greater in certain diseases, such against the reperfusion injury that follows. as atherosclerosis, diabetes mellitus, and hyperlipidemia, Treatment with either L-NAME or SPT increased where there is evidence of impaired endothelium-depen CPP, although the increment reached statistical signifi dent NO-mediated vasodilatation. Thus, it is possible cance only after L-NAME (see Fig 2). These data that in hearts affected by these diseases, the concentra support an important role for NO but not adenosine as tion of adenosine in the coronary perfusate is higher a determinant of coronary artery tone in the unstressed than that in healthy hearts. Because adenosine preserves isolated, perfused heart.®-^ However, during the reper myocardial blood flow during reperfusion^ and inhibits fusion period that followed 45 minutes of regional neutrophil accumulation,we hypothesize that in ischemia, CPP was higher in both L-NAME- and SPT- creased adenosine release could explain a recent obser treated groups compared with control (see Fig 2), which vation that streptozotocin-induced non-insulin-depen- indicates that both NO and adenosine contribute to dent diabetes mellitus protects the rabbit heart from vasomotor tone during reperfusion. Therefore, the con ischemia-reperfusion,'*" although this observation was centration of adenosine present in the coronary perfu not repeated in the diabetic dog."^' sate during reperfusion may have been increased in Reperfusion after 5 minutes of global ischemia results L-NAME-treated hearts, which could have further con in a substantially greater amount of adenosine being tributed to myocardial protection.^ washed out into the coronary perfusate than does treat There is basal release of ATP from the isolated, ment of the whole heart with L-NAME. (Branch coro perfused heart that is metabolized by extracellular ecto- nary artery occlusion, which affects a smaller portion of phosphatases to adenosine, which vasodilates the coro the myocardium than global ischemia, would obviously nary circulation35'36 (see previous discussion). The re lead to the washout of less adenosine into the perfusate, lease of adenine nucleotides, principally from although the affected myocardium would have been endothelial cells but also from cardiac myocytes and exposed to a similar interstitial concentration.) In view purinergic nerves, increases substantially during isch- of this observation, we suggest that a relation may exist emia.^^^^ In the present study, in which L-NAME- between the amount of adenosine washed out into the treated isolated hearts were perfused at a constant rate coronary effluent and the extent of cardioprotection. of 35 mL/min, the increased concentration of adenosine The data do not permit us to comment as to whether it in the coronary perfusate was not associated with in is the peak concentration of adenosine achieved or the creased myocardial lactate levels, which suggests that total released (ie, either the peak or the area under the these hearts were not ischemic. Although it is possible curve on Fig 3) that determines the extent of protection. 1550 Circulation Vol 91, No 5 March 1, 1995 One aim of the present study was to investigate whether 2. Walker DM, Yellon DM. Ischemic preconditioning: from mech overproduction of NO during reperfusion might contribute to anisms to exploitation. Cardiovasc Res. 1992;26:734-739. 3. Liu GS, Thornton J, Van Wrinkle DM, Stanley AWH, Olsson RA, myocardial injury. This question would not have been prop Downey JM. Protection against infarction afforded by precondi erly addressed if IGNAME had been added to the perfusate at tioning is mediated by A, adenosine receptors in the rabbit heart. the beginning of reperfusion since NO release from the Circulation. 1991;84:350-356. occluded coronaiy circulation would not have been immedi 4. Gruber HE, Hoffer ME, McAllister DR, Laikind PK, Lane TA, ately inhibited. Therefore, we chose to include L-NAME in Schmid-Schoenbein GW, Engler RL. Increased adenosine concen tration in blood from ischemic myocardium by AICA riboside: effects the perfusate from before ischemia until 15 minutes after the on flow, granulocytes, and injury. Circulation. 1989;80:1400-1411. start of reperfusion. As a result, IGNAME caused vasocon 5. Zhao ZQ, McGee DS, Nakanishi K, Toombs CF, Johnston WE, striction that persisted for the first 30 minutes of reperfiision Ashar MS, Vinten-Johansen J. Receptor-mediated cardiopro (see Fig 2), but despite this, infarct sizes were smaller in tective effects of endogenous adenosine are exerted primarily during reperfusion after coronary occlusion in the rabbit. L-NAME-treated hearts. We believe that the adverse vaso Circulation. 1993;88:709-719. constrictive effects of I-NAME during reperfusion were mit 6. Palmer RMJ, Rees DD, Ashton DS, Moncada S. L-Arginine is the igated by the constant flow of coronaiy perfusate and the physiological precursor for the formation of nitric oxide in endo compensatory release of adenosine. It remains possible that thelium-dependent relaxation. Biochem Biophys Res Commun. 1988;153:1251-1256. the benefits conferred by these additional factors obscured a 7. Moncada S, Higgs A. The L-arginine-nitric oxide pathway. N Engl minor degree of myocardial protection that resulted from J Med. 1993;329:2002-2012. inhibition of overproduction of NO during the first 15 to 30 8. Ameczua JL, Palmer RMJ, De Souza BM, Moncada S. Nitric oxide minutes of reperfiision. synthesized from L-arginine regulates vascular tone in the coronary Progressive loss of myocardial function with decreas circulation of the rabbit. Br J Pharmacol. 1989;97:1119-1124. 9. Kelra M, Schrader J. Control of coronary vascular tone by nitric ing LVDP and increasing ventricular diastolic pressure oxide. Circ Res. 1990;66:1561-1575. is a feature of the isolated, perfused heart preparation. 10. Radomski MW, Palmer RMJ, Moncada S. Endogenous nitric oxide However, the decreases in LVDP were more marked in inhibits human platelet adhesion to vascular endothelium. Lancet. control hearts and in the three groups of hearts pre 1987;2:1057-1058. 11. Kubes P, Suzuki M, Granger DN. Nitric oxide: an endogenous treated with SIT, which reflects the larger infarcts modulator of leukocyte adhesion. Proc Natl Acad Sci USA. sustained by the hearts in those groups in comparison to 1991;86:4651-4655. those treated with L-NAME, IP, or both (ie, IP plus 12; Kubes P, Granger DN. Nitric oxide modulates microvascular per L-NAME). Similarly, increases in diastolic pressure meability. Am J Physiol. 1992;262:H611-H615. were more marked in those hearts that had sustained 13. Cronstein BN, Levin RI, Belanoff J, Weissmann G, Hirschhorn R. Adenosine: an endogenous inhibitor of neutrophil-mediated injury larger infarcts (ie, control, L-NAME plus SPT, SPT, IP to endothelial cells. J Clin Invest. 1986;78:760-770. plus L-NAME plus SPT) in contrast to the smaller- 14. Woditsch I, Schror K. Prostacyclin rather than endogenous nitric infarct groups (ie, L-NAME, IP, IP plus L-NAME). oxide is a tissue protective factor in myocardial ischemia. A m J Our results contrast with those of Vegh et al,^^ who Physiol. 1992;263:H1390-H1396. 15. Rees DD, Palmer RMJ, Schulz R, Hodson HP, Moncada S. Char have demonstrated that NO generation contributes to acterization of three inhibitors of endothelial nitric oxide synthase the antiarrhythmic effects of IP in the in situ canine in vitro and in vivo. Br J Pharmacol. 1990;101:746-752. heart. This may reflect either a disparity between ar- 16. Patel VC, Y ellon DM , Singh KJ, Neild G H, W oolfson RG. Inhi rhythmogenesis and infarct size as measures of myocar bition of nitric oxide limits infarct size in the in situ rabbit heart. dial injury or that L-NAME may inhibit the opening of Biochem Biophys Res Commun. 1993;194:234-238. 17. Pohl U, Lamontagne D, Bassenge E, Busse R. Attenuation of preformed collaterals in the canine coronaiy circula- coronary autoregulation in the isolated rabbit heart by endotheli tion^3 that are absent in the rabbit heart. um-derived nitric oxide. Cardiovasc Res. 1994;28:414-419. In conclusion, results of the present study support our 18. Kostic MM, Schrader J. Role of nitric oxide in reactive hyperemia previous report that inhibition of NO synthesis protects of the guinea pig heart. Circ Res. 1992;70:208-212. 19. Park KH, Rubin LE, Gross SS, Levi R. Nitric oxide is a mediator of against ischemia-reperfusion injury in the rabbit heart. hypoxic coronary vasodilatation. Circ Res. 1992;71:992-1001. We propose that the mechanism responsible for this 20. Yamabe H, Okumura K, Ishizaka H, Tsuchiya T, Yasue H. Role of cardioprotection is an increase in release of adenosine, endothelium-derived nitric oxide in myocardial reactive hyperemia. which has cardioprotective properties. This adenosine- Am J Physiol. 1992;263:H8-H14. mediated mechanism is similar to that implicated in IP 21. Lefer AM, Aoki N. Leucocyte-dependent and leucocyte-independent mechanisms of impairment of endothelium-mediated vasodilation. except that the stimulus for adenosine release after Blood Vessels. 1990;27:162-168. inhibition of NO synthesis is not myocardial ischemia. 22. Ma X, Tsao PS, Viehman GE, Lefer AM. Neutrophil-mediated Our results do not exclude the possibility that some vasoconstriction and endothelial dysfunction in low-flow perfusion- myocardial protection results from inhibition of NO reperfused cat coronary artery. Circ Res. 1991;69:95-106. 23. Ma X -L Wqyrich AS, Lefer DJ, Lefer AM. Diminished basal nitric release during early reperfusion; however, we conclude oxide release after myocardial ischemia and reperfusion promotes that most of the benefit is derived from the release of neutrophil adherence to coronary endothelium. Circ Res. 1993;72: adenosine. Finally, we suggest that a concentration- 403-412. response relation exists between adenosine concentra 24. Johnson G, Tsao PS, Mulloy D, Lefer AM. Cardioprotective effects tion in the coronary perfusate and the extent of protec of acidified sodium nitrite in myocardial ischemia with perfusion. J Pharm Exp Ther. 1990;252:35-41. tion against subsequent injury. 25. Siegfried MR, Erhardt J, Rider T, Ma X-L, Lefer AM. Cardiopro tection and attenuation of endothelial dysfunction by organic nitric Acknowledgments oxide donors in myocardial ischemia-reperfusion. J Pharm Exp This work was supported by grants from the British Heart Ther. 1992;260:668-675. Foundation and the Hatter Foundation. 26. Weyrich AS, Ma X-L, Lefer AM. The role of L-arginine in amelio rating reperfusion injury after myocardial ischemia in the cat. Circulation. 1992;86:279-288. References 27. Beckman JS, Beckman TW, Chen J, Marshall PA, Freeman BA. I. Murry CE, Jennings RB, Reimer KA. Preconditioning with Apparent hydroxyl radical production by peroxynitrite: implications ischemia: a delay of lethal cell injury in ischemic myocardium. for endothelial injury from nitric oxide and superoxide. Proc Nall Circulation. 1986;74:1124-1136. A cad Sci USA. 1990;87:1620-1624. Woolfson et al NO Inhibition Reduces Ischemia Reperfusion Injury 1551 28. Geng Y, Hansson GK, Holme E. Interferon -y and tumor necrosis 36. Ralevic V, Bumstock G. Roles of Pj-purinoceptors in the cardio factor synergize to induce nitric oxide production and inhibit mito vascular system. Circulation. 1991;84:1-14. chondrial respiration in vascular smooth muscle cells. Circ Res. 37. Borst MM, Schrader J. Adenine nucleotide release from isolated 1992;71:1268-1276. perfused guinea pig hearts and extracellular formation of 29. Matheis G, Sherman MP, Buckberg GD, Haybron DM, Young H, adenosine. Circ Res. 1991;68:797-806. Ignarro LJ. Role of L-arginine-nitric oxide pathway in myocardial 38. Vial C, Owen P, Opie LH, Posel D. Significance of release of reoxygenation injury. Am J Physiol. 1992;262:H616-H620. adenosine triphosphate and adenosine induced by hypoxia or 30. Buisson A, Plotkine M, Boulu RG. The neuroprotective effect of a adrenaline in perfused rat heart. 7 Mo/ Cell Cardiol. 1987;19:187-197. nitric oxide inhibitor in a rat model of focal cerebral ischaemia. Br 39. Bumstock G. Vascular control by purines with emphasis on the J Pharmacol. 1992;106:766-767. coronary system. Eur Heart J. 1989;10(F):15-21. 31. Mulligan MS, Hevel JM, Marietta MA, Ward PA. Tissue injury caused 40. Liu Y, Thornton JD, Cohen MV, Downey JM, Schaffer SW. by deposition of immune complexes is L-arginine dependent. Proc Natl Streptozotocin-induced non-insulin-dependent diabetes protects A cad Sci USA. 1991;88:6338-6342. the heart from infarction. Circulation. 1993;88:1273-1278. 32. Jenkins JR, Belardinelli L. Atrioventricular nodal accommodation 41. Forrat R, Sebbag L, Wiemsperger N, Guidollet J, Renaud S, de in isolated guinea pig hearts: physiological significance and role of Lorgeril M. Acute myocardial infarction in dogs with experimental adenosine. Circ Res. 1988;63:97-116. 33. Hohorst H-J. Tissue lactate analysis. In: Bergmeyer H, ed. M ethods diabetes. Cardiovasc Res. 1993;27:1908-1912. of Enzymatic Analysis. New York, NY: Academic Press Publishers; 42. Vegh A, Szekeres L, Parratt J. Preconditioning of the ischaemic 1963:266-270. myocardium: involvement of the L-arginine nitric oxide pathway. Br 34. Daly JW, Padgett W, Shamim MT, Butts-Lamb P, Walters J. 1,3- J Pharmacol. 1992;107:648-652. Dialkyl-8-(p-suIfophenyl) xanthines: potent water-soluble antag 43. Yamamoto H, Tomoike H, Shimokawa H, Nabeyama S, Nakamura onists for A,- and A;-adenosine receptors. J Med Chem. M. Development of collateral function with repetitive coronary 1985;28:487-492. occlusion in a canine model reduces myocardial reactive hyperemia 35. Paddle BM, Bumstock G. Release of ATP from perfused heart in the absence of significant coronary stenosis. Circ Res. during coronary vasodilation. Blood Vessels. 1974;11:110-119. 1984;55:623-632. Cardiovascular Research LSEVIER Cardiovascular Research 31 (1996) 148-151 Pre-conditioning with adenosine leads to concentration-dependent infarct size reduction in the isolated rabbit heart R.G. Woolfson *, V.C. Patel, D.M. Yellon The Hatter Institute for Cardiovascular Studies, Department of Academic Cardiology, University College London Medical School, Gower Street, London WCJE 6AU, UK Received 14 December 1994; accepted 1 February 1995 Abstract Adenosine (ADO) has a cardioprotective effect in ischemia-reperfusion injury when administered both prior to ischemia and during re perfusion. ADO has also been implicated in the mechanism of ischemic pre-conditioning. The aim of this study was to investigate whether there was a concentration-response between the administration of ADO prior to ischemia-reperfusion and reduction in subsequent infarct size. Rabbit isolated perfused hearts were subjected to 45 min ischemia and 180 min reperfusion following pre-treatment with either Krebs Henseleit buffer alone or buffer containing ADO at a range of concentrations (3 ^tM-100 /jlM) for 5 min followed by 5 min perfusion with buffer. Infarct/risk ratios were significantly reduced in hearts pre-perfused with higher (> 3 /xM) concentrations of ADO (Control, 58.5 + 1.5%; 3 fiM ADO, 51.6 ± 3.0%; 6 /xM ADO, 44.1 + 2.0%; 10 /xM ADO, 33.3 ± 1.9%; 20 /xM ADO, 26.6 + 0.9%; 50 /xM ADO, 21.6 + 3.5%; 100 /xM ADO, 23.0 ±0.6%). We conclude that pre-treatment with ADO leads to a concentration-dependent reduction in infarct size. Keywords: Adenosine; Myocardial infarct size; Rabbit, heart 1. Introduction reperfusion can increase tolerance against subsequent sus tained ischemia-reperfusion [2]. This phenomenon, termed Coronary artery occlusion followed by reperfusion leads “ ischemic pre-conditioning” , is associated with both pres to infarction of a proportion of the ischemic area. The ervation of function and an anti-arrhythmic action, using extent of injury depends on the availability of collateral both in vivo and in vitro models of myocardial ischemia blood flow, the duration of ischemia, the adhesion, activa (see reference [3] for review). The release of adenosine tion and extravasation of neutrophils together with the from ischemic cells and its subsequent washout during the generation of free radicals. There is continuing interest in pre-conditioning cycle has been implicated in the mecha the development of strategies to mitigate against is nism of ischemic pre-conditioning [4]. The effector path chemia-reperfusion injury. way of adenosine-induced protection in pre-conditioning During ischemia, the release of adenine nucleotides remains unclear but appears to involve A,/A3 (and not from endothelial cells, cardiac myocytes and purinergic A j) receptors on the cardiac myocytes. nerves increases substantially [1]. Nucleotides are rapidly In addition to the cardioprotective effect resulting from degraded by extracellular ectophosphatases to adenosine exposure to adenosine prior to sustained ischemia-reperfu which accumulates in ischemic tissue and is progressively sion as observed for pre-conditioning, the presence of washed out during reperfusion. Interest in the role of increased concentrations of adenosine during reperfusion is adenosine during ischemia and reperfusion has increased also beneficial and further augmentation of adenosine lev following the observation that a brief period of ischemia- els during reperfusion offers protection against injury [5,6]. The mechanism of the cardioprotection offered by adeno sine in this respect may involve A 2-mediated coronary * Corresponding author. Tel. ( + 44-171) 380-9888; Fax ( + 44-171) arteriolar vasodilatation [l], inhibition of local release of 388-5095. vasoconstrictors such as noradrenaline [7] and endothelin 0008-6363/96/$ 15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI 0008-6363(95)00185-9 R.G. Woolfson et a l./Cardiovascular Research 31 (1996) 148-151 149 ^[8 ] and the inhibition of activation and aggregation of group, was monitored for an additional 1 0 min before . circulating platelets and granulocytes [5,9]. being subjected to ischemia and reperfusion. The remain We have recently reported a relationship between ele der of the hearts (Groups 2 to 7) were perfused with Krebs vated adenosine concentration in the coronary perfusate of Henseleit buffer containing adenosine at a fixed concentra the rabbit isolated heart and infarct size reduction by tion (3 )U,M, 6 fxM, 10 )lcM, 20 fxM, 50 /iM or 100 /xM) comparing two different experimental techniques which for 5 min and then with Krebs Henseleit buffer alone for 5 ^have adenosine-dependent mechanisms [10]. In the light of min prior to ischemia and reperfusion. In each experiment, those findings, we have gone on to investigate whether ischemia lasted 45 min and was followed by 180 min of there is a concentration response between the administra reperfusion. tion of adenosine prior to ischemia-reperfusion and subse quent infarct size. 2.3. Measurement of infarct and risk area At the end of each experiment, the heart was flushed 2. Experimental method with room temperature saline for 1 min. The coronary branch was then reoccluded and 1 - 1 0 /rm fluorescent zinc 2.1. The isolated heart model cadmium sulfide particles infused into the perfusate until the risk area could be visualized with UV light. The ligature was then released and 2-3 ml of 1% triphenyl Male New Zealand White rabbits (2.0-3.0 kg) were tétrazolium chloride (TTC) were injected through the side ^ anesthetized with intravenous sodium pentobarbital (40 arm and the heart immersed in Krebs Henseleit buffer at mg/kg) following cannulation of a marginal ear vein. A 37°C for 2 min. (TTC stains viable tissue deep red.) The tracheostomy was performed under local anesthetic (3 ml heart was weighed and frozen. While frozen, the heart was of 2 % lidocaine) and the rabbit was ventilated mechani cut into 2 mm transverse slices. Tracings were made of the cally with 100% oxygen. The chest was opened through a risk zones (lacking fluorescence under ultraviolet light) left thoracotomy and the pericardium incised to expose the and the infarct zones (TTC-negative tissue) and the areas heart. A 3-0 silk suture on a small round-bodied needle were determined by planimetry of the tracings. The vol was passed through the myocardium underneath a proxi ume of infarct and risk tissue was calculated as the product mal branch of the left coronary artery and the ends were of area (cm^) and 0.2 cm (thickness of slice). This experi threaded through a small vinyl tube to form a snare which was used to occlude the artery as required. The rabbits mental method is well established [4,6]. ( were given 1 0 0 0 units heparin intravenously, then the heart was rapidly excised by cutting the great vessels, placed in 2.4. Chemicals room temperature saline, and mounted on the Langendorff apparatus within 1 min. Adenosine was purchased from Sigma Chemicals Ltd., The hearts were perfused retrogradely at a constant flow UK. rate of 65 ml/min in a non-recirculating system using a All studies were in accordance with the United King roller pump. The perfusate was Krebs Henseleit buffer dom Home Office regulations for the care and use of ' equilibrated with 95% O; and 5% COj at 37°C which laboratory animals (Project Licence No. 70/01759). contained (mM) NaCl 118.0, KCl 4.7, MgSO^ • 7 H 2O 1.2, CaClg - 2 H 2O 1.8, NaH 2 P0 4 14.6, KH2 PO4 1.2 and glu- 2.5. Data analysis cose 11.0, pH 7.4. All hearts were electrically paced at 180 beats per minute via the right atrium. Left ventricular All results are expressed as mean ± standard error pressures were measured by means of a fluid-filled latex (s.e.m.). Statistical analysis between groups was performed ' balloon, connected by polyethylene tubing to a pressure using ANOVA followed by Bonferroni’s multiple compar transducer and inserted into the left ventricle via an inci isons test. sion in the left atrial appendage. The balloon volume was adjusted to give an initial diastolic pressure of 10 mmHg. Coronary perfusion pressure was measured via a side arm 3. Results in the aortic cannula using a pressure transducer. The heart was allowed to stabilize for at least 30 min before the 3.1. Infarct size data experiment was begun. Pre-treatment with adenosine led to concentration-de 2.2. Experimental protocols pendent reduction in infarct/risk ratio which was highly significant (P < 0 .0 1 ) at concentrations of 6 /xM and There were seven experimental groups with rabbits above. Mean risk volumes were not significantly different being assigned sequentially to each. Group 1, the control between groups (see Fig. 1 and Table 1). 150 R.G. Woolfson et al. / Cardiovascular Research 31 (1996) 148-151 min perfusion with buffer plus adenosine led to a dose-de pendent reduction in coronary perfusion pressure (signifi cant at concentrations of 20 ^t,M and greater) which had Infarct / Risk disappeared 5 min later. Adenosine had no effect on (%) ventricular performance as judged by left ventricular sys tolic and diastolic pressures (see Table 2). At the end of reperfusion, coronary perfusion pressure was the same in all groups. Left ventricular systolic pres sures were significantly greater in hearts treated with 0 3 6 10 M 30 100 higher concentrations of adenosine. Conversely, left ven [Adenosine] in perfusate (|iM) tricular systolic pressures tended to be lower and diastolic pressures higher in groups which had sustained larger Fig. 1. Ratio of infarct volume to risk volume expressed as percentages for 7 groups of isolated perfused rabbit hearts. Hearts were perfused for 5 infarcts (see Table 2). min with buffer + adenosine (0 -1 0 0 /a M) followed by 5 min washout, 45 min ischemia and 180 min re perfusion. 4. Discussion 3.2. Hemodynamic data This study demonstrates that pre-treatment with adeno Hemodynamic data relating to ventricular performance sine leads to a concentration-dependent reduction in infarct for all experiments are presented in Table 2. There were no size in the rabbit isolated perfused heart. These results differences between groups in the hemodynamic variables support the findings of an earlier study in which we had measured following the period of stabilization. However, 5 reported a correlation between adenosine concentration in Table 1 Infarct/risk ratios and risk volume data for 36 isolated perfused rabbit hearts exposed to 45 min ischemia and 180 min reperfusion. Seven groups were studied: hearts perfused with buffer for 10 min prior to ischemia-reperfusion (Control) and hearts perfused with buffer + adenosine (3 /itM-lOO ptM) for 5 min, then buffer for 5 min prior to ischem ia-reperfusion Group Control 3 /iM ADO 6 /xM ADO 10 /xM ADO 20 /AM ADO 50 /AM ADO 100 /a M a d o n 7 7 5 4 5 4 4 I/R (%) 58.5 + 1.5 51.6 + 3.0 44.1+2.0 33.3 ±1.9* 26.6 ±0.9 * * 21.6 ±3.5 ' * 23.0 ±0.6 * * Risk volume (cm^) 1.0 ±0.4 0.9+ 0.5 0.9+ 0.6 1.0 ± 0 .4 1.1 ± 0 .6 1.2 ± 0 .2 0.9 ± 0 .5 P < 0.01; * * P < 0.001 vs. corresponding values from Control hearts. Table 2 Hemodynamic data for 36 isolated perfused rabbit hearts exposed to 45 min ischemia and 180 min reperfusion.Seven groups were studied: hearts perfused with buffer for 10 min prior to ischemia-reperfusion (Control) and hearts perfused with buffer + adenosine (3 /xM-lOO fiM) for 5 min, then buffer for 5 min prior to ischem ia-reperfusion Group Control 3 /a M ADO 6 ADO 10 /AM ADO 20 /A M ADO 50 /AM ADO 100 /a M a d o n i l 5 4 5 4 4 Time = 0 min (end of stabilization) CPP 100 + 3 98 ±1 103 + 2 1 0 0 ± 3 105 ± 1 9 9 ± 3 100±2 LVDP 118±3 116 + 4 115 + 3 122±4 118±5 115±2 120±6 DP 8 + 2 7 + 2 10+1 8±3 6±2 10± 1 7±1 Time = 5 min (end of adenosine) CPP 101 + 2 97 + 3 95 + 5 88 ± 1 82±5 * * 85±2* 83 ± 4 * * LVDP 116 + 4 115±3 115 + 3 120±3 120 ±5 114±5 120 ±2 DP 10 + 2 7 + 3 10+1 10 ±3 6±2 10± 1 8±1 Time = 10 min (i.e. 5 min ADO + 5 min buffer) CPP 100 + 3 102 + 4 108 + 3 95±3 108 ±3 100 ±2 105 ±3 LVDP 118 + 4 115 + 6 118 + 4 116±3 118±2 116±3 120±4 DP 8 + 2 10 + 2 8 + 2 10± 1 1 + 2 12±2 10± 1 Time = 180 min reperfusion (i.e. conclusion of experiment) CPP 148 + 5 148 + 4 145 + 4 150±6 [32 + 1 1.35 ± 5 145 ± 3 LVDP 74±5 79 + 3 85 + 4 87 ±3 91 + 2 * * 90±3 98 ± 4 * * * DP 28 + 4 23 + 2 22 + 3 18± 1 14±2 18±2 16±3 P < 0.05; * * f < 0.01; * * * F < 0.001 vs. corresponding values from Control hearts. I/R, infarct volume as a percentage of risk volume. CPP, coronary perfusion pressure (mmHg). LVDP, left ventricular developed pressure (mmHg). DP, diastolic pressure (mmHg). R.G. Woolfson et a l./Cardiovascular Research 3 1 (1996) 148-151 151 the coronary perfusate and the degree of infarct size reduction in subsequent infarct size following ischemia- reduction [10]. reperfusion in the rabbit isolated perfused heart. The graded Cardioprotection was noted following perfusion with response to adenosine occurs over a narrow concentration adenosine concentrations of 6 jxM and the effect was range (one order of magnitude) and this may account for maximal at a concentration of 50 /iM. Thus the graded the supposition that ischemic pre-conditioning has an “ all- cardioprotective response was present over a narrow dose or-none” action. ■ range, only one order of magnitude. The degree of protec tion noted at higher doses was similar to that which we have previously reported following ischemic pre-condition References ing in the rabbit isolated perfused heart and which could be abrogated by prior treatment with 8-p-sulfophenyl-theo- [1] Ralevic V, Bumstock G. Roles of Pg-purinoceptws in the cardio phylline (8-SPT), an adenosine receptor antagonist. vascular system. Circulation 1991;84:1-14. Although there is one report of “dose-dependency” of [2] Murry CE, Jennings RB, Reimer KA. Preconditioning with is chemia: a delay of lethal cell injury in ischemic myocardium. protection by ischemic pre-conditioning against ischemia- Circulation 1986;74:1124-1136. induced arrhythmias in the rat isolated heart, this has not [3] Walker DM, Yellon DM. Ischemic preconditioning: from mecha been reported in other species [11]. Optimal protection nisms to exploitation. Cardiovasc Res 1992;26:734-739. against myocardial necrosis can be achieved in dogs and [4] Liu GS, Thornton J, Van Wrinkle DM, Stanley AWH, Olsson RA, rabbits with a single pre-conditioning cycle of 5 min Downey JM. Protection against infarction afforded by precondition ing is mediated by A, adenosine receptors in the rabbit heart. ischemia and 5 min reperfusion and there appears to be no Circulation 1991;84:350-356. additional protection from further cycles [12,13]. Although [5] Gruber HE, Hoffer ME, McAllister DR, Laikind PK, Lane TA, the detailed mechanisms which underly the anti-arrhythmic Schmid-Schoenbein GW, Engler RL. Increased adenosine concentra and anti-ischemic effects of ischemic pre-conditioning may tion in blood from ischemic myocardium by AICA riboside: effects well be different, it remains possible that a graded re on flow, granulocytes, and injury. Circulation 1989;80:1400-1411. [6] Zhao ZQ, McGee DS, Nakanishi K, Toombs CF, Johnston WE, sponse between pre-conditioning and myocardial necrosis Ashar MS, Vinten-Johansen J. Receptor-mediated cardioprotective could be demonstrated in the rabbit or dog if the pre-condi effects of endogenous adenosine are exerted primarily during reper- tioning cycles were sufficently brief. This conclusion is fiision after coronary occlusion in the rabbit. Circulation consistent with our observation that graded adenosine-de 1993;88:709-719. pendent cardioprotection can be demonstrated over a rela [7] Richardt G, Waas W, Kranzhomig R, Mayer E, Schomig A. Adeno sine inhibits exocytotic release of endogenous noradrenaline in rat tively narrow range of concentrations of adenosine. heart: a protective mechanism in early myocardial ischemia. Circ ( Treatment with adenosine led to a brief fall in coronary Res 1987;61:117-123. perfusion pressure with no change in the indices of ventric [8] Velasco CE, Jackson EK, Morrow JA, Vitola JV, Inagami T, ular performance. No chronotropic effects of adenosine Forman MB. Intravenous adenosine suppresses cardiac release of were identified because all hearts were paced electrically endothelin after myocardial ischaemia and reperfusion. Cardiovasc Res 1993;27:121-128. at 180 bpm. At the conclusion of reperfusion, we noted no [9] Cronstein BN, Levin RI, Belanoff J, Weissmann G, Hirschhorn R. differences in coronary perfusion pressure between the Adenosine: an endogenous inhibitor of neutrophil-mediated injury to groups. endothelial cells. J Clin Invest 1986;78:760-770. ' Progressive loss of myocardial function with falling [10] Woolfson RG, Patel VC, Neild GH, Yellon DM. Inhibition of nitric ventricular systolic and increasing ventricular diastolic oxide synthesis reduces infarct size by an adenosine-dependent mechanism. Circulation 1995;91:1545-1551. pressure is a feature of the isolated perfused heart prepara [11] Lawson CS, Coltart DJ, Hearse DJ. ' ' Dose''-dependency and tem tion. However, the decreases in ventricular systolic pres poral characteristics of protection by ischaemic preconditioning sure were more marked in Control hearts and there was a against ischaemia-induced arrhythmias in rat hearts. J Mol Cell correlation between infarct size and systolic pressure in Cardiol 1993;25:1391-1402. adenosine-treated hearts. Similarly, infarct size correlated [12] Li GC, Vasquez JA, Gallagher KP, Lucchesi BR. Myocardial protec tion with preconditioning. Circulation 1990;82:609-619. with increases in ventricular diastolic pressure. [13] Van Winkle DM, Thornton J, Downey JM. Cardioprotection from In conclusion, this study demonstrates that pre-treat ischemic preconditioning is lost following prolonged reperfusion in ment with adenosine leads to a concentration-dependent the rabbit. Cor Art Dis 1991;2:613-619.