Adenosine Release and Functional Hyperemia in an Isolated Guinea Pig Heart Preparation
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ADENOSINE RELEASE AND FUNCTIONAL HYPEREMIA IN AN ISOLATED GUINEA PIG HEART PREPARATION by Gerard Ho 1mes Submitted to the Faculty of the School of Graduate Studies of the Medical College of Georgia in Partial Fulfillment of the Requirements for the. Degree of Doctor of Philosophy December 1983 ADENOSINE RELEASE AND FUNCTIONAL HYPEREMIA IN AN ISOLATED GUINEA PIG HEART PREPARATION This dissertation submitted by Gerard Holmes has been examined and approved by an appointed committee of the faculty of the School of G·raduate Studies of the Medical College of Georgia. The si-gnatures which appear below verify the fact that all required changes have been incorporated and that the dissertation has received final approval with reference to content, form and accuracy of presentation. This dissertation is therefore accepted in partial fulfillment of the requirements for the degree of Doctor of Philosophy. May 18, 1984 Date ACKNOWLEDGEMENTS The author wishes to express his sincerest appreciation to the members of his dissertation committee for their valued suggestions and to Dr. V.T. Wiedmeier for his advice, support and confidence, without which his dissertation would not have been pqssible. Special thanks go to Mrs.· E.J. Jones for her expert·technical assistance and for sharing her irrepres s i b1 e sense of human dignity. Finally the author a 1so wishes to thank the faculty of the School of Graduate Studies for their help in the construction of a sound foundation in the basic sciences and t.o his friends and family for their encouragement during difficult times. iii TABLE OF CONTENTS Page I. INTRODUCTION .................................................. 1 A. Statement of ·Prob 1em ..•.•..• ·• • • • • • . • . • . • . • • . • . • • • . • • • • • • . • 1 B. Literature Review .••••••• ,••••.••.••...•••••••••.••.•••..••••• 6 II. MATERIALS AND METHODS .•....••..••..••••••..•••••••.••.••.•.••• 36 A •. Apparatus •.• : ••.••..••••.••.••••••••••••.•.•..•• ·.~············ 36 B. Isolation and Perfusion of Hearts ••..•.••••••••••.••••.••••.•• 38 C. Protoco·l s . ..................................... ~ ....· ... ~ . 39 D. Analytical Procedures •.•.•.•••..••..•.....••.•...•••..•.••.•. 43 I I I . RESULTS • •.•..........•... ~•............•........ ~ . • • . 45 A. Protoco 1 s 1 .and 2 ...: .............•....•......•....•.•....•.. ~ 45 B. Protocol 3 ............. ·...................................•.. 48 C. Protocol 4 ........ ·........................................... 51 I V. DISCUSS I 0 N. .. .. • . .. 57 A. Characterization of Preparation ••.••..•...••••...•.•.••..•••.. 57 B. · Effects of Theophylline •...••..•••...... ~ . • . • . • . • . • 61 V. SUMMARY •.•.... ~ .......•••.....•.....•........•..•..•.••.• ~ ... 65 VI. REFERENCES ....•..•..•.•.....•..•..•• ~ .•....•••..•...••.••..... 67 iv LIST OF TABLES · Table Page 1. Summary of protocols ............ o o o. o •• 0 •••• o ••• o .• 0. 0 ••• 0 0 40 2o Means and standard error for ·parameters monitored in protocols 1·and 2. 0 0 o •• o o o o. o o •• o 0. 0. o o o •• 0. o 0. 0. 0. 0 0 o, 46 3. Percent change in parameters from no work to work in protocols 1 ·and 2o. o o o. o. o. o o. o ••• o o o. o o .'o o o. o o o o o ••• o 47 4. Correlation coefficient among variables in protocols 1 and 2o.ooo.oooo•oo•••ooooooo0000000'0000000000 49 5. Means and standard error for measured parameters in protocol 3o.oo••o••oooo•oo~ooooo•o•••oo•·oooo•o••ooooo 52 6o Means and standard errors for measured parameters in protocol 4. o o o. o. o •• o ••• o o o 0 o o 0 o 0. 0. 0 o o. 0. 0 o. 0 0 ••• 0 ••• 54 7. Change from non-work to work periods in parameters " measured in protocol 4 •... oo•·o•o • .- •• ·o•o•····o••••o••· o• 56 v LIST OF FIGURES Figure Page 1. Perfusion apparatus ........................... -. ............ 37 2.· Effect of increasing a fterl oad on aortic hemodynamics, flow and adenosinerelease ..... ~ .......... -................. 50 3. Effect of theophylline on ventricular functions, oxygen utilization, coronary flow and adenosine release .... 55 vi 1. INTRODUCTION A. Statement of the Problem· The role of adenosine in the local regulati'on of coronary flow has been the subject of intense investigation since 1963 (21, 69) and much existing evidence is consistent with the concept that this nucleoside is intimately involved in (the mediation of) the matching of circulato~y oxygen delivery to myocardial metabolic needs (see literature review or published reviews 23, 24, 133, 15, 61, 191). Most recent studies have focused on the feasi bi 1i ty of adenosine mediation of coronary functional hyperemia, as it must be considered more physiologic than the ischemic or hypoxemic stresses employed in earlier investigations. Thus, adenosine levels have risen appropriately in the myocardium (129, 181), in pericardial infusates (138, 222, 9), and in coronary sinus effluent (131) when cardiac effort and myocardial oxygen 0 utilization (MV0 2) were elevated by a var_iety of means, including normal physiological stimuli (131, 222, 9). These reports support but do not prove adenosine involvement in coronary functional hyperemia. Direct proof requires documentation that interstitial adenosine concentration in the periarteriolar region varies inversely with vas~ular resistance. Unfortunately, no satisfactory method for quantitating interstitial adenosine is presently available (155). One possible way in which the i-nvolvement of adenosine in coronary functional hyperemia may be tested, without actually measuring interstitial levels, is through the use of pharmacological antagonists of adenosine's vascular activity. The methylxanthines, aminophylline and theophylline, appear to possess the property of competitive antagonism of adenosine vasodilation at the level of the vascular myocyte (38, 152) and have been used extensively to examine adenosines' role in reactive hyperemia (102, 1 2 27, 46, 103, 58, 194, 136, 131). They have also been used, less exten sively, in studies of functional hyperemia, but the results are inconsis tent (122, 130, 101). Lammerant and Becsei (122) infused aminophylline into the left circumflex coronary arteries of anesthetized dogs at a constant rate of 250 ug/.mi n and compared the changes in oxygen metabo 1ism and coronary blood flow with those observed in a control group in which no aminophylline was given. Because aminophylline infusion resulted in a 64% decrease in flow and decreased coronary sinus oxygen tension compared to control when heart rate was elevated to 153 beats/min by electrical pacing, the authors. concluded that adenosine mediation of metabolic flow regulation was supported·. McKenzie ·et al. (130) infused theophylline continuously at the rate of 19 uMoles/min into the left main coronary artery in a similar preparation. Although they reported no change in the magnitude of decrease in coronary resistance with isoproterenol injection, tissue and coronary sinus adenosine levels were increased 2-3 fold over c·ontrol. They concluded that these results indicated that the lack of flow inhibition was due to a contemporary increase _in interstitial adenosine levels. Most recently, Jones et al. (101) found no significant change in hyperemic response, oxygen utilization or tissue adenosine levels in response to norepinephrine infusion when 100 mg of aminophylline was given previously has an intravenous bolus. Since sufficient time was reportedly alloted for the alterations in cardiovascular parameters produced by this dose of aminophylline to return to control while the vasodilator effect of infused adenosine remained inhibited, these authors concluded that adenosine mediation of norepinephrine induced functional hyperemia was not indicated. 3 Interpretation of these varied results must take into account all potential cardiovascular effects of theophylline at the concentrations obtained in these studies. Although plasma theophylline levels during the test active hyperemic responses in these studies were not measured, they were likely to approximate 10- 5M or less. Available evidence indicates that besides competitively antagonizing adenosine induced coronary vaso dilation, theophylline may affect the cardiovascular system by: increasing sympathetic outflow via central nervous system stimulation (162, 180), decreasing extraneuronal inactivation of catacholamines (104), augmenting catacholamine stimulated cAMP through phosphodiesterase inhibition (8), and through direct·effects on cardiac and.vascular·myocytes th~ough cAMP· independent mechanisms (127, 116, 205, 82, 164, 180). · Rutherford et al. (180) have studied the effect of aminophylline infusion on the cardiovascular system at rates which. produce productive plasma levels not exceeding 50 uM. In conscious dogs intravenous infusion at low rates caused coronary vasoconstriction through stimulation of sympathetic outflow to the heart at the level of the CNS, whereas this vasoconstriction did not occur in anesthetized dogs or when equivalent doses were infused arterially. When the rate of intravenous infusion was doubled in the conscious animal the vasoconstriction also failed to occur. This caused the authors to conclude that, at this increased rate of theophyl- line .infusion stimulation of sympathetic outflow caused myocardial oxygen utilization to increase to a level at which metabolic vasodilation was able to override theophylline-induced sympathetic vasoconstriction. These results suggest that the vascular effect of theophylline infusion in doses productive of plasma levels not exceeding 10- 5M is dependent on the rate