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 •...••..•••...... ~ ...... • . • . • . • . . . . • 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 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, and theophylline, appear to possess the property of competitive antagonism of adenosine at the level of the vascular myocyte (38, 152) and have been used extensively to examine ' 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 i- 4 an;d route of infusion, the level of consciousness of the animal and the I rate of myocardial oxygen utilization. Considering the potential dependence of the expression of th,eophylline-induced sympathetic vasoconstriction on myocardial oxygen utilization,i it is also important to note that metabolic conditions may vary substantially in the myocardium even at the same level. of oxygen utilization, depending on the natur~ of the stimulus to MV0 2• Thts is because many stimuli to t4V0 2 have also direct vascular effects and .qualita­ tive and quantitative differ'ences in the balance of direct vascular effects and indirect vascular effects secondary to the. metabolic effects apparently exist among different means of stimulating MV0 2. Smith et al. (202), for example, found that norepinephrine produced a smaller decrease in coronary resistance than did isoproterenol at doses which produced equivalent . levels of MV0 2 in constant pressure perfused hearts of open chest dogs. They also found a significant time course lag between decreases in coronary sinus oxygen content and vascular resistance with norepinephrine but not with isoproterenol to this study. Manfredi and Sparks (126) also. found evidence for important differences among means of augmenting MV0 2. -They demonstrated that over the same range of change in coronary blood flow and . MV02 the calculated ratios of the ch~nge from control values .in adenosine release vs. coronary. conductance, coronary blood flow, and MV0 2 respectively,- were greater when MV0 2 was stimulated by norepinephrine infusion than when stimulated by atrial pacing. This suggests that adenosine production at . the same rate of MV0 2 was greater with norepinephrine infusion because norepinephrine also produces an alpha adrenergic vasoconstrictive effect while isoproterenol produces a beta adrenergic vasodilatory effect. 5

Finally, Foley et al. (65). demonstrated a significant transmyocardial adenosine gradient when MV0 2 was stimulated with isoproterenol and aortic pressure dropped due to peripheral beta adrenergic vasodilation but found . no gradient when MV0 2 was stimulated by proximal aortic constriction and proximal aortic pressure increased. This underscores the importance of perfusion pressure in the determination of myocardial adenosine metabolism during functional hyperemia. Keeping these observations in mind, there are many potenti·a 1 explanations for the varying results that have been reported in the studies of the effect of theophylline on functional hyperemia. They may be due to differences in the cardiovascular effects of theophylline secondary to the different routes and methods of infusion used, to differences in the level of consciousness of the animals, or to potential drug interactions at various sites. They may also be due to differences in the ·nature of the . stimulation to MV0 2 regarding direct vascular effects or changes in perfu- sion pressure alone or in combination with differences in the cardiovascular effects of theophylline. Isolated hearts have the advantage of being removed from extrinsic neurohumeral influences and afford conveni~nt control over hemodynamics. Thus, many potentially confusing extracardio-coronary drug actions can be eliminated in the isolated heart. Furthermore, isolated working hearts have been used extensively in the study of myocardial metabolism. Addi­ tionally, Wiedmeier and Morris (~1orris Masters Thesis~, unpublished observa­ tions) found that theophylline infusion only significantly affected the reactive hyperemic response to flow occlusion in the isolated guinea pig heart after 15 or more minutes of continuous infusion. These considerations 6 led us to test the effects of long-term theophylline infusion ·on coronary flow, adenosine release, and oxygen metabolism in isolated guinea pig hearts made to perform external pressure-volume work in hope of shedding some light on these discrepancies and on the role of adenosine in functional hyperemia. Before this was attempted, however, it was necessary to exten­ sively character'1ze the determinants of adenosine release during functional hyperemia in this preparation.

B. Literature Review 1. General Principles Termination of oxygen delivery to the myocardium results in a detectable decrease tn mechanical function with 8 sees. or 8 beats (117). This is followed by appreciable decreases in creatine phosphate at 8-10 · sees. and ATP, as·well as the other over the next few minutes (17, 62, 119). The diminished ATP is also associated with a decrease in mechanical performance (168) and is refractory to correction (168, 62). Thus, the myocardium is unable to maintain mechanical function or adequate energy metabolism in the absence of oxygen. Fortunately, ci-rculatory oxygen delivery .is able to meet myocardial oxygen need over a wide range of conditions (128, 88, 20, 157, 123) such that function limiting tissue hypoxia· occurs only in ischemia or in severe hypoxemia. This may be partly attributed to the tremendous energy generat­ ing capacity of myocardial oxidative phosphorylation, which is manifest even at very low oxygen tension (72). However, most of the ability to match oxygen delivery to oxygen need in the myocardium is undoubtedly due to the existence of potent mechanisms regulating circulatory oxygen delivery in this tissue. Indeed, in one study (43), tissue oxygen tension, as 7

determfned by cardiac myoglobin saturation using the tracer carbon monoxide technique, was found to be relatively constant (4-6 mm Hg) and no net lactate formation occurred at arterial oxygen tensions as low as 30-35 mm Hg, suggesting ae'robic metabolism even at these low P0 levels . . 2 Coronary flow is well carrel a ted with MV0 over a wide range of 2 conditions (128, 88, 201-, 57, 123) and cardiac oxygen extraction is rela­

tively high even in the absence of myocardi~l stress. Therefore,- altera­ tions in circulatory oxygen delivery are thought to be primarily dependent on changes in vascular resistance and flow. Too often, however, the tacit assumption is made that oxygen delivery cannot be significantly changed by changes in oxygen extraction. This is not correct, as an increase in extraction of up to 86-90% has been reported after maximal flow was achieved . during MV02 stimulation by dobutamine or pacing in the cons~ant pressure perfused dog heart without exceeding the cardiac aerobic limit (223). Moreover, measurement of intracapillary. distance in hypoxemia (33, 84, 85) and diffusional areas in increased MV0 2 (172) have shown capillary recruit- ment to be significant under these conditions and have suggested that it may occur with some degree of independence from alterations in arteriolar resistance. Thus, increased oxygen extraction secondary tocapillary recruitment may significantly enhance oxygen delivery to the myocardium. The increased extraction may not be reflected in coronary sinus blood . o2 because: 1) coronary sinus blood is a mixed venous blood, 2) regional heterogenieties may exist in the myocardium drained by this vessel (19, 224, 81, 110, 139, 227) and 3) flow redistrib,ution may occur (227, 12, 1_3, 14, 10, 86). Obviously, independent control. of the precapillary sphincter, if it occurs, adds a complicating consideration to the study of local . ·metabolic circulatory regulation. 8

2. Oxygen Cardiac oxygen metabolism may be related to coronary circulatory regulation through a direct effect of oxygen on the vasculature. For this to occur, oxygen tension in the vicinity of the coronary resistance vessels must be in the range at which these vessels are responsive to oxyg~n. Isolated coronary microvessels maintain tone until the P0 is less than or 2 equal to 5 mm Hg (67). Since coronary sinus oxygen tension is usually 20· mm Hg, many investig~tors have considered that a direct effect of oxygen is unlikely-to account for local metabolic circulatory regulation. This conclusion is based on the idea that coronary sinus oxygen tension is a measure of tissue oxygen tension. Data suggesting that this is not so has a 1 ready been mentioned. Furthermore, when tissue P0 2 has been measured directly wtth platinum electrodes a population of P0 2 values -has been found. to exist in the myocardium with the mode below 5 mm Hg (192, 125, 135). As significant oxygen loss may occur along the precapillary resistance ves.sels (53) it is not inconceivable that periarteriolar Pa 2· may be within the vasoactive range, although it would seem more likely that it would be closer to the arterial P0 2 than to tissue P0 2• Thus, a direct effect of low oxygen on arteriolar res-istance cannot be ruled out and certainly not a direct effect of low oxygen on capillary sphincter tone under physiologi­ cal conditions. Hypoxia would obviously be more likely to affect these functions_directly in ischemia or hypoxemia.

Meta bo 1 i tes Oxygen delivery may also be matched ,to myocardial oxygen need through the acti-on of a- secondary messenger whose metabolism is somehow related to myocardial oxygen metabolism. This concept is known as the metabolite hypothesis. Several different lists of criteria have been 9 proposed-to aid in the evaluation 9f the ca'ndidacy of potential mediator substances (156, 24, 61). That which follows is an adapted list of criteria which seem to be necessary properties of putative mediator. 1) The proposed mediator metabolite must be vasoactive. 2) The biochemical apparatus for the production of the mediator must be present in the heart. · 3) The mediator must be rapidly inactivated once_its vascular effects are produced as reoxygenated coronary sinus blood fails to produce significant vasodilation. 4) The mediator must be present in the heart under normal (non- stressed) conditions. ' 5) Mediator levels must change appropriately with changes in myo­ cardial oxygen metabolism and/or cardiac effort. 6) Mediator levels and time course changes are appropriate to pro­ duce relevant changes in vascular smooth muscle tone under phy­ siological conditions. 7) Vascular smooth muscle must have a for the mediator and the receptor must be able to effect appropriate changes in tone. 8) The action of various inhibitors and blocking agents on mediator synthesis, release, inactivation and vascular smooth muscle activity can be shown to be consistent with its hypothetical function as ·a circulatory regula tor. a) Carbon Dioxide Carbon dioxide is an end product of aerobic oxidative catabolism in cardiac muscle and its release has been correlated with coronary blood

flow ( 61). It. is therefore possible that it may mediate. changes in arteri- olar resistance in the matching of flow to myocardial metabolic need. To determine the ·feasibility of this hypothesis, the effect of arterial hypercapnia on the. coronary circulation has been studied. However, concomi- tant changes in _Mvo 2 and_perfusion pressure preclude a clear interpretation. of results in most cases. In those studies in which changes in MV0 2 and perfusion pressure were minimal, coronary vasodilation has usually been 10 seen (56, 6), indicating an independent vasodi.lating effect of hypercapnea. In at least one simila.r study, however, no change in coronary blood flow was seen even when arterial PC0 2 was doubled (213). Since coronary sinus­ oxygen tension increased at a constant sinus oxygen content in this study, it was_postulated that a vasoconstricting effect of increased tissue oxygen availability, due to a rightward shift in the oxyhemoglobin satura­ tion curve, had masked the vasodilation that might have been produced by the hypercapnea. Obviously, the vasodilatory tendency of the carbon dioxide level utilized in this study was small at best. As levels equal to two times normal arterial levels were used in this study and similar co2 levels in those studies finding altered vascular resistance produced relatively little vasodilation, C0 2 must be cons-idered a weak vasodilator. This means that unless periarteriolar PC02 significantly exceeds at least twice the normal arterial levels, carbon dioxide probably would have little effect on coronary vascular resistance. Since these levels are probably not obtained under physiological conditions, carbon dioxide most likely does not act as a physiological mediator of the matching of coronary flow to myocardial metabolic need. Studies of the effects of hypercapnea on intracapillary distance suggest that co 2 also does not mediate changes in·capillary recruitment (32). b) Hydronium Ion

Isolated coronary vessels a~e responsive to changes in ambient pH ( 171). Si nee organic acids are a1 so produced by energy metabo 1ism in the heart and increases in cardiac work are accompanied by decreases in venous pH (199), it is possible that hydronium ion changes play a role in the mediation of metabolic circulatory regulation. 11

Studies of the effects of a 1tera'ti ons in arteria 1 pH on the coronary circulation have yielded inconsistant results. This is probably . . because changes in o2 availability, co 2 levels and MV0 2, which usually occur concomitant to changes in blood pH, and also effect vascular resis­ tance, were not equal. In. a study in which hydrochloric acid was given intravenously and PC0 2 and MV0 2 were maintained constant, no change in coronary vascular resistance occurred until pH was decreased to 7.12 (208). Furthermore, a

greater decrease in vascul~r resistance was produced when pH was lowered

to a lesser extent by ventilating with 5% co 2, indicating that co 2 is a more powerful dialator than hydronium ions. This conclusion is, of course, limited in value by the fact that oxygen availability may have been greater during hydrochloric acid infusion because of a more accentuated rightward ·shift in the oxyhemoglobin saturation curve. Shifts in the oxyhemoglobin saturation curve may also account for the observations that alkalosis can result in decreased coronary vascular resistance (132).' Thus, alkalosis induced vasodilation should not be considered evidence against a vasodilatory effect of low pH. Even if an independent vasodi-lating effect of low pH is accepted, however, it would seem that physiologically relevant changes in pH produce changes in coronary vascular resistance far too sma 11 to account for a major portion of meta­ bolic flow regulation. c ) Po tass i um Relaxation .of isolated coronary vascular· strips has been observed

with elevations in suffusate K+ up to 15 rrfl1 (44, 115, 68, 146) and coronary vasodilation has occurred when perfusate K+ conce_ntration was moderately elevated in several preparations (109, 197, 39). Moreover, myocardial K+ 12

release and coronary sinus K+ levels are increased when heart rate is increased and when preload is increased (77, 141, 184). In one case (141) it was calculated that interstitial K+ concentration rose high enough to account for half of the active hyperemic response to increased heart rate. This evidence is consistent with mediation of a significant amount of + . + metabolic circulatory regulation by K • However, the release of K from the myocardium with increased effort is only a temporary phenomenon (70, 75, 206) while increased flow persists, and in most instances this transient K+ release is small.· This makes K+ mediation of the steady state active hyperemic response unlikely, although it may help produce the initial vasodilation. Release of K+ is also increased after 30 sees. of occlusion (199) / and the reactive hyperemic response to 40 sees. of occlusion is reduced by ouabain pretreatment, indicating that it may also contribute to the reactive hyperemic response when flow is occluded for 30 sees. or greater. d) Prostaglandins Several observations are consistent with a role for prostaglandins in the metabolic regulation of the coronary circulation. They are: 1) prostaglandins are known to be vasoactive, 2) the enzymes necessary for their synthesis are present in the coronary microvessels (71), 3) infusion of the prostaglandin precursor, arachidonic acid, produces coronary vasa-: dilation which is blocked by cyclo-oxygenase inhibitors (79, 89), and 4) they have been recovered from coronary venous b1 ood ( 29). Because pretreatment with cycl o-loxygenase i nhi bi tors has no -effect on reactive hyperemia or hypoxia-induced hyperemia they are thought not to be important under hypoxic conditions (18, 32). This is intuitively 13"

consistent with the fact that oxygen is required in their synthesis. ·However, it does not rule out a role in circulatory regulation under non­ hypoxic conditions. Data concerning prostaglandin metabolism during functional ·hyper­ emia under normoxic conditions is lacking. This is probably because they. are evanescent and vasoactive in extremely low levels. ·Thus, no definitive conclusion can be drawn concerning their role in physiological coronary circulatory regulation at this time. e) Adenosine Adenosine is a potent coronary dilator when infused into the coronary arteries. It has a large potential source in the adenine nucleo­ tides and, unlike the nucleotides, which are also vasoactive, may freely permeate cell membranes. Moreover, it is rapidly inactivated. These observations led investigators to begin the examination of the role of adenosine in the local metabolic control of the coronary circulation (21). e.l) Presence in Normal Tissue A compound must be present in the perivascular region in the normal, unstressed myocardium if it is to mediate the local metabolic regulation of the coronary circulation. Early attempts to detect adenosine in the unstressed myocardium were unsuccessful due to the low sensitivity of the assay method used and the presence of degradative enzymes in tissue 'and blood (97, 105). The ·imaginative perfusion of the pericardium in the open chest dog with a significant volume of saline overcame some of these

limi~ations and allowed the first indication of the presence of adenosine in the normal heart (176).· Following the development of more sensitive assay techniques, the presence of adenosine in the tissue under non­ stressed conditions was confirmed in the hearts of open chest dogs (149, 14

65, 129, 1~1) in hearts of open chest rats {22, _64) and in the guinea pig heart, in which systolic levels were found to be higher than diastolic levels (209). e.2) Adenosine and Hypoxia Because hypoxemia was known to be one of the most powerful stimuli to coronary flow, _i ni ti a 1 experi mentation concerned the effects of hypoxemia _ on adenosine metabolism. Again, using relatively insensitiv,e assay methods by present day standards, early investigators were able to demonstrate increased coronary sinus and levels following 15 min periods of hypoxic perfusion in the isolated Tyrodes perfused cat heart in following similar ·periods of asphyxia in the .i!!. vivo dog heart (21). As these compounds are metabolites of adenosine, through the actions of the enzymes adenosfne deaminase and nucleoside phosphorylase. respectively, this study was interpreted as providing indirect evidence for increased adenosine release in severe tissue hypoxia. However, alternate pathways for their production from IMP were also known to exist. To prove that these metabolites were produced from adenosine breakdown, the effects of the adenosine deaminase inhibitor 8-azag-uanine was studied in isolated rabbit hearts subjected to 20 min of hypoxic perfusion (169). While no adenosine was detected in non-hypoxic, 8-azaguanine treated hearts, or in hypoxic hearts not treated with this compound, appreciable amounts of adenosine were detected in the sinus effluent of hypoxic, 8-azaguanine treated hearts; indicating- that adenosine release was increased in long­ term hypoxic perfusion but that it was rapidly inactivated as previously proposed. In subsequent studies, in which an improved assay technique was employed, adenosine levels were shown to increase in the tissue (48, 49, 15

178, 189), in the coronary sinus effluent (20), and in pericardial washings ( 176) with 1ess severe degr.ees of tissue hypoxia. Furthermore, the amount of adenosine release was found to·be related to the degree of arterial hypoxia in the isolated saline perfused guinea pig heart (178). This suggests that adenosine produc~ion and release parallels the degree of tissue hypo xi a. e.3) Adenosine and Ischemia Studies of the effect of ischemia on adenosine metabolism have progressed along liAes parallel to. those involving hypoxemia. Early efforts demonstrated that tissue adenosine and adenosine metabolites were increased only after relatively long periods of ischemia {50-60 min.) (69, 22, 97) and a more recent study found increased adenosine after far shorter periods of occlusion (5-15 sees.) (149). A linear relationship between tissue adenosine levels and the number of heart beats during occlusion, was also obtained in this later study, suggesting that adenosine production in ischemia is related to energy utilization by the contractile process - ( 149). Another recent study found significant subendocardial to subepi­ cardial adenosine gradients when flow was reduced to 50% of control (65). Since the subendocardium is more susceptible to ischemia, this later observation provided further evidence that tissue adenosine production is quantitatively related to the degree of ischemia. These ischemic studies along with the previously mentioned studies of hypoxemia indicate that adenosine production in the myocardium is related to the degree of tissue hypoxia. e.4) Reactive Hyperemia and Adenosine If a metabolite is responsible for local coronary circulatory regulation its coronary sinus levels might parallel post occlusive reactive 16

hyperemic flow rates. Adenosine produces vasoconstriction in the kidney. Using it as an assay organ, vasoconstriction occurs upon its perfusion with coronary sinus effluent blood following 20 to 40 sec coronary artery occlusions and this vasoconstriction is prevented by pretreatment with adenosine deaminase (200, 198) .. This indicates that adenosine is present in the reactive hyperemic effluent in vasoactive amounts when the coronary artery flow is occluded for greater than 20 sees. This observation has been confirmed by direct measurement of adenosine in sinus blood (177) and extended to occlusions as short as 5 sees (T53). In this later study, coronary sinus adenosine levels were found to decrease exponentially with reactive hyperemic flow. These studies are consistent with the mediation of at least a portion of reactive hyperemic flow by adenosine when occlusion is greater than or equal to 5 sees. e.5) Adenosine and Functional Hyperemia Interstitial adenosine concentration should increase in parallel with vascular conductance when cardiac energy utilization increases relative to oxygen delivery, if adenosine mediates. functional hyperemic vasodilation . Myocardial energy utilization and MV0 2 may be increased by a number of different means. One of these is through cardiac adrenergic stimulation by pharmacological agents such as epinephrine, norepinephrine and isoproterenol. Epinephrine infusion in Langendorf perfused cat (105) and . guinea pig hearts (226, 49) has resulted in increased adenosine release into coronary sinus effluent (105, 226) and in increased tissue adenosine

levels (49). Similar results h~ve been obtained in norepinephrine infusion in Langendorf perfused hearts (226, 105) and in intact blood perfused dog hearts (126) and also in isoproterenol infusion in this later preparation ( 130). 17

Cardiac performance and· energy utilization may also be increased by increasing left ventricular afterload. When left ventricular afterload was increased by proximal aortic constriction in the rat (64) and dog (130) increased blood flow (64) and increased vascular conductance (130) were accompanied by increased tissue adenosine 1eve 1s ~ Similarly, adenosine. concentration incre~sed in pericardial infusate fluid when MV0 2 was augmented by stellate ganglion stimulation (138, 222) and tissue adenosine increased during pacing in the dog heart- ( 181). Finally, recent reports-demonstrated increased coronary effluent adenosine levels in treadmill exer.cise (222) and increased adenosine concentration in pericardial was~ings in treadmill exercise and in excite~ ment caused by loud noises or feeding (10) in the dog. As these means of elevating cardiac energy utilization are relevant physiological stimuli, these studies indicate that increased myocardial adenosine production and release may mediate increases in vascular conductance in functional hyper­ emia when the myocardium is normally oxygenated and thus may mediate the physiological matching of oxygen delivery to myocardial oxygen need. e.6) Quantita·tive Evaluation of Adenosine Mediation The literature thu·s far reviewed shows that: 1) adenosine is vasoactive, 2) it is present in the normal, non-stressed heart, 3) it is rapidly inactivated in the heart, 4) levels in tissue, pericardial infusates and/or coronary sinus levels change in a directionally appropriate manner in response to increased energy utilization and to hypoxemic and ischemic stresses, and 5) the magnitude of these changes is related to the severity of the stress imposed. 18

However, a quantitative determination of the role of adenosine as the physiological mediator of the matchfng of circulatory oxygen delivery to myocardial oxygen need requires that interstitial adenosine levels, which bathe the vascular smooth muscle, be shown as appropriate to account for the changes in tone occurring in physiological states. To do this the dose response relationship between adenosine concentration and vascular tone and the interstitial adenosine levels during physiological states must be known. e.6.a) Dose Response Relationships

Infusion of adenosine in isolated guinea pig, ra~bbit, cat and rat hearts causes bradycardia (113, 161, 186, 225, 36) and decreased contrac­ tility (35, 76, 100, 212, 220, 225). Given intravenously in intact animals it can produce bradycardia and decreased contractility (35, 76, 100, 212, 225) or tachycardia (4, 90, 121, 120, 175) and increased contractility .(90, 121, 120). In cases where tachycardia and increased contractility were reported these were thought to be secondary reflex effects caused by adenosine-induced peripheral vasodilation, as they were lessened by intra­ coronary infusion and were blunted by beta adrenergic blockade (90, 120). This 'means that the direct cardiac effects of adenosine are negative chronotropism and negative inotropism. Adenosine infusion uniformly produces coronary vasodilation. This vasodilation occurs in. excess of any that might result from reflex stimulated increases in MV0 2, as coronary sinus oxygen content increases sharply with infusion (121, 165, 175); and is opposed to any flow reductions which might occur, as a result of its direct negative chronotropic and inotropic effects. It produces vasodilation in isolated guinea pig hearts 19 in concentrations 1/100 of that which is·required to cause bradycardia (186). These data show that adenosine's direct coronary effects are stronger than its direct or reflex stimulated cardiac effects, as must be the case for the endogenous mediator of local metabolic circulatory control. Adenosine infusion has no appreciable effect on small vessel blood volume {45). As most of this volume consists of capillary volume, adenosine probably has little or no effect on capillary recruitment in the heart. There is, however, evidence that it may effect capillary recruitment in skeletal muscle {92). Adenosine infusion produces maximum flow rates approximating peak reactive hyperemic flow in a plasma concentration of approximately 0.56 uM (177, 221, 154). Since adenosine infusion results in a more potent vaso­ dilation in_ the presence of reduced pH or elevated· PC0 2 (166), interstitial adenosine concentration during peak reactive hyperemic flow should approxi­ mate or be somewhat lower than 0.56 urn. As maximum vasodilation probably occurred in peak reactive hyperemia in these studies, interstitial adenosine might be expected to be proportionally less in conditions in which vascular dilation is proportionally less. This, of course, assumes that exogenous and endogenous adenosine act with equal efficacy. e.6.b)- Interstitial Adenosine The perivascular adenosine concentration is dependent on its rate of delivery and its rate of removal from that compartment. The rate of delivery will be dependent on the rate and location of adenosine production and the permeabilities of any barriers that exist between the compartment where adenosine is produced and the perivascular compartment. The rate of removal will be dependent on the rate and location of inactivation or uptake, the rate of circulatory washout and the permeabilities of any 20 barriers which separate the perivascular compartment from the compartments where these respective activities take p.lace. e.6.c) Adenosine Production The enzyme 5' nucleotidase can catalyze the formation of adenosine from AMP. Its location in the heart may be approximated by histochemical studies in which tissue is incubated with AMP and inorganic phosphate release is trapped by lead. Such studies have yielded inconsi·stent results and interpretations; with some reporting 5' nucleotidase activity in myocardial T- tubules,_ sarcolemma, intercalated discs, and flattened sarcoplasmic reticulum (231), some indicating it resides on capillary endothelial cells on the luminal side (142) and some reporting it most prominently in the vascular pericyte (30, 31). Some investigators have proposed that the vascular pericyte serves as the sou.rce of mediator adenosine (30) based on their histochemical studies, but this proposal has not been well received, nor well pursued. It is interesting to note, however, that coronary endothelial cells grown in culture release adenosine during hypoxia and thus may contribute to overall cardiac adenosine release in hypoxia. This means that coronary sinus adenosine release should be somehow corrected for endothelial release before it is used to estimate adenosine levels at the perivascular· compa-rt­ ment in hypoxic conditions. More recently, evidence has been presented that cardiac 5' nucleo­ tidase is an ectozyme, acting on substrate outside of the cell membrane, and that inhibition of this enzyme with alpha, beta methylene adenosine 5' diphosphate (AOPCP) does not decrease adenosine release or coronary vasodi­ lation in hypoxia in isolated guinea pig hearts (195). These observations ·21 suggest that ectozyme 5• nucleotidase is not responsible for hypoxic adenosine production.

Failure of ectozyme 5 1 nucleotidase .inhibition by AOPCP to inhibit hypoxic adenosine release has been confirmed in other studies (190, 195). Since AOPCP does not enter the cell and since it was also noted in these studies that inhibition of nucleoside transport by 4-nitrobenzyl thioinosine (NBMPR) decreased both adenosine release and mYOcardial loss, it was concluded that the adenosine released in hypoxia was produced intracel­ lularly via either cytosolic 51 nucleotidase or from dissociation from the S-adenosylhomocysteine hydrolase complex. The potential adenosine produc­ tion via dissociation from S-adenosylhomocysteine hydrolase was proposed to be determined by the extent of cellular methylation. Lastly, it was concluded in these studies that tissue adenosine levels do not necessarily reflect the free and active concentration of this nucleoside due to signifi­ cant intracellular sequestration of adenosine by S-adenosylhomocysteine hydro 1ase. Adenosine sequestration by S-adenosylhomocystein hydrolase has been confirmed in a·n additional study (155). However, further evidence was also presented in this study mitigating against production of adenosine ·in hypoxia via dissociation from this complex. Extracellular adenosine was esti rna ted to be only 6% of tota 1 tissue 1e~el s in .this study. It is unclear at this time, however, if the ratio between intra-cellular and extracellular.adenosine is constant or conditionally variable; precluding utilization of this percentage to estimate interstitial adenosine levels based on tissue levels.- 22

e.6.d) Adenosine Inactivation

Adenosine inactivation requires its cell~lar uptake from the interstitium. Following this, it may be rephosphorylated to AMP by adeno­ sine kinase, sequestered by S-adenosyl homocysteine·· hydrolase,. or deami na ted to inosine by adenosine deaminase. Adenosine enters cells by simple and facilitated diffusion. Thi-s has been observed in red blood cells (114, 172, 188, 211), and myocardial cells (42, 151, 150). Since adenosine deaminase is ubiquitous in the heart, being p~esent in cardiac, endothelial, and red blood cells (11 ~ 41, 170) and since adenosine levels are small in the coronary effluent, one might expect that uptake and deamination by the vascular endothelium and red.cells would account for most of adenosine inactivation. However, the major mode of adenosine inactivation in the heart appears to be uptake into cardiac cells (45, 150, 174) followed by phosphorylation to AMP (124, 143), although recent evidence indicates that sequestration in the S-adenosylhomocysteine hydrolase complex may be quantit~tively significant {190, 195).· e.6.e) Other Measures of Interstitial Adenosine

The evidence cited ~hus far has implied that tissue adenosine levels may not be an accurate measure of adenosine -activity at the level of vascular smooth muscle and that coronary venous adenosine levels are at least partially determined by endothelial adenosine release in hypoxia. Other means of estimating interstitial adenosine levels have therefore been sought. Use of cardiac lymph has been found to underestimate these levels because of the presence of adenosine deaminase in lymphatic endothelium ( 24). Infusion of fluid val umes into the peri cardia 1 space has a 1so been 23

tried, .but this method has been shown to yield quantitatively useful results only when the infusate fluid is allowed to equilibrate for 30 min. or longer (155). However, it seems reasonable that lesser periods of· equilibration could be useful in detecting qualitative changes. e.7) Adenosine Vascular Receptor and Mechanism of Action Adenosine analogues have been used to study how adenosine might affect vascular smooth muscle tone {5, 6, 42, 60, 167, 193, 203, 228). Their pattern of potency suggests a specific receptor (154) that is similar in ligand specificity to adenylate cyclase associated Ra receptors (156) and is different than the adenosine carrier in facilitated diffusion. This, along with the observations that adenosine relaxation in isolated coronary vascular strips is correlated with cAMP concentration (118) and is potentiated by phosphodiesterase inhibitors (118, 144) when suffusate adenosine concentration is 0.1 nf.'1 has been interpreted as supporting a cAMP mediation of adenosine relaxation. Feigl (61), however, has ·rightly pointed out tha.t interstitial adenosine concentration is more likely in , the micromolar range.and has questioned whether the correlation between adenosine action and cAMP represents a specific action involved in coronary vasodilation or a nonspecific action found with high adenosine levels. An alternative hypothesis is that ade.nosine relaxation occurs through blockage of.entry of Ca++ into the vascular smooth cell. This is supported by studies which show that adenosine blocks the slow inwa.rd current of vascular myocytes and that adenosine blocks radioisotopic Ca++ entry but not egress from rat aortic myocytes (156). e.8) Adenosine Inhibitors and Blocking Agents As stated above, the effects of various inhibitors and blocking agents on the coronary circulation must be consistent with their effects 24 on mediator synthesis, release, inactivation and v~scular smooth muscle activity if the putative mediator functions as a circulatory regulator. Such compounds have been used to evaluate adenosine mediation of .local circulatory regulation and have included , lidoflazine, adeno­ sine deaminase, and the theophylline based methylxanthenes. e.8.a) Dipyridamole Dipyridamole (DP) retards the entrance of adenosine into RBCs (27, 118, 204), platelets (204), endothelial cells (153), and myocardial cells (94, 93, 112) and to a lesser extent vascular smooth muscle cells.· (153). It also inhibits adenosine deaminase (27, 50, 51, 157), but only in concentrations far greater than those required to inhibit transport

(61). This.is consistent with the observ~tion that DP prevents adenosine degradation to a far··greater extent in whole blood than in hemolyzed blood. DP relaxes isolated coronary vascular strips in the absence of myocardial adenosine (61). Therefore, unless it enhances vascular adenosine formation (for which there is no evidence.), it must have an independent vascular effect. Pretreatment with DP enhances the vasodilator response to ad·enosine infusion (1, 34, 80, 147). This may be secondary to DP enhancement of· perivascular adenosine levels due to inhibition of cellular uptake or may be a result of synergism at the level of the smooth muscle cell. Intracoronary DP infusion produces marked vasodilation (61). Possible mechanisms proposed to account for this effect must take into Gonsideration the aforementioned possible independent vascular effects, effects on perivascular adenosine activity, as well as possible adenosine DP synergism. Similarly, adjudication of adenosine mediation of various potential manifestations of local metabolic flow regulation mandates consideration of these possible DP actions. 25

DP infusion has been found to increase the reactive hyperemic response to octlusions greater than 30 sees in duration (25, 47~ 103, 140) but appears not to affect it when occlusion is 10 sees. or less (26, 158, 104). This may mean that adenosine is not involved_ in the reactive hyper­ emic r~sponse when occlusions are_lO sees. or less. A clear interpretation, however, cannot be made because these studies gave no indication of how adenosine ·levels were changed· by these procedures. Thus, it is possible that DP has no significant effect on perivascular adenosine activity after short occlusions but causes an increase in these levels after longer occlusions. It is also possible that the same dose of DP enhances the vascular response to higher perivascular adenosine levels than to ·lower levels. Adenosine mediation of reactive hyperemic vasodilation after short periods of occlusion cannot-be ruled out on the basis of present evidence because of this. Infusion of DP in doses thought not to i'nhibit adenosine deaminase results in augmentation of hypoxic increase in tissue adenosine (47, 69) and slows the rate of hydrolysis of ATP (91). It also has been reported to reduce venous adenosine, inosine, and hypoxanthine release in response to hypoxia (235). This has been implied to be inconsistent with adenosine mediation of hypoxic vasodilation, as one would have to postulate a DP dependent rectification of transcapillary adenosine diffusion in order to account for this result, as well as DP potentiation of the vasodilator response to infused adenosine, and this diffusion is thought to occur through water-filled clefts in between capillary endothelial cells (61). However, it will be recalled that endothelial cells may be responsible for a signif~cant portion of the intravascular adenosine content in hypoxia 26 and DP may inhibit adenosine transport out of endothelial cells. At least some of the reduction in venous adenosine levels in hypoxia when DP is infused nay be accounted for on this basis. DP had no effect on flow augmentation caused by catacho)amine infusion (160). Again a c-lear interpretation of this result must await a clearer understanding of the actions of DP and on determination of inter­ stitial adenosine levels. e.8.b) Lidoflazine Li dofl azi ne (LF) is thought to be simi 1a r to DP ( 61). It retards the entry of adenosin·e into RBCs (187, 213) and into isolated guinea pig hearts (232) and inhibits the degradation of this nucleoside in whole blood (2, 214). It also potentiates the vasodilator effect of infused adenosine (2, 27, 99, 187) and has a vasodilatory effect when infused into the coronary vessels (98, 185, 207; 210, 217, 219). Similar considerations of potential actions of LF, as were made for DP, must-be entertained before conclusions can be drawn from studies of the effects of LF infusion on manifestations of local metabolic control of coronary flow. The effects of LF on reactive hyperemia has been studied but results are conflicting. LF pretreatment increased reactive hyperemic flow in response to occlusions greater than or equal to 10 sees. in pigs (223) and greater than 30 sec. occlusions in dogs in at least one report (104). However, another report failed to detect any increase in reactive hyperemic response to 30-120 sec. occlusions with LF in the dog (21). Yet another study found that LF infusion had no effect on vascular response to hypoxemic or occlusive stresses (3). Obviously, further study is needed before conclusions concerning adenosine mediation of these events may be rightly made. 27

e.8.c) Adenosine Deaminase Infusion of the enzyme· adenosine deaminase results in a 35% reduction· in rective hyperemic flow volume after occlusions lasting from 5-30 sees {182). As radiolabelled adenosine deaminase has been detected in cardiac lymph nodes after arteria 1 infusion of the tagged enzyme, significant adenosine deaminase activity was likely introduced into the interstitium by this procedure (186). This suggests that at least 35% of the reactive hyperemic flow after these periods of occlusions is adenosine· mediated. Baseline coronary flow was not appreciably altered in response to enzyme infusion in this study, however. This means that either adenosine is not vasoactive under resting conditions in the preparation used ~r that the adenosine deaminase had a proportionally greater effect on the vaso­ active adenosine in reactive hyperemia. This may be explained if one considers that the myocardium's reserve capability for producing and releasing more adenosine is likely to be greater under resting conditions than after coronary artery occlusion. In any case, measurement of tissu'e or coronary sinus adenosine levels during these procedures would be helpful. e.8.d) Methylxanthines Theophylline is a methylxanthine compound. Aminophylline is a theophylline derivative with superior aqueous solubility because of the addition of an ethylene diamine group, which is thought to be pharmacologi­ cally inert. These compounds have a great diversity of pharmacological­ effects and have received widespread clinical and experimental utilization. They apparently affect the cardiovascular system at multiple levels including: 1) increasing sympathetic outflow through CNS stimulation (160, 180) or through increased transmission in sympathetic ganglia (52, 28

95), 2) increasing catacholamine levels at the effector through decreased extraneuronal inactivation (104), 3) augmenting catacholamine dependent increases in cAMP through phosphodiesterase inhibition (8, 180), andA) by direct effects. on cardiac and vascular IT\YOCytes through cAMP independent mechanisms (8, 180, 116, 205). These compounds are also known to retard adenosine uptake in some tissues (17, 163, 230) but not in cardiac tissue (232) inhibit sarcolemal s• nucleotidase at concentrations greater than or equal to 10- 5M (87) and not to affect adenosine inactivation in whole blood or heart-homogenates (4).

e.8~d.l) Determinants of Cardiov~scular Effects Theophylline has been noted to produce increased contractility in isolated papillary muscles in doses as low as 2.5 uM (127). However, this. effect is largely dependent on the presence and release of endogenous beta adrenergtc agoni sts, as it is reduced by beta b1 ockade or prior reserpinization. The part of the increase in contractility blocked by reserpinization or beta antagonists may be caused by a theophylline induced

decrease in extraneuronal degradation (104), or throug~ its phosphodiesterase i nhi bi ti on and augmentati-on of ca tachol amine induced increases in cAMP (8). The rest must be due to direct effects ·on the cardiac myocyte. At doses of 0.1 rrM, theophylline has been shown to augment the atrial response to norepinephrine in the rabbit (139). Similar doses have been found to increase cAMP and cAMP-dependent protein kinase activity, independent of catacholamines, in the Langendorf perfused guinea pig heart,

. but catacholamine independent increases in contractfl ity require 1 rrM - levels in this preparation (8). The increased contractility seen at this 29

higher dose has been dissociated from increases in cAMP (8) and is accom­ panied by an increase in ca++ influx (116) during the cardiac action potential and decreased sarcolemal Ca++ transport {205). It may be depen­ dent on Ca ++ stimulation of phosphorylase activity (218) and phos-phorylation of troponin I resulting in increased Ca++ sensitivity (180). The coronary circul.ation in isolated guinea pig hearts exhibits its full range of vasodilator response to adenosine over the doses of 10- 7 to 10- 5M (38). Theophylline also produces vasodilation in this preparation, . -4 -3 . but the dose required 1s 10 to 10 M, and the response is not as ~reat as to adenosine (38). When theophylline is infused- in less than vasodila­ tory doses, (10- 6 to 10- 5M), it inhibits the vasodilator effect of infused adenosine in a competitive manner {38). Similar competitive inhibition of adenosine dilation by theophylline has been found when these.compounds were linked to large molecules to produce compounds too large to penetrate cell membranes (152). This suggests that these compounds may compete for the same receptor on the surface of the vascular myocyte. Inhibition of adenosine dilation by theophylline is also seen in blood perfused hearts in doses as low as 200 ug/min (160). Studies of the effects of adenosine and theophylline on isolated hog aorta vascular strips contracted with norepinephrine or K+ have demon­ strated· a Ca++ sensitive vasodilation (10- 6, 10- 5M respectively) in doses not producing appreciable alterations in cAMP levels (83). This suggests that the vasodilation produced by these compounds does not occur via a cAMP mediated mechanism. Interestingly, however, rather than a competitive inhibition of adenosine relaxation when these compounds were present simultaneously, augmented relaxation was seen. This may mean that theophyl- 30

line inhibition of adenosine dilation occurs at a level other than at vascular smooth muscle. Also of interest are the observations that doses of theophylline in the 10- 5M range have also been observed ~o augment norepinephrine cont,raction. in the aorta and coronary arteries (104), presumably due to decreased extraneuronal catacholamine uptake. The reason for relaxation in one instance and contraction in other instances at approximately the same dose of theophylline is not clear but may be due to difference in species or differences in the preparations used. The effects of infusion of aminophylline intravenously in doses thought to produce systemic levels not exceeding 50 uM have been studied in the conscious dog ( 180). When aminophylline was i n.fused at a rate of 1 ·

mg/kg/min for 10 min, increased cardiac performance, a small incre~se in heart rate and coronary vasoconstriction resulted. Since the increase in

_cardiac performance and heart rate ~as partially blocked by propranolol or prior reserpinization and the vasoconstriction was blocked by phenotolamine, beta and alpha adrenergic mechanisms were thought to be respectively responsible. Theoretically, this could have been due to increased sympathe­ tic outflow because of a CNS stimulation (160), increased transmission in sympathetic ganglia (52, 95"), increased catacholamine availability via a direct effect at the effector level (104), or augmentation of catacholamine induced cAMP. Because arterial infusion at a rate thought to be equivalent produced vasodilation in another vascular bed which was also constricted by intravenous infusion, stimulation of the ANS was thought to be the mechanism responsible for the vasoconstriction. Lack of complete blockage of theophylline induced increased contractility in doses not .31

thought to stimulate cAMP was thought to suggest that a portion of the increased performance was due to a direct,. cAMP independent, theophylline effect. When infused at 2.5 mg/kg/min, ·more pronounced increases in cardiac performance ·and heart rate were produced and vasodilation resulted. This implies a dose dependency of cardiovascular effects of theophylline in the 10- 6 to 10- 5M range in consci.ous animals. Presumably through accentuation of effects at different points controlling cardiovascular function at different doses. Since doses roughly equivalent to 1 mg/kg/min in. anesthetized animals (180), produce increased cardiac performance and vasodilation, aminophylline vasoconstriction in awake animals would seem to be mediated through stimulation of sympathetic outflow at the level of the CNS. The cardiovascular effects of theophylline are temporary, lasting from 20 to 30 min (61). The reason for this tachyphylaxis is not known. The above data show that the cardiovascular effects of the methyl­ xa nthi nes may be dependent on the sum tota 1 of their effects at many different levels and that these may at times be in conflict. Factors· affecting the final outcome are: the species and preparation used, presence .or absence of anesthesia, dose, route and method of administration, adrener­ gic .status during drug infusion, and time. These factors should be ·taken into account in the interpretation of studie.s attempting to evaluate the prospective candidacy of adenosine as a mediator of local flow regulation based on theophylline competition with adenosine at the vascular level. e.8.d.2) Reactive Hyperemia As mentioned above, one of the actions of theophylline or amino- phylline is a competitive type inhibition of the vasodilator action of 32 infused adenosine, presumably at the level of a vascular smooth muscle receptor. Infusion of these compounds has therefore been used to evaluate the proposed role of-adenosine in the mediation of several manifestations of local coronary flow regulation. The effects of theophylline or aminophylline infusion on the reactive hyperemic response to arterial occlusion has been most frequently studied. However, reports are 'conflicting. Some have found the reactive hyperemic response to be diminished (46, 194, 136). In one of these studies the reduction in reactive hyperemia was comparable to that seen with adenosine infusion (46), but most cases reporting a diminution of reactive hyperemia found it to be less than that seen with adenosine infusion (194, 136). Most investig-ators have found reactive hyperemia unchanged by theophylline (102, 27, 103, 74) and one study (58) reported a time dependent increased response. It is difficult to reconcile these differences in terms of divergent drug actions, as experimental conditions varied widely. Also, unfortunately, circulating drug concentration (102, 27, 46, 103, 58, 74), and cardiac, vascular (27, 46) or metabolic (103, 27, 46,, 58, 74) effects of drug infusion, at the time of occlusion and afterward, were inadequately documented in many cases. Thus, other pharmacological actions of these compounds, which might potentially effect the reactive hyperemic response to occlusion cannot be ruled out in many instances. Also, based on an extensive description of reactive hyperemic responses to variable periods of occlusion (103), the reactive hyperemic response was i-nsuffi­ ciently characteriied, in at least one instance (27) to determine the drug effect on this response. Furthermore, the effects of drug infusion on 33 adenosine metabolism during occlusion or the hyperemic response has never been reported. This means that the results of studies published to date should not be regarded as evidence against adenosine mediation as changes in adenosine metabolism may have occurred which might account for them. It should also be again noted that other factors, such as H+, K+, co , o , 2 2 and even myogenic factors, may play a significant role in this response. Indeed, they may potentiate the effects of adenosine but not theophylline inhibition {136, 137). The effect of continuous long-term (40 min) intracoronary infusion of 25 ug/ml of aminoph~lline on the reactive hyperemic response in anesthe­ tized dogs was recently studied while heart rate, arterial blood pressure, left ventricular. pressure and dP/dt, and coronary flow were continually monitored and MV0 2 calculated by the Bretscheiden method {194). Although this rate of infusion produced no change in .the measured indices of cardiac performance, coronary flow, or calculated MV02, there was a twenty percent decrease in the hyperemic~response to coronary artery occlusions lasting 10 beats or more. This must be considered as the most complete study of the effect of aminophylline in coronary reactive hyperemia published .to date. It is consistent with unpublished observations (Wiedmeier and Mor.ris) that long-term tnfusion of theophylline in doses without appreciable cardiac or coronary effect results in significant diminution of reactive hyperemia. e.8.d.3) Hypoxemic Hyperemia Fewer studies have examined the effects of aminophylline or theophylline on hypoxemic hyperemia (234, 137). One early study is parti­ cularly complete (234). In this study, heart rate, arterial blood 34 pressure, cardiac .output, coronary sinus flow, arterial and coronary sinus· ~2 content, and MV0 2 were .continuously monitored and the effects of amino- phylline on these parameters during control and hypoxic ventilation in the anesthetized dog determined. Aminophylline (7.5 mg/kg) was given as a bolus via the right atrium. The cardiovascular effects of this infusion were allowed to return to control and remain that way for approx-imately 15 min before hypoxic ventilation was repeated. No significant change in peak hypoxemic coronary flow was seen after aminophylline. However, several other findings bear mention. Although hemodynamic parameters were not significantly changed in control ventilation in. the presence of aminophyl-' line, oxygen extraction was increased as was MV0 . Additionally, heart 2 . rate, blood pressure, cardiac output, oxygen extraction and were MV0 2 increased in hypoxic .ventilation in the presence of aminophylline. Since blood pressure and MV0 2 were increased in this instance, flow should have also increased, unless the vascular bed was maximally dilated in hypoxic ventilation without aminophylline. It certainly was not maximally dilated

in control ventilation, however, and coronary sinus o was also de~reased . 2 and increased with aminophylline in this instance. Although a sympa- MV0 2 . thetic vasoconstriction might have occurred, accounting for the decrease in oxygen content, it seems unlikely as anesthetized animals were used. Thus, these data suggest that. aminophylline decreased the proportionality between coronary flow and MV02. This means that there was a relative inhibition of flow in the presence of this compound. Perhaps failure to document an actual decrease in flow was due to the use of peak hypoxemic flow rather than volume flow during. hypoxic ventilation. The other studies reported to date have also failed to find hypoxemic hyperemia to be affected by aminophylline or theophylline (137). 35 . However, oxygen extraction and MV0 2 were not monitored in either of these studies and cardiac performance was not well characterized in one case (137). These shortcomings and the lack of characterization of experimental effects on adenosine metabolism preclude definitive conclusions relevant to adenosine mediation of hypoxemic vasodilation._· e.8.d.4) Functional Hyperemia Similarly, conflicting results have been reported from studies of ·the effects of aminophylline on coronary functional hyperemia (122, 130, · 101). These studies have been reviewed in the introduction. II. MATERIALS AND METHODS A. Apparatus The isolated working heart preparation used was essentially a modification of the previously described preparations of Neely et al. (145) or Bunger et al. (39). This modification (Figure 1) consisted of a system of two one-way ball valves and three columns of oxygenated perfusion fluid. Alterations in left ventricular afterload, minimum diastolic aortic pressure, and 1eft ventri cu.l a r fi 11 i ng pressure were achieved by varying the height of fluid in columns B, A, and C respectively. Because of the ball .valve in column A, cardiac output could not be ejected through column A and intraventrictilar pressure had to exceed. that existing in column B before ejection could occur. The volume of fluid in this column was kept constant as was the volume of air in the aortic "Windkessal" chamber. This meant that the component of left ventri­ cular afterload due to fluid inertia was constant in the face of alterations in the height of column B and that changes in the height of column B changed the pressure against which cardiac ejection had to occur and also altered the compliance characteristics of the outflow tract for ejection. Increasing the height of col.umn B, for example, increased this pressure and decreased the compliance of the outflow tract for the cardiac output; thus increasing left ventricular afterload. Because of the ball valve in column B', diastolic aortic pressure could fall below the pressure in this column to a minimum level equal to that in column A. At a constant afterload, left ventricular filling pressure was determined by the height of column C.

36 Figure 1. Perfusion apparatus. Filling pressure of left heart chambers determined b hei ht of fluid in "venous reservoir" column C . Pressure against which left ventricle had to eject fluid determined by height of column Band minimum possible diastolic aortic pressure determined by height of column A due to presence of one-way ball valves 38

The check valves consisted ·of glass balls 4 mm i·n diameter and glass seats. They were obtained from L/I Repipetes and were connected together arid to an aortic cannula by a 11 Y11 -shaped tube with an inner diameter of 4 mm rrade -of pyrex glass. The smallest diameter in the outflow tract for the cardiac output was 1.5 mm in the aortic cannula. Total volume in the apparatus between the aortic semilunar valves and the valves in the perfusion apparatus was 1 ml. Electromagnetic flow probes (Statham model M 4001) positioned as depicted in Figure 1 permitted the continuous monitoring of cardiac output and coronary flow. Pressure in the aortic cannula was measured via a side arm connected to a Statham model P23Dc pressure transducer in protocols 1 and 2. This pressure transduc·er was connected to a 20 gauge needle inserted

into the left ventricle through the apex in protocols_3 and 4, thus permi~ ting the measurement of left ventricular pressure and dP/dt in these protocols. Input from the pressure transducer was channeled through a differentiator (Grass polygraph model 7P20C) for the measurement of dP/dt. -Flow and pressure parameters were continuously recorded (Grass model 7D _polygraph recorder). Heart rate was determined-from these pulsatile tracings. B. Isolation and Perfusion of Hearts Guinea pigs of either sex, weighing between 200-400 g, were stunned by a blow to the back of the head, the heart and lungs removed as a unit, and placed in an i-ced bath of saline to reduce the asphyxial effects of isolation. While in the bath the pulmonary veins were quickly ligated. The aorta was then cannulated and modified Langendorf perfusion

begun at a pressure of 45_ em H20. 39

The perfusion fluid was Krebs-Henseleit solution containing: 118 mM NaCl; 4.6 mM KCl; 27 mM NaHC03; 2.4 mM CaC1 2; 0.5 mM MgS04; 0.2 mM KH 2Po4; and 5.5 rrM glucose., equilibrated with a 95% 02, 5% co 2 gas mixture at pH 7.4 and 37C. This identical fluid occupied columns A, Band C. Once perfusion was initiated the lungs were removed distal to the ligatures on the pulmonary veins and the pulmonary artery incised to permit venting of the right heart. A polyethylene cannula, filled with perfusion fluid and connected to column C was then inserted through a small incision in the left atrial appendage and securely tied in place. In protocol 4 an additional polyethylene catheter was positioned in the coronary sinus and tied in place. Total time elapsed from the blow on the head until initiation of perfusion was one minute or less. Elapsed time to completion of the entire preparation was under ten minutes. C. Protocols 1. Protocol 1

Parameters me~sured and a time frame diagram for the different protocols used are summarized in Table 1. Hearts prepared as above were allowed to equilibrate for 25 minutes in order to recover from the short period of asphyxia. Coronary effluent was then collected, in an ·ice cold receptical for five minutes and saved for later analysis. This period, during which no external cardiac work was done, was defined as the non­ work period. At the end of this period a clamp on the line connecting column C (venous reservoir) to the left atrial cannula was released allowing filling of the left chambers of the heart and the ejection of fluid from the left 40

Table 1 Summary of Protocols Protocol 1 Protocol 2 Protocol 3 Protocol 4 (vary B) (vary B) (vary A) (Thea.) Column Heights (in em of water) A 45 45 45/60 45 ( mi n • press . ) B 70/90 50/100 100 100 (a fterl oad) c 10 10 10 10 ( fi 11 i ng press.) Measured Parameters Aortic Pressures yes yes no no Ventricular Press. no no yes yes dP/dt no no yes yes Oxygen Utilization no no no Yes Coronary Fl ow yes yes yes yes Adenosine Release yes yes yes yes Time 1 i rie (min.) 0 ------ISOLATION------15 Start Thea

25 ------NON- WORK PERIOD BEGUN------30 ------NON-WORK PERIOD ENDS------35 ------WORK PERIOD BEGUN------40 ------WORK PERIOD ENDS------45 ------NON- WORK PERIOD BEGUN----- 50 ------NON-WORK PERIOD ENDS------55 ------WORK PERIOD BEGUN----- 60 ------WORK PERIOD ENDS------41 ventricle. After a five minute equilibration period, during which hemodynamic parameters stabilized, coronary effluent was again collected for five minutes, with timed collection of cardiac output for one minute in midperiod to permit calibration of the flow probes.. Experiments were terminated follow1ng this work period. The· height of column C was set at a constant 10 em during these procedures. Minimum possible aortic pressure was set by a constant height of 45 em in column A. In twelve hearts the height of column B was set at 70 em during the work period and in another eight hearts the height was set at 9.0 em. 2. Protocol 2 Hearts were treated identically as in protocol 1 except that experiments were not terminated following the work period. Instead, the clamp on the line from column C was replaced causing external cardiac work to cease. After an additional five-minute equilibration period, collection of a second· "non-work 11 sample of coronary effluent was made for the same duration as was the first. Following this period, the clamp was once again removed and work at a second different afterload was begun. Equilibration, collections and measurements were made in this second work period in a manner identical to the first. Eight hearts were subjected to protocol 2. In four hearts the height of column B was 50 em during the first work period and 100 em during the second work period. In four hearts this afterloading sequence was reversed. This was done to rule out time dependent changes. Columns A and C were set as in protocol 1. Following termination of the collection of coronary effluent in the second work period, the 42 height of column C was raised above existing levels with column B set at 100 em to assure that the hearts were operating on the ascending portion of the Starling curve (i.e., non-failing). 3. Protocol 3 This protocol was undertaken to determine the effects of altera­ tions in minimum diastolic aortic pressure, as determined by the height of column A, on ventricular performance, coronary flow and adenosine release. This protocol is identical to protocol 2, except that instead of altering the height of column B and thereby changing afterload, the height of column B remained set at 100 em and the height of column A was varied between 45 em to 60 em. Collections in this protocol were identical to protoco 1 1 . ' '4. Protocol 4 In this protocol, preparation of hearts was identical to that previously described. However, in eight experiments, beginning at 15 minutes after excision, perfusion fluid containing 1 X 10- 5M theophylline was used. A 30-minute equilibration period followed the initiation of theophylline infusion. Coronary effluent was then collected for five minutes and in mid-collection a small sample withd~awn from the coronary sinus catheter and co"ncurrently from the aortic cannula for measurement of P0 • This period was designated as the theophylline non-working period. 2 Upon termination of this period external cardiac work was initiated by removing the clamp on the line from column C. The height of column B was 100 em, that of column A 45 em, and that of colUmn C 15 em at this time. Following a five-minute equilibration period a. five-minute collection of effluent was made. Cardiac output was collected as previously described 43 and concurrent sampling of coronary sinus effluent and aortic perfusate was also made in midperiod. This period' was designated as the theophylline working peri ad. In eight additional hearts an identical protocol was followed except that theophylline was not infused. ·These hearts served as a control group. D. Analytical Procedures Adenosine analysis To each sample of coronary effluent collected, 25 mg of acid washed charcoal (Norit A) was added and the mi~ture shaken for 1 hr, during which time adenine derivatives were adsorbed by the charcoal. It was then centrifuged at 15,000 g for 15 minutes, the supernatant discarded and the charcoal then shaken with 2·0 ml of 10% pyridine in 50% for 2 hrs to remove adenine compounds from the charcoal. Following centrifuga­ tion at 15,000 g for 15 minutes, the supernatant was air dried and resus­ pended. in 1.5 ml of stock 0.05 M potassium phosphate buffer. To this mixture, 0.5 ml of hydrated chloroform was added and mixed.by vortex agitation. This was followed by centrifugation at 5,000 g and resulted in a precipitation of contaminating proteinacious matt~r. From this 1.0 ml of aqueous contaminant free solution was taken for analysis. With samples obtained in protocols 1, 2 and 3, conversion of adenosine to was accomplished by the sequential addition of the enzymes .oxidase, nucleoside phosphorylase and adenosine deaminase. All enzymes were obtained from the Boehringer Mannheim Corporation and 2 ul contained 0.016 U, 0.05 U and 0.08 U of the enzymes listed above respectively. Enzyme reactions were followed by the increase in absorbance 44

at 292 nm using a dual beam spectrophotometer (Beckman, Acta CIII).

Conversion of standard adenosine solutions to u~ic acid provided a concen­ tration absorbance curve from which sample adenosine content was calculated. Adenosine was quantitated using a model 334 HPLC (Beckman Inst.) with an Ultrashere -ODS column (5u, 4x6x250 mm) and a precolumn in protocol 4. Samples (20-50 ul) of the previously described phosphate buffer solu- tions containing adenosine released· by hearts in respective non-work and· work· periods were loaded on a loop (50 ul capacity}, injected onto the column and eluted with 20 rrM sodiu,m phosphate buffer (95%)- acetonitrile (5%}, pH 5.0. Absorbance was measured at 255 nm using a variable wavelength spectrophotometer (Hitachi, model 100-10) equipped with a flow cell.

Elution times were compared to those of standards chroma~ographed under the same conditions. Oxygen tension

Coronary inflow and coronary ,sinus effluent P0 2 were determined using a Radiometer model BMS 3 MK2 blood gas analyzer (Radiometer/ Copenhagen). P0 2. was converted to oxygen content using known solubility relationships. MV0 2 was calculated via the .Fick method. Statistical analysis Student's t-Test was used to determine the significance of changes ·or differences between parameters under different conditions, using the 95% confidence interval (p less than or equal to .05). III. RESULTS A. Protocols 1 and 2

Protocols 1 and 2 were undertaken to determine-., the effects of work at increased afterload on aortic hemodynamics, flow and adenosine release in the preparation used. Means and their standard errors for all parameters measured in the first two protocols for·each of the four work­ loads, as well as for each respective paired non-work period are given in Table 2. In addition to the directly measured parameters, values for the product of heart rate and peak aortic pressure (HRxPAP) and the ratio of this product to the end diastole aortic pre~sure (HRxPAP/EDP) were calcu-· lated for_ use as indices of myocardial oxygen consumption and the level of stress imposed on myocardia 1 oxygen ba 1a nee by a given work 1oad ( exp 1a i ned in the discussion). These parameters are also included in Table 2. There was no significant difference in any of the monitored parameters between non-work periods in the same protocol or between protocols. Values for all parameters were greater in work peripds than in non-work periods at all workloads, with the exception of_end diastolic aortic pressure at the lowest afterload (B=50 em). Increases, expressed as percent of non-work are given in Table 3. There was a,general trend for greater changes from non-work values for peak aortic pressure (PAP), end diastolic aortic pressure (EDP}, pressure pulse· (PP}, and HRxPAP to occur as the afterload level in the work period was increased. However, - the maximum change from non-work values for HRxPAP/EDP and·adenosine release occurred when the afterload was 70 em. Changes in these parameters at higher afterloads were not significant. Increases in heart rate from non-work to work periods did not vary with afterload.

45 Table 2 Means and standard error of the mean for parameters monitored in protocols 1 and 2

Protocol 1 Protocol 2 n=12 n=8 n=8 n=8 8=70 em H 0 8=90 em H 2 em H2o 8=50 em H2o 8=100 2-o NW w Nl~ w NW w NW w Heart Rate 180 234 198 228 180 210 180 840 ( beats/min) ±6 ±12 ±12 ±6 ±2 ±12 ±2 ±6 Peak Aortic Pres. 40 79 39 91* 40 53 40 92* (mm Hg) ±1 ±1 ±1 ±1 ±1 ±1 ±1 ±1 End Diastolic Pres. 33 37 33 43* 33 33 33 48* (Aortic, mm Hg) ±2 ±2 ±2 ±3 ±2 ±2 ±2 ±2 Pressure Pulse 7 42 6 48 7 20 7 45* (mm Hg) ±2 ±2 ±2 ±4 ±2 ±1 ±2 ±2 HRxPAP 6.84 184.8 7.62 208.8* 6.90 111.0 6.90 195.0* (x 10 2) ±.31 ±5.2 ±.36 ±9.0 ±.78 ±5.0 ±.21 ±4.1 HRxPAP/EDP 208 515 228 507* 209 336 209 408* ±11 ±13 ±11 ±44 ±2 ±18 ±2 ±12 Cardiac Output 0 12 0 9* 0 16 0 10* ' (ml/min) ±1 ±1 ±1 Coronary Fl ow 200 420 200 480 280 400 270 660* (ml/min/100 g) ±20 ±40 ±20 ±20 ±40 ±40 ±40 ±60 Adenosjne Release 118 270 91 224 64 114 76 200* n.moles/min/100g) ±19 ±42 ±15 ±49 ±10 ±20 ±12 ±40

All measured parameters i ~ work periods si gni fi cantly changed from non-work va 1ues except end diastolic pressure at the lowest afterload (50 em). ~ There was no difference in any of the non-work parameters. 0'\ * = Significant change from protocol paired work value. 47

Table 3 Increase in monitored parameters in protocols 1 and 2 in going from periods of no external work to work periods expressed as percentage of respective values from non-work periods

Afterload in work period 8=50 8=70 8=90 8=100

Heart Rate 12 ± 5 33 ± 7 . 15 ± 6 15 ± 4

Peak Aortic Pres. 13 ± 1 96 ± 5* 123 ± 3* 142 ± 2*

End Diastolic Pres. 12 ± 6* 30 ± 8 45 ± 6

Pressure Pulse 200 ± 35 566 ± 75* 898 ± 84* 803 ± 47

HRxPAP 61 ± 8 174 ± 16* 176 ± 11 182 ± 12.

HRxPAP/EDP 61 ± 8 152 ± 20* 118 ± 16 95 ± 7

Coronary Flow 50 ± 12 152 ± 41* 146 ± 11 162 ± 22

Adenosine Release 86 ± 28 287 ± 144 212 ± 108 177 ± 37 * Significant change from next 1ower a fterl oad setting. 48

Work period values for key parameters are shown in Figure 2. This shows that there is a genera 1 trend for PAP, EDP, PP, HRxPAP and flow to be increased and cardiac output to be decreased as afterload was increased. However, maximum HRxPAP/EDP occurred at 8=70 em and decreased thereafter, with the calculated value· corresponding to work at the highest afterload setting being less than that of the next two lower afterloads.

Adenosine release also reaches a maximum value in the work period wh~n the afterload was 70 em. Changes from this maximum value at higher afterloads were not significant. To determine which parameters varied most closely with flow and adenosine release, regression analysis was done among the measured para.;. meters using mean values for all periods (n=8). Correlation coefficients from such analysis are given in Table 4. Adenosine release was best correlated with HRxPAP/EDP but was also significantly correlated with all other measured parameters with exception of EDP and coronary flow. Coronary flow was best correlated with EDP but was also significantly correlated with PAP and HRxPAP. B. Protocol 3

Protocol 3 was undertaken to determine if pressure-flow autoregu- 1 lation occurred in the present preparation. Work periods were included in a schedule similar to protocol 2 and rule out metabolic stresses being present in hearts in which the effects of increased work were studied that weren•t present in those hearts· in which autoregulation was studied. Means and standard error of the mean for parameters measured in protocol 3 are listed in Table 5. Significant differences were found between values for coronary flow and dP/dt max in the non-work period at 49

Table 4 Correlation coefficients obtained from regression analysis of mean values for monitored parameters in protocols 1 and 2, including all non-work and work periods N=8

HR PAP EDP pp HRxPAP HRxPAP/EDP Flow

Flow .424 .731* .823* .781 .770* .605* Adenosine .790* .794* .466 .848* .742* .890* .457 * = Significantly correlated p = .05 Figure 2. Effect of increasing afterload on aortic hemodynamics, flow and adenosine release. a) peak aortic pressure b) end diastolic aortic pressure (EDP) c) aortic pressure pulse d) cardiac output e) coronary flow f) ~roduct of heart rate and ~eak aortic _ressure (HRxPAP) g) HRxPAP/EDF h) adenosine release Triangles indicate significant·change from next lower afterload 51 the different settings of column A, indicating that pressure flow autoregu­ lation did not occur to an appreciable extent. There was no significant difference in any of the other measured parameters in this comparison or in any of the measured parameters when values from work periods at these different settings were compared. Lack of difference in measured parameters at different settings of co 1umn A in the work period was expected a,s the results of protocol 2 indicated that the end ·diastolic aortic pressure at the afterload used was greater than the highest level of column A (60 em) used in this protocol. Once again, all parameters were significantly greater in work periods compared to non-work periods, for both settings of column A, with the exception of heart rate. C. Protocol 4 Protocol 4 was undertaken to determine the effect of long-term theophylline infusion on cardiac function, coronary flow, oxygen utilization and adenosine release in non-working and in working hearts and on the functional hyperemic response to changing the conditions of no external work to conditions of external work. Means and respective standard errors for both non-work and work periods for parameters measured in protocol 4 are listed in Table 6. Values for selected parameters are also graphically represented in Figure 3. Arterial oxygen tension and content were constant. in all cases. Heart rate, peak ventricular pressure, dP/dt max, and MV0 2 were not changed by theophylline infusion in the non-work period. However,. coronary sinus oxygen content was 30% less, coronary flow 33% less, adeno­ sine release 30% less, and oxygen extraction 24% greater in the non-work period in the group given theophylline than in the control group in this period. 5_2

Table 5 Mean and standard errors for measured parameters in protocol 3

Minimum p6ssible aortic pressure A=45 A=60 em Ho NW W2 NW

Heart Rate 204 ± 18 228 ± 6 216 ± 6 216 ± 6 ( beats/min)

Peak Vent. Press. 44 ± 3 87 ± 3 50 ± 2 82 ± 3 ( mm Hg) dP/dt 470 ± 41 958 ± 43 635 ± 39 945 ± 61 (mm Hg/sec) ** Cardiac Output 7 ± 1 9 ± 1 ( ml /min)

Coronary Fl ow 320 ± 20 . 580 ± 40 460 ± 40 640 ± 60 ( ml /mi n/100g) ** Adenosine Release 48 ± 4 160 ± 44 72 ± 12 164 ± 32 (n.moles/min/100g) All measured parameters in work periods were significantly changed from non-work values with exception of heart rate. ** = Significantly di-fferent than the non-work pair. 53 . Similarly, heart rate, peak ventricular pressure, dP/dt, and MV0 2 were not changed by theophylline infusion in the work period. Cardiac output was also unchanged. Coronary sinus oxygen content was 26% less and

I oxygen extraction 24% greater in the group getting theophylline than the control group. These changes do not differ from those described in the non-work period. However, there was no significant change in coronary flow (.1 ~ p > .5) and adenosine release was 236% greater in the theophyl­ line group than in the control group. As coronary flow, coronary sinus oxygen content, oxygen extraction and aden·osine release were less in the non-work period during theophylline infusion, changes between non-work values and work values were expressed as percent of non-work values to facilitate comparison of the hyperemic responses. These are given in Table 7. The normalized change from non­ work to work was less for dP/dt and greater for adenosine release in hearts given theophylline than in control hearts. The relative increase in flow was unchanged by theophylline. 54

Table 6 Means and standard errors for measured parameters in protocol 4

Control Theophylline NW w NW w

Heart Rate .150 ± 12 222 ± 12* 144 ± 18 204 ± 18* ( beats/min)

Peak Vent. Press. 41 ± 2 94 ± 2* 40 ± 3 90 ± 2* (mm Hg) dP/dt 450 ± 42 1096 ± 57* 560 ± 42 945 ± 45* (mm Hg/sec)

Cardiac Output 10.8 ± 7 11.8 ± 1.6 (ml/min)

Arterial P0 410 ± 5 417 ± 10 400 ± 7 408 ± 6 (mm Hg) 2

Arterial o 2ont. 1. 28 ± .. 02 1.34 ± .03 1.26 ± .03 1.27 ± .02 (ml/ml X l-)6 Sinus P0 172 ± 13 187 ± 10 128 ± 4** 137 ± 6** (mm Hg) 2

Sinus 0~ Con~. 0.56 ± .04 0.60 ± .03 O.o39 ± .01** 0.44 ± .03** (ml /ml 10- ) a-v 0 0.71 ± .05 0.74 ± .05 0.88 ± .03** 0.88 ± .04** (ml/m~ x 10- 2)

Coronary Fl ow . 204 ± 16 572 ± 100* 136 ± 12** 394 ± 26* (ml/min/lOOg)

0~ Uti 1i za t i on 1.78 ± .18 5.16 ± 48* 1.94 ± .20 5.38 ± .70* ( l/min/100g)

Adenosine Release 8.2 ± .14 130 ± 30* 5.8 ± .2** 4.38 ± 114** *

* = Significant change from non work pair. ** = Significant change from control pair. Figure 3. Effect of theophylline on ventricular function, oxygen uti 1i za ti on, coronary flow, and adenosine release t a) peak ventricular pressure b) rate of pressure development c) heart rate d) oxygen content, arterial and venous e) flow f) ~en utilization g) adenosine release h) cardiac output Solid line- control group Dotted 1i ne - theophylline group Triangles indicate significant change from control gorup Abbreviations: NW no external work W external work 56

Table 7 Change from non-work to work periods in parameters measured in protocol 4, expressed as percent of non-work.value

Control Theophylline

Heart Rate 59 ± 2 49 ± 13

Peak Vent. Press. 136 ± 15 135 ± 17 dP/dt max. 297 ± 35 166 ± 10*

Coronary Flow 174 ± 39 206 ± 31

163 ± 13 177 ± 13 02 Utilization Adenosine Release 1485 ± 37 7452 ± 283*

* = Significant change from control. 57 IV. DISCUSSION A. Characterization of preparation Herein a mod.ified isolated working guinea pig heart preparation has been described. The peak developed aortic pressure increased along with left ventricular afterload toward a rraximum level (Figure 3) in this modification in accordance with the Starling relationship, as has also been documented in intact hearts, (183) heart lung preparations (111), and previously described isolated working heart preparations (145, 40). The

decreased cardiac output which w~s observed at· high ventricular afterload has been reported in other isolated working heart preparations (145, 40). Coronary flow a 1so increased as 1eft ventricular a fterl oad was increased in a manner similar to that in previously described isolated working hearts (145, 40). Myocardial oxygen utilization at the highest afterload

setting, measured in protocol 4 was 5.4 ml 02/min/100 gww and is at the lower end of the range of 4.2 to 12.9 02/min/lOOg utilized by isolated working guinea pig hearts performing pressure- v.o 1ume work reported by others (40). The level of oxygen utilization of hearts working at the highest afterload in the current preparation approximated that used by hearts working at a mean arterial pressure of 55 mm Hg in a previous

preparation (40). This preparation differs from previously describe~ isolated working heart preparations in.that the one way ball valves (Figure

1) placed above the a~rti~ cannula, permitted aortic dia~tolic pressure to fall below the component of left ventricular afterload usually attributable to the end diastolic aortic pressure. Thus, in this preparation the ·coronary inflow pressure in the work periods at the onset of diastole was equivalent to the hydrostatic component of the left ventricular afterload 58

(i.e., the height of column B). It then fell to-a minimum level equal to the end diastolic aortic pressure. Figure 3 illustrates that end diastolic pressure increased in an essentially linear manner as afterload was increased and that the difference between it and the beginning diastolic inflow pressure (height of column B, open circles) also increased at higher work loads. This means that although mean diastolic inflow pressure (not measured) increased at higher afterloads, it increased in a manner that would be described by a line between the line connecting end diastolic pressure points and the line connecting beginning diastolic inflow pressure points. Thus, its increase at higher afterloads did not strictly parallel the hydrostatic element of the left ventricular afterload as would be the case in the absence of the ball valves. In the absence of significant aortic semilunar valvular stenosis, proximal aortic pressure during ejection does not differ greatly from ventricular pressure. There was no significant· difference between the peak developed ventricular pressure (measured in hearts in protocols 3 and 4) and maximum aortic pressure in hearts (measured in protocol 2) at the same work load. As the product of peak ventricular systolic pressure and heart rate has been well correlated with oxygen utilization in isolated working hearts (66), the product of peak aortic pressure and heart rate may justifiably be used as an index of oxygen utilization in interpreting the results of the first two protocols. It might therefore be expected that changes in coronary flow would most closely parallel .changes in this index, as coronary flow has been well correlated.with MV0 2 over a wide range of hemodynamic conditions (128, 88, 201, 57, 123) and coronary flow is thought to be prinBrily 59 controlled by local metabolic events. However, it must be recalled that coronary flow is also affected by physical factors such as driving pressure (61). The results of protocol 3 indicate that driving pressure may have exerted a significant influence on coronary flow in the present preparation as appreciable pressure flow autoregulation was not observed when inflow pressure was increased. This is corrected with the concept that pressure flow autoregulation is only seen in isolated guinea pig hearts in the presence of pyruvate (61}, as pyruvate was not included in the present perfusion fluid. Lack of pressure flow autoregulation would explain the observation that coronary flow was best correlated with end diastolic aortic pressure although it ~as also correlated with HRxPAP, and PAP, (determinants of oxygen consumption).

Myocardial cellular P0 2 is dependent on the balance between oxygen utilization and delivery. Oxygen delivery to the myocardium is largely dependent on coronary flow, especially in the saline perfused heart because of the absence of hemoglobin. As coronary flow in this preparation was significantly affected by inflow pressure, the magnitude of stress exerted on myocardial oxygen balance, in the present preparation would be expected to be proportional to ~he product of HRxPAP and inversely proportional to diastolic inflow pressure. Since diastolic inflow pressure was not measured, the ratio of HRxPAP/EDP was used to estimate the stress to myocardial oxygen balance produced by a given workload in the present prepa ration. Inspection of Figure 3 reveals that the shape of the curve of adenosine release versus afterload closely resembled the curve relating HRxPAP/EDP to afterload. This close relationship between HRxPAP/EDP and 60 adenosine release i-s confirmed by the high correlation coefficient that was found to exist between these parameters. This suggests that in this preparation adenosine release is dependent on the degree of stress to myocardial oxygen balance and is consistent with the proposal of Berne ( 24).

Coronary sinus P0 2 did not change from non-work levels in the work period in the control group in protocol 4. This, along with the observation made in preliminary experiments that developed pressure and cardiac output did not diminish appreciably for well over an hour when hearts ~ere made to work at the highest afterl oad, suggests that an oxygen deficit did not occur at this workload. This means that local metabolic flow regulation occurred when cardiac effort was increased by changing

I from non-work to work at this afterload. Thus, these results are consistent with adenosine mediation of functional hyperemia in this preparation. Lack of significant correlation .between adenosine release and coronary flow was most likely due to the fact that inflow pressure dependent increases in flow occurred. Thus underscores the importance of considering all determinants of coronary flow in studies of local metabolic flow regulation and suggests that pressure flow autoregulation and metabolic flow regulation are achieved through different mechanisms in the guinea pig heart. ' Isolated working hearts have been criticized as being ischemic (216). Since the diastolic inflow pressure in this preparation fell below that usually seen in isolated working hearts, ischemia may be particularly suspect in this preparation. The increased adenosine release at higher workloads may therefore have been caused by decrements in ATP induced by ischemia. The above mentioned observations regarding the lack of appre­ ciable decreases in peak developed pressure for over 1 hr when hearts were 61 made to continuously work at the highest work load and the failure of coronary sinus P0 2 to fall below non-work P0 2 at this work load are incon­ sistent with significant ischemia, at least at this particular workload in this preparation. Regional ischemia, however, cannot be ruled out and no conclusions concerning the presence of ischemia may be drawn for work at 8=70 em and 8=90 em, as preliminary studies of the stability of the pre­ paration did not include work at these afterloads and as the calculated index of stress to myocardial oxygen balance were greater·and flow less than values for work at 8=100. B. Effects of theophylline Competitive blockade of the coronary vasodilator effect of adeno­ sine by theophylline may be used to test adenosine mediation of active hyperemia, if other, confusing and complicating cardiovascular and metaboli~ effects of this drug are accounted for or eliminated. The present study attempted to eliminate some of the extraneous actions of the methylxanthenes through the use of an isolated heart. . Although intraventricular pressure, dP/dt, heart rate, and MV0 2 were not changed in the non-work period by theophylline infusion, coronary flow and coronary sinus o2 tension were less, as was adenosine release. The simplest explanation for this is that adenosine-induced metabolic vasodilation was significant in this period in this preparation due to.tne low perfusion pressure used and that theophylline fnhibited this vasodilation. The decreased adenosine release might be explained in this scheme by postulating that since adenosine release into the capillaries is dependent on its transcapillary gradient and this is partially determined by flow rate, release may be lessened at low flow rates. 62

Alternate explanations, however, are also possible. The decreased flow and adenosine release may have been due to inhibition of 5'nucleotidease by theophylline as such an inhibition has been shown to be produced by theophylline at the concentration used in these studies (87), Similar results were obtained in the work period, except that flow was not significantly less and adenosine release was increased many fold. Despite the lack of significance in the change in coronary flow in the work period, one is likely to have occurred as·coronary. sinus oxygen tension was reduced from control at an equivalent MV0 2 and as the relative magnitude of the hyperemic response occurring upon changing from non-work to work conditions was not greater in hearts given theophylline .. The lack of significance may be attributable to the wide variance in flow during cardia~ work in this preparation. It may also be partially due to the fact that diastolic inflow pressure was dependent on the tone of the coronary vasculature in this preparation. Thus at elevated levels of tone, shown to exist in the non-work period when theophylline was infused, diastolic inflow pressure increased. Since pressure flow autoregulation did not oc~ur in the present preparation, this may have caused coronary flow to be higher than it might otherwise have been in the work period in the group given theophylline, mitigating any theophylline induced fl_ow decrement in the work period. The percent of flow decrement from control did not differ between non-work and work periods (32% decrease), but adenosine release was decreased in the non-work period and increased in the work period compared to control. Moreover, the functional hyperemic response to changing from non-work to work was not lessened by theophylline infusion, while the associated 63

release of adenosine was increased many fold. This suggests that theophyl­ line had no effect on functional hyperemia because of a compensatory increase in mediator adenosine, but caused a constant flow decrement (when expressed as percent control) which was dissociated from changes in adeno­ sine release. This suggests that theophylline infused at a dose of· 10- 5M produced a vascular effect other than through adenosine antagonism. The finding of no change in the active hyperemic response and increa-sed adeno­ sine release with theophylline infusion is consistent with the results of McKenzie et al. (130). The increased adenosine levels in active hyperemia in the presence of theophylline is in opposition_ to the results of Jones et a 1 . ( 101) . In the present study, isolated hearts have been used, precluding cardiovascular effects of theophylline. seco~dary to extracardiac neurohumeral actions. Cardiac effort and MV0 2 were stimulated by physical means, other than electrical pacing·, thereby eliminating the potentially confusing effects of drug interactions, and release of catacholamines· via inadvertent nerve stimulation from consideration. Heart rate, however, was found to increase when left heart chamber filling was allowed (Table 4). This may have been due to the release of endogenous catacholamines because of stretch of the walls of the heart chambers. If s.o, the increase in heart rate should have been proportional to the level of afterload. The results of protocols 1 and 2 indicate that thi·s was not the case (Table 2). Since a thermal jacket around the heart was not used in these studies the increase in heart rate observed when going from non-work to work periods was likely due to warming of the tissue by the fluid introduced into the chambers. Considering these facts, a possible explanation for the decreased flow observed is that although endogenous catacholamine release was not 64 likely increased, long-term infusion may have amplified the vascular effects of endogenous catacholamines through decreased extraneuronal inactivation as well as through phosphodiesterase inhibition. Evidence for a vasoconstrictive action for norepinephrine strong enough to oppo~e metabolic dilation includes the fac~ that coronary sinus oxygen content is reduced with its infusion (61) .. Norepinephrine vasoconstriction is possible in the present case even though no increase in heart rate occurred with theophylline infusion as the vascular effects of norepinephrine occur at lower doses than its cardiac effects. Theophylline-induced decrements in catacholamine degredation and concurrent phosphodiesterase inhibition could also potentially account for the decreased flow in response to pacing reported in the anesthetized dog by Lammerant and Becsei (122), as well as for other instances in which theophylliDe or aminophylline resulted in reduced vascular responsiveness to a metabolic challenge. This suggests that these drugs should not be utilized to test adenosine•s role in metabolic flow· regulati-on in the absence of alpha adrenergic blockade. SUMMARY Adenosine has previously been strongly implicated in the mediation of coronary blood flow regulation. The present studies were designed to further delineate the role of adenosine in functional hyperemia. To that end, a modified isolated working heart preparation was developed and characterized. Important features of this modification included: 1) diastolic coronary inflow pressure and left ventricular afterload were not synonymous; 2) coronary inflow pressure was not held constant but peaked in systole and fell to a minimum in diastole; 3) pressure-flow autoregulati,on did not occur. Steady state adenosine release was measured at five different conditions of left ventricular afterload and coronary inflow pressure. It was found to be well correlated with peak developed aortic pressure (PAP) and the product of heart rate and PAP (HRxPAP), a measure of myocardial oxygen utilization, but was most highly correlated with the calculated ratio of HRxPAP to end diastole aortic pressure (EDP ; end diastolic coronary inflow pressure), a measure of myocardial oxygen balance. Coronary flow, on the other hand, while also being correlated with PAP and HRxPAP, was most highly correlated with EDP, but was not correlated with adenosine release. This data suggests that adenosine release during functional hyperemia was primarily dependent on the status of myocardial oxygenation, as determined by the balance of supply and demand, and that pressure flow autoregulation and metabolic flow regulation may not be equivalent in the prepa ration. If adenosine mediates alterations in coronary vascular resistance during functional hyperemia, interstitial levels should vary in accordance

65 66 with flow demand. Sin.ce interstitial levels may not be adequately reflected by venous, tissue or pericardial fluid levels, a second series of experiments I were undertaken in which the effects of long-term {<30 min.) infusion of the competitive adenosine antagonist, theophylline on functional hyperemia in the isolated wo,rking heart was studied. Infusion of doses without direct cardiovascular effects {10- 5M) decreased baseline coronary fl~w and coronary sinus oxygen tension, but did not affect the functional hyperemic response to increase work load, while increasing adenosine release many fold. It was concluded that the decreased baseline flow may have been secondary to theophylline induced augmentation of catacholamine activity. It was also concluded that increased adenosine release in functional hyperemia in the face of a constant concentration of a competitive antagonist was consistent with adenosine mediation of functional hyperemia in the guinea pig heart. REFERENCES

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