ADENDSINE CORONARY DILATION AS INFLUENCED BY PERFUSATE HYDROGEN ION ACTIVITY AND THEOPHYLIJNE Dissertation for the Dame of WI. D. MICHIGAN STATE UNIVERSITY GARY F. MERRILL 1975 This is to certify that the thesis entitled Adenosine Coronary Dilation as Influenced by Perfusate Hydrogen Ion Activity and Theophylline presented by Gary F. Merrill has been accepted towards fulfillment of the requirements for Ph.D. _degree in PhYSiQIng fl Moo-or prororror J Date Noveniber 14, 1975 0-7639 W In ._ BUUK BINUE Y INC. LIBRARY BINDERS smueropt. mama! ____.- ABSTRACT ADENOSINE CORONARY DILATION AS INFLUENCED BY PERFUSATE HYDROGEN ION ACTIVITY AND THEOPHYLLINE BY Gary F. Merrill Adenosine has been proposed as a mediator of reactive hyperemia and hypoxic coronary dilation. This hypothesis is based on the finding that myocardial levels of adenosine increase during myocardial ischemia and hypoxia. However, while theophylline, a competitive antagonist of adenosine, attenuates the coronary dilator response to exogenously administered adenosine, it fails to affect greatly the mag- nitudes of reactive and hypoxic hyperemias. The hydrogen ion concentration also increases during myocardial ischemia and hypoxia. Therefore, an objective of the present study was to determine whether or not the inability of theophyl- line to attenuate reactive hyperemia is related to an inter- action of hydrogen ion with theophylline and/or adenosine. An isolated perfused guinea pig heart was used in all experiments. The coronary vascular bed was perfused at constant pressure (65 cm H20) and temperature (38°C) from a reservoir with a modified Ringer's solution gassed with Gary F. Merrill 95% 02-5% CO2 (pH 7.42 i 0.01). Coronary inflow was measured with an electromagnetic flowmeter and coronary out- flow with a graduated cylinder and stopwatch. Adenosine and theophylline were added volumetrically to the reservoir, and pH was varied by altering the PCO of the perfusate. 2 Coronary flow increased as a function of the adenosine concentration over the range of 10—8 to 10-6M. When the perfusate pH was reduced to 7.36 and 7.20, coronary flow increased but the level achieved with lO-GM adenosine was the same as at pH 7.43. However, at pH 6.89 the vasodilator activity of adenosine appeared to be enhanced, as it was when hearts were initially perfused at pH 7.20 following stabilization. When perfusate pH was subsequently inoreased to pH 7.69, coronary flow failed to change and the vasodila- tor activity of adenosine appeared to be suppressed. At pH 7 . M adenos1ne was 7.20, the vasodilator activity of 8x10- sustained for at least 30 minutes; a finding previously observed at a perfusate of pH 7.4. If adenosine is more ‘active in the presence of acidosis, this could in part explain why theophylline fails to greatly modify reactive and hypoxic hyperemias. Theophylline was without effect on coronary flow and heart rate in concentrations up to 10-4M (at higher concen- trations coronary flow and heart rate increased). —=A-*— Gary P. Merrill The vasodilation produced by 8x10—7M adenosine was attenu- ated by theophylline (10'4M) at both pH 7.42 and 7.20. Theophylline, 5x10-5M, was essentially without effect on the reactive hyperemia seen following 30 seconds of coronary inflow occlusion. Since the potassium and hydrogen ions have also been suggested as mediators of hypoxic and ischemic dilation and of autoregulation, ouabain (1.4x10’7 M): a blocker of potassium vasodilation, and alkalosis (perfus- ate pH 7.69) were combined with the theophylline in an attempt to modify these manifestations of local regulation by minimizing the contribution of potassium and hydrogen ions and adenosine. Hypoxic dilation was unaffected. Both the volume and duration of hyperemic flow were reduced some- what but failed to return to control after normalizing the perfusate. Autoregulation, while modified, still occurred. These studies suggest that an increase in hydrogen ion concentration enhances adenosine's coronary action and a decrease in hydrogen ion concentration diminishes adenosine's coronary action. They also show that reducing the perfusate pH has little effect on theophylline's ability to inhibit adenosine induced coronary dilation. Further, adenosine, at a low pH, has the ability to maintain coronary dilation over an extended period of time. ADENOSINE CORONARY DILATION AS INFLUENCED BY PERFUSATE HYDROGEN ION ACTIVITY AND THEOPHYLLINE BY ht Gary FfoMerrill A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Physiology 1975 DEDICATION To my wife Marlene for all that she is. ii ACKNOWLEDGMENTS I wish to express appreciation to the members of my guidance committee: Drs. J. M. Dabney, F. J. Haddy, J. B. Scott, B. H. Selleck, and J. L. Stickney for the time and effort they have spent in my behalf. I have benefited greatly from the perception and persistence shown by each. iii TABLE OF CONTENTS Page INTRODUCTION AND SURVEY OF THE LITERATURE............ l I. Local Blood Flow Regulation: Definition, Forms of and Chemical Participants........... 1 A. Involvement of Oxygen in Local Flow RegulatiOnOCOOOOOOCCOOOOOOOOOOQOOOOOOOOOOO 3 B. Participation of Hydrogen Ion and Carbon Dioxide in Local Flow Regulation.......... 4 C. Participation of Metabolites Other Than 02' and C02 and pHOOOOOOI...0.000.000.0000 5 D. Simultaneous Contribution of Oxygen and HYdrOgen101.10.00.00...OOOOOOOOOOOOOOOOOCOO 6 II. Adenosine Hypothesis for the Local Regulation Of coronary BlOOd FlOWOOOOOOOOOO0.00.00.00.00 7 A. Support for and Opposition to Adenosine's Involvement in Hypoxic Coronary Dilation.. 12 B. Support for and Opposition to Adenosine's Involvement in Coronary Reactive Hyperemia 16 III. A Statement of Objectives.................... 21 EXPERIMENTAL METHODSOOOOOOOOOOOOOOOOOOOOOOOCOOOIOOOOO 23 I. Preparation of the Isolated Guinea Pig Heart (Figure 3)OI...O0.0..OOOOOOOOOOIOIOOOOOOOOOOO 23 II. Experimental Equipment and Calibration....... 28 / III. Stabilization and Tests of Coronary Respon- \SiveneSSOOO-OO0.000000000000000000000.0.0.0... 29 A. Stabilization...00.00....I0.00000000000000 29 B. Coronary Responsiveness................... 29 iv TABLE OF CONTENTS--continued IV. Experimental Protocols..... ....... ........... A. Coronary Flow as Affected by Adenosine at Different Levels of Perfusate pH (n = 40). Effect of Perfusate pH on Coronary Flow in the Absence and Presence of Adenosine (8x10-7M) (n = 7)......................... Time Course of the Response to Adenosine (8x10-7M) at pH 7.20 (n = 5).............. The Effect of Theophylline on Coronary Flow (n = 12)............................. Effect of Perfusate pH on Theophylline Attenuation of the Coronary Response to Adenosine (8x10‘7M) (n = 18).............. The Effect on Reactive Dilation, Autoregu- lation and Hypoxic Dilation of Concurrent Theophylline (leO'SM), Ouabain (1.4x10'Afl and Alkalosis (Perfusate pH 7.69) (n = 15) RESULTSOICOCOOCOOOOOIOOOOOOOOOCOOOIOO00.000.000.00... I. Coronary Responsiveness to a 250 pg Bolus of Adenosine and Following Release of a 30 Second Inflow Occlusion...................... II. Coronary Flow Responses to Adenosine (10‘8-10‘5M) in Perfusates of Different Hydro- gen Ion ActiVitieSOIOOOOOCOOOOIOOOOOOOOOOOOOO A. B. C. Effect of Lowering the Perfusate pH on the Coronary Flow Response to Adenosine....... The Effect of First Perfusing the Coronary Vessels with Perfusate of pH 7.20 on Adenosine's Dilating Action............... The Effect of Increasing Perfusate pH on the Coronar Flow Response to Adenosine (10-8-5x10- M)............................ III. Coronary Flow Response to Adenosine (8x10‘7M) at Perfusate pH 7.20 for 30 Minutes.......... IV. The Effects on Coronary Flow of Changing the pH of the Perfusate in the Absence and Presence of a Single Concentration of Adeno— sine (5x10‘7M)............................... Page 30 33 34 35 36 39 39 41 41 52 57 59 67 TABLE OF CONTENTS--continued VI. VII. VIII. IX. The Effects of Theophylline (10—6-10-3M) on Coronary Flow and Heart Rate.. ......... ...... 6 4 Perfusate pH and Theophylline's (10- -10- M) Capacity to Attenuate the Coronary Dilation Produced by Adenosine (8x10'7M).............. Attenuation of Adenosine (8x10-7M) Dilation by Theophylline When Perfusing Initially with Solution of pH 7.20.......................... The Effect of Concurrent Theophylline, Ouabain and Alkalosis (Perfusate pH 7.69) on Reactive Dilation and Autoregulation......... Effect of Concurrent Theophylline (leO-SM), Ouabain (1.4x10'7M) and Alkalosis (Perfusate pH 7.69) on Hypoxic Coronary Flow............ DISCUSSION0000000000.000000000000000.0000.0.0.0...O00 I. II. III. IV. v. VI. VII. Adenosine Coronary Dilation as Affected by Perfusate Hydrogen Ion Concentration......... Adenosine's Coronary Action at a Low pH for an Extended Period of Time................... Effect of Variable Perfusate pH on the Coro- nary Action of a Single Concentration of AdenOSine.0000.00.00.00000000 ..... 0.0.0.0000. Theophylline Attenuation of Adenosine Coro- nary Dilation as Affected by Perfusate pH.... Coronary Reactive Hyperemia as Affected by Theophylline and Perfusate pH................ Effect of Concurrent Theophylline, Ouabain and Alkalosis on Hypoxic Coronary Dilation... Effect of Concurrent Theophylline, Ouabain and Alkalosis on Coronary Autoregulation..... SUMMARY0000000000000.00000000000.000. ......... 0.00.00 BIBLIOGRAPHYO00000.0000.00.00.00000000...000. ....... 0 vi Page 74 84 86 88 100 104 105 115 117 118 120 123 125 127 131 TABLE OF CONTENTS--continued Page APPENDICES A. STATISTICS AND ANOVA TABLES. 0 . 0 . 0 0 0 0 0 0 0 0 0 0 0 0 0 140 B0 RAW DATA.....000000000000.00000.00.00.00.00.. 148 vii LIST OF TABLES TABLE Summary of 69 individual experiments (9 series of experiments) showing the magnitude of coro- nary flow in the isolated guinea pig heart following 30 seconds of inflow occlusion or a 250 Hg bolus of adenosine in the pre- and post- experimental states............................. The effect of lowering the perfusate pH on the capacity of a series of adenosine concentrations to affect flow and heart rate of the isolated guinea pig heart................................ Coronary flow and heart rate in response to adenosine in perfusates of two different [H+]... Adenosine's effects on mean, diastolic and sys- tolic coronary flow (ml/min) and on spontaneous heart rate. Hearts were initially perfused at pH 7.42 followed by subsequent perfusion at pH 7.69....000000.000.000.00.000000.0.00.00.00.0000 Effect of adenosine on coronary flow and heart rate over a 30 minute period during which the heart was perfused with solution of pH 7.20..... Effect of perfusates of differing pH (6.89-7.69) on coronary flow (ml/min) and heart rate in the absence and presence of adenosine (5x10‘7M)..... Effect of theophylline (M) on steady-state coro- nary flow in two groups of hearts (top and middle panelS).00....0000......0......0.....00.. Effect of theOphylline on the coronary vasodi- lation produced by adenosine in perfusates with two different [H+]000.000.000.00000000000000.0.0 viii Page 40 42 58 60 65 68 75 83 LIST OF TABLES--continued TABLE 9. Effects of theophylline on heart rate and adeno- 10. 11. sine-induced coronary dilation when hearts were initially perfused with solution of pH 7.20 followed by a perfusate with a pH of 7.43....... Effect of concurrent theophylline (5x10 5M), ouabain (1.4x10'7M) and alkalosis (perfusate pH 7.69) on coronary flow following release of a 30 second inflow occlusion...................... Effect of concurrent theophylline (5x10_5M), ouabain (1.4x10‘7M) and alkalosis on coronary flow (ml/min) and calculated resistance in response to increasing and decreasing perfusate pressure....................................... ix Page 87 93 97 LIST OF FIGURES FIGURE Page 1. Schema illustrating adenosine hypothesis for regulation of coronary blood flow.............. 9 2. Schematic diagram of myocardial tissue illus— trating formation, fate, and site of action of adenosine coming from intracellular ATP........ 10 3. Apparatus for experiments; diagram of the non- recirculating perfusion system................. 26 4. The effect of adenosine (10-8-10—6M) on coro- nary flow in the isolated, spontaneously beat- ing guinea pig heart using two perfusates of different hydrogen ion activities.............. 44 5. The effect of adenosine (10 8-10 6M) on coro- nary flow in the isolated, spontaneously beat- ing guinea pig heart using two perfusates of different hydrogen ion activities.............. 45 6. The effect of adenosine (10 8--5x10 7M) on coro- nary flow in the isolated, spontaneously beat- ing guinea pig heart using two perfusates of different H+ activities........................ 47 7. Regression analysis computed from experiments in which guinea pig hearts were perfused with solutions of two H activities................. 51 8. Effect of adenosine on coronary flow in the isolated, spontaneously beating guinea pig heart (hearts perfused first with solution of pH 7.20)....................................... 54 9. This figure illustrates data taken from Figures sand 6.00.0.0.0000...00...0000..00.0.0.0000... 56 LIST OF FIGURES--continued FIGURE 10. ll. 12. 130 140 15. 160 17. 18. 19. 20. 21. This figure compares the effects on coronary flow (ml/min) of adenosine (10’8-5x10‘7M) at perfusate pH 7.42 and 7.69.................... Adenosine (8x10-7M) on coronary flow for 30 minutes at perfusate pH 7.20.................. Coronary flow as affected by adenosine (8x10'7M) over a period of 30 minutes at per— fusate pH 7.20. N = 5.. ..... ................. The effect of perfusate pH on coronary flow in the absence and presence of 5xlO'7M adenosine. Linear regression of coronary flow on per- fusate pH; method of least squares used for computation, linearity tested with an analysis of variance................................... Effect of theophylline (M) on coronary flow and spontaneous heart rate at two levels of perfusate pHOO0.000.000.000000......0...0...0. The effect of theophylline (5x10-5M) on reac- tive dilation000.0.00.00.00.00000.0.0.0...0... Effect of theophylline on coronary flow and spontaneous heart rate when hearts were per- fused With SOlutj—on Of 7.43....0.......0000..0 Effects of theophylline on coronary flow dur- ing the treatment with 8x10'7M adenosine...... Effect of theophylline on coronary flow during treatment with 8x10‘7M adenosine. Hearts initially perfused with solution of pH 7.20... Effect of theophylline on the coronary dila- tion produced by adenosine (8x10’7M). Data are from Figures 18 and 19.................... Effect of concurrent theophylline (5x10 5M), ouabain (1.4x10'7M) and alkalosis (perfusate pH 7.69) on reactive dilation following re- lease of 30 second occlusions............ ..... xi Page 62 64 66 71 73 77 80 82 85 89 91 95 LIST OF FIGURES--continued FIGURE 22. 230 Page Effect of concurrent theophylline (5x10—5M), ouabain (1.4x10’7M) and alkalosis (perfusate pH 7.69) on coronary autoregulation............ 99 Effect of concurrent theophylline (5x10 5M), ouabain (1.4x10'7M) and alkalosis (perfusate pH) on coronary flow in response to hypoxia (perfusate equilibrated with 20% 02-5% C02- balance N2)............................... ..... 102 xii INTRODUCTION AND SURVEY OF THE LITERATURE I. Local Blood Flow Regulation: Definition, Forms of and Chemical Participants The phrase 'local regulation of blood flow' refers to that regulation which is intrinsic to the organ. Specifi- cally excluded is regulation secondary to remote influences which alter vasoconstrictor or vasodilator nerve activity or the concentrations of vasoactive substances in inflowing blood (Haddy and Scott, in press). The main forms of local regulation are: l) autoregulation--the ability of an organ to maintain a relatively constant blood flow despite changes in arterial perfusion pressure, 2) active hyperemia--an increase in blood flow above control levels in response to increased metabolic activity, and 3) reactive hyperemia--a transient increase in flow to a level greater than control following a period of reduced flow. One hypothesis for the local regulation of blood flow is the metabolic hypothesis, which proposes in its simplest form that changes in metabolism or blood flow are accom- panied by alterations in the concentrations of oxygen and vasodilator metabolites in the tissue fluids. Such altera- tions result in active vasomotion which adjusts flow to a level more appropriate to the metabolic rate (41). Gaskell (34) was the first to report that artificially exercising skeletal muscle by electrically stimulating the motor nerves to the muscle, produced an increase in blood flow through the active muscle. Since Gaskell's report, numerous others (31,43,54,77,88) have verified that skeletal muscle has the capacity to adjust its blood flow to meet metabolic needs, and still others have expanded the findings to most of the major vascular beds (6,7,17,25,30,32,42,46, 52,75,90,93). Amongst the vascular beds able to regulate their blood flow is the coronary bed. For example, study- ing the dog heart-lung preparation, Hilton and Eichholtz (47) reported that coronary vessels are extremely suscepti- ble to changes in blood oxygen tension, a fall of oxygen saturation below 20 per cent causing maximal dilation. Cross gt_gl. (14) studied autoregulation in isolated heart preparations and in intact, innervated dog hearts, and demonstrated this organ's ability to maintain a relatively constant flow over a broad range of arterial pressures. Driscol, Moir and Eckstein (20) studied coronary autoregula- tion in the anesthetized dog and reported that coronary flow responses to changes in perfusion pressure are not sig- nificantly affected by collateral flow. They found that pressure-flow responses (examined by perfusing one of the first major branches of the left circumflex coronary) were the same with and without perfusing simultaneously the left common coronary artery at the same pressure, i.e., the absence of a pressure gradient between the branch of the left circumflex and its collaterals did not effect the coronaries ability to autoregulate. More recently Bunger §E_§1. (11) using an isolated guinea pig heart found that in the presence of pyruvate (2.0 mM) plus glucose (5.5 mM) this preparation displayed typical autoregulatory pressure- flow responses over the pressure range of 20-90 cm H20. Reactive dilation, following release of occlusion, was also observed and was found to be typical of reactive hyperemic responses reported for the in_vigg_heart. A. Involvement of Oxygen in Local Flow Regulation It is evident that those conditions which reduce tissue oxygen tension via increased oxygen utilization and reduced oxygen delivery favor an increase in blood flow to meet the metabolic needs of the tissue (5,12,13,24,26,39,48,58). That oxygen is directly or indirectly involved in exercise hyperemia (51,61,70), reactive hyperemia (61,65,70) and autoregulation (53,54) in cardiac and skeletal muscle is well documented. Studying the effect of blood oxygen satur— ation on autoregulation in the dog hindlimb, Guyton gt_31. (39) found that blood flow progressively increased as the per cent oxygen saturation of arterial blood is decreased. Flow increased on the average of two and one—half times as oxygen saturation was decreased from 100 per cent to 30 per cent. Carrier and co-workers (12) using short segments of isolated dog femoral artery, and Detar and Bohr (l9) studying helical strips of rabbit aortas, found that reduc- ing the oxygen tension of perfusing/bathing solutions re— sults in relaxation. Daugherty and his associates (16) demonstrated a reduction in coronary resistance when the oxygen tension of blood perfusing the coronaries was de- creased below 40 mm Hg. The effect of oxygen content on coronary blood flow was studied by Guz et_al. (40) while perfusing the isolated rabbit heart with a hemoglobin solu- tion. They found that reducing the arterial oxygen content by diluting the perfusate with Ringer-Locke's solution increased coronary flow; whereas, increasing the arterial oxygen content decreased coronary flow. B. Participation of Hydrogen Ion and Carbon Dioxide in Local Flow Regulation Several studies have shown that local exposure of blood to increased PCO2 or local administration of acid, reduces resistance to blood flow through skeletal muscle (16,37), cerebral (29,35) and coronary (l6) vascular beds, while reduction of the local carbon dioxide tension or administra- tion of alkali, increases resistance to flow through the forelimb (16,66), brain (29,35), and heart (16,38,63). CE ir. io Investigating the effect of hydrogen ion concentration on coronary flow in an isolated rabbit heart, Smith and co- workers (86) found that a decrease in perfusate pH from 7.68 to 7.38 increased flow 7 per cent. When pH was further reduced to 7.0, coronary flow was augmented 80 per cent relative to that at pH 7.68. McElroy, Gerdes and Brown (63) reported, from a study using an isolated, perfused guinea pig heart, that only when changes in HC0 and PCO produced 3 2 alterations in pH was coronary flow affected. In these and similar studies the effects of rather large changes in pH were studied, while more direct evidence (18,37,61,81,82) indicates that local regulation may be accompanied by marked changes in resistance with very small or no change in venous blood pH occuring. Hence, factors in addition to hydrogen ion and carbon dioxide must play a part in local flow regulation. C. Participation of Metabolites Other Than 02LCO2 and pH Bioassay and chemical analysis of venous blood support the argument that metabolites other than oxygen, hydrogen ion and carbon dioxide are involved in local regulation of blood flow (41,75,8l,82). Scott §E_al. (85) reported that chemical analysis of venous effluent blood from an exercis- ing dog gracilis muscle showed an elevation in potassium ion concentration. However, the importance of potassium in the regulation of coronary flow is questionable, i.e., hyper- kalemia may transiently increase coronary flow (64) but has been shown to produce an increase in coronary resistance (60). Hypokalemia decreases coronary flow (44). Other chemicals such as lactate, pyruvate, inorganic phosphate and Krebs cycle intermediate metabolites have been shown to increase in skeletal muscle venous blood with exercise and reactive hyperemia (41,42) and during cardiac hypoxia (5). Although many of these factors are capable of eliciting some degree of vasodilation in skeletal and car- diac vasculature, most fail to meet the criteria necessary for a true physiological mediator of blood flow (41). D. Simultaneous Contribution of Oxygen and Hydrogen Ion Several investigators have attempted to assess the simultaneous contribution of oxygen lack and increased hydrogen ion to the regulation of local blood flow. In one study, Scott et_gl, (85) selectively lowered the oxygen tension of blood flowing into the resting hindlimb to a level which produced a low venous oxygen tension. The sub- sequent fall in vascular resistance was noted. Arterial inflow oxygen was then selectively increased to a high level and exercise initiated. Venous oxygen tension did not fall to the level seen during hypoxia but vascular re- sistance fell to a level which was lower than that during hypoxia alone, thus demonstrating that chemicals other than oxygen were producing vasodilation. In another study, Stowe and co-workers (89) pumped venous blood from a dog gracilis muscle through a hollow fiber gas-exchange perme- ator into the artery of an assay gracilis. During electric- al stimulation of the regulatory muscle's motor nerve, calculated resistance of both muscles decreased. P02 and pH of the blood perfusing the assay muscles both decreased. When PO2 or pH were individually corrected to control values, calculated resistance returned toward but did not reach control values. However, if both P02 and pH of blood flowing into the assay muscle were successfully corrected to pre-exercise levels, dilation in the assay muscle disap- peared. II. Adenosine Hypothesis for the Local Regulation of Coronary Blood Flow Changes in regional myocardial metabolism are associ- ated with alterations in coronary blood flow which are immediate and marked in effect (68). There is loss of stored ATP (68,78,80) and creatine phosphate (68) within seconds of an occlusion. Hydrolysis of stored triglyceride can result from activation of myocardial lipase (67). In addition it has been shown more recently that associated with myocardial hypoxia, is the release of adenosine (7,76, 78,79), a major degradative product of ATP. In 1963, some 40 years after Drury's (21,22) pioneering demonstration that adenosine is a potent coronary dilator, Berne (5) introduced the current adenosine hypothesis for the metabolic regulation of coronary blood flow in which he proposed a mechanism whereby coronary flow increases during hypoxia. Berne suggested that any condition which tends to lower cardiac tissue oxygen tension, e.g., increased meta— bolic activity, reduced coronary flow, and/or hypoxemia would result, via net adenine nucleotide degradation, in the enhanced production and release of adenosine from the myo- cardial cell (Figures 1 and 2). Adenosine could subse- quently pass through the interstitial fluids and act upon the coronary resistance vessels to dilate them. Since 1963, much evidence has accumulated in support of Berne's "adenosine hypothesis" (6,7,8,15,58,78,84); however, for some time this hypothesis was viewed with skepticism because investigation failed to reveal adenosine in the effluent perfusate of isolated (5,49,76) or intact heart preparations (5). The failure to find adenosine in the venous effluent prompted the undertaking of studies aimed at determining if the proper catabolic enzymes were present in the myocardium whereby adenine nucleotides could be hydrolyzed to adenosine. In one experiment, using pig heart muscle extracts, Goldthwait (36) reported that adenosine kinase, adenosine Acodmmasuoa an .mme .mam .som .Hoammnm .h .a< scum couscOHQom. .30: nooB 30:88 co c2639: .2 £85an 36058 3:252: 0623 M. 8:91.. $8.554 i 33.. $4288 806 $5245 $4288 50:2; 9 “12.8234 \ 895382 93... 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O o O 0‘0 II 'II ‘ 342.53 3.3234 0 (DIL41LJHJ -:EP-uafl£dfl--4--euomom mo “0385c «.2 .> can .>H .HHH .H muumm Bonn mucosauomxo onuaocw can .maooououm Hmucoawuomxm .>H coauoom Scum coxmu who sumo .mucoswuomxo mo mowuom comm cw chHumofiHmou .mmumum Hmucosauomxoumom can loam ecu ca ocwmococm mo msdon on 0mm o no cowmsaooo zoawcfl mo mpcooom om mewsoaaom uuomn mam museum causaomw on» cw 30am mumcouoo mo ocsuflcmme on» mewsonm Amazoawuomxo Mo mowuom my mucoaauomxo Hmavw>wccw mm mo humsfiam .H menus 41 Examination of the table shows that the preparations se- lected met the above requirements. II. Coronary Flow Responses to Adenosine (lO-B-lO-GM) in Perfusates of Different Hydrogen Ion Activities A. Effect of Lowering the Perfusate pH on the Coronary Flow Response to Adenosine In experiments conducted on three groups of hearts the perfusate pH was reduced by increasing its PCO The 2. effects of only two perfusate pHs were studied in each experiment. In each group of hearts an adenosine dose- response relation was first established at a perfusate pH of 7.43 i 0.01 and this was subsequently compared with an adenosine dose-response relation at a pH other than 7.43 (Table 2, Figures 4, 5 and 6). In the first group of hearts adenosine (lo-6M) increased coronary flow to a level which was quantitatively similar at both perfusate pH 7.43 and 8 7 7.36. At pH 7.36, control flow and flows at 10- and 10- M adenosine were approximately 19 per cent greater than respective flows at pH 7.43. The difference is significant (P < 0.05). In a second group of hearts in which the second dose— response relation was conducted at pH 7.20, the coronary response to 10-6M adenosine was not different than that produced by the same concentration of adenosine at pH 7.43. II 42 Table 2. The effect of lowering the perfusate pH on the capacity of a series of adenosine concentrations to affect flow and heart rate of the isolated guinea pig heart. Hearts in all panels were initially perfused with a perfusate equilibrated with 95% O -5% CO ). Values are expressed as means :_their respec- tive S.E.M.,2N=9 in panels 1 and 2 and N=6 in panel 3; (*) denotes statistical significance (P<<0.05) with respect to pre-experimental values. Coronary_flow (ml/min) Heart rate N = 9 Perfusate pH Perfusate pH 7.42 7.36 7.42 7.36 Pre-exper. 6.2:0.39 7.4:0.26 260:7.0 266:7.1 m 10'8M 6.210.35 7.5:p.29 260:7.0 265:6.8 -§ 10'7M 7.1:p.37 8.5:0.29 264:7.6 267:5.7 g lO-6M 10.9:p.31* 10.9:p.35* 263:].4 26716.0 3 rPostexper. 6.3:0.30 268:5.7 Perfusate pH Perfusate pH N = 9 7.42 7.20 7.42 7.20 Pre-exper. 5.8:0.24 7.719.26 258:10.5 255:8.4 g lO-BM 5.710.24 8.0:0.35 253:8.9 25618.7 '§ 10-7M 6.6:0.24 9.9:p.41* 259:s.2 256:8.5 .g lO-GM ll.3:0.66* 12.1:p.58* zeois.4 258:8.1 Postexper. 6.0:0.66 258:8.0 Perfusate pH Perfusate pH N = 6 7.43 6.89 7.43 6.89 Pre-exper. 5.2:0.16 7.4:0.l8 260:2.5 263:5.0 lO-BM 5.2:0.l6 7.6:0.20 262:2.0 262:5.7 E leO-BM 5.310.22 9.110.36 26012.5 25814.1 é 10'7M 5.9:p.27 10.519.32* 262:3.7 258:4.1 fi 5x10—7M 8.9:0.63* 13.7:o.ss* 27212.9 26015.1 Postexper. 4.9:0.15 260:5.1 Figure 4. 43 The effect of adenosine (10-8-10-6M) on coronary flow in the isolated, spontaneously beating guinea pig heart using two perfusates with dif- ferent H+ activities. Hearts were initially perfused with solution of pH 7.42. Subsequently, hearts were switched to a perfusate of pH 7.36 and a second adenosine dose-response relation was studied. *Denotes statistical significance (P:<0.05) when compared with respective control (C). N=9 All values are displayed as the mean + S.E.M. CORONARY FLOW (ml/min) 44 --- 92% 02 - 8%CO2 pH736 — 95% 02 ' 5% CO2 c ‘ IO'BM IO'7M IO'GM (ADENOSINE) Figure 4 45 .63. Ia .0 8:28 5.... 5: 33:8 3.35 too... 9a 85:0 0583 zmaomcscoamfimhsor of 5 so: focoeou :0 37.058 .6 Born. ..m 959... «mz_mozmaHuoommoH us money 30am some Uo>uomno u 0V .o>udo map so c3ocm mum .z.m.m_H some .muz .mao.oun .ummcHH ma m>uso c0fimmmumou ecu umcu Uocwsuouoc cosmewm> mo mflmmamcm cm was .cOHumusmEoo ca poms mos moumsvm ummoa mo vogue: .o~.> no mo mummsmnmm spas consumed maucosvmeSm mums can >HHMHuficfl m¢.> mm mo mummsmuom ou oomomxm ouo3 muumom .mofiuw>fluom +3 03D mo coausHOm cues Ummswuom ouo3 munmoz mam mmcflsm cowcz ca mucoEHuomNm Eoum cousmfioo mamMHmcm coammoumom .n musmflm 51 F 952m :33 ”12.8234 new new 5a 0 1 seem. r v 90.0 r a... 3.5.. K ow r m seem I, , N. m. (usw/Iw) M015 Aavnoaoo 52 An analysis of variance revealed significant linearity of the curve. The regression curve illustrates (when the curve is linear) that if one were to use adenosine concen- trations greater than 2.67 ug/l but less than 267 ug/l, flow would increase as adenosine concentration increased and that the pattern of the response would be similar to that observed in Figure 7. B. The Effect of First Perfusingpthe Coronary Vessels with Perfusate oprH 7.20 on Adenosiners Dilat- ing Action Early in the adenosine studies, the question was posed: Does one see the same coronary flow response to adenosine when hearts are perfused initially with solution of a pH other than 7.42 then subsequently perfused with solution of pH 7.42? To investigate this question coronary vasculature was exposed to control perfusate (pH 7.42) during the stabilization period. Upon reaching the steady state flow, hearts were immediately switched to a reservoir with per— fusate of pH 7.20. The previously established stabiliza- tion flow (pH 7.42) was regularly increased by approxi- mately 25 per cent (5.710.33 to 7.1:0.5 ml/min) by per- fusate of pH 7.20. Altering the order in which hearts were perfused had a marked effect on the coronary action of adenosine. Figure 8 shows that the flow response to adenosine (lo-GM) at pH 7.42 was similar to responses seen in our previous experiments using the same concentration 53 of adenosine and the same perfusate pH (Figures 4 and 5). However, the response to adenosine (lo-GM) at pH 7.20 (Figure 8) was approximately 30 per cent greater than that produced when hearts were perfused secondly at pH 7.20 (Figure 5), and 34 per cent greater than that at pH 7.42 in the present study. Note that 10‘7M adenosine (pH 7.42) had no effect on coronary flow whereas the same concentra- tion of adenosine at pH 7.20 increased mean flow markedly above its respective control. An interesting finding is that the flow responses to adenosine (5x10-7 and 10-6M) at pH 7.20 are not significantly different from each other and flow appears to be reaching a plateau at 10-6M. Adenosine 7 (5x10— and lO-6M) responses at pH 7.42 are significantly (P‘<0.05) different from each other and flow gives no signs of reaching a plateau at 5x10—7M adenosine. To facilitate inspection of adenosine's coronary action at pH 7.20 as affected by varying the pH perfusion order, the top curves from Figures 5 and 8 were used to construct Figure 9. Inspection of control flow rates (7.7 and 7.1 ml/min) in each curve in Figure 9 suggests that the difference in the flow responses to 10-6M adenosine cannot be accounted for by the magnitude of difference (0.6 ml/min) in respective control flows, but rather must be related to an effect of the order of perfusion on adenosine's dilating capacity. 54 muz 0.58 at; 00.60E00 c023 3:005:20 60.8.8.0 0200:0566 48.1.30 «0 .mom N In e0 5:200 5:: Eu... 0002.00 0:00.: too; oi 005:0 0:000 23000200090220». 0.: c_ 30: E00200 :0 0500500 e0 80.5 ”meson. wz_mozmo< ztfbma 3-0. rub. my 0 re . m .«. will)? «51a .0. «00.3- «0 3.8 II. 09:3 r 0« «004.0. - «0.4.00 I I u (“I'll/I'M) MO'L-l AHVNOUOO 55 ma u z .50 H.H u A v usmflm3 pummn came .80 N.H n AIIIIV usmflm3 unmmn cam: .z.m.m.H some may mum w>uso £000 :0 mucfiom was .mv.n mm um cofimsmumm an UmonHOM om.h mm um umuflm vmmSMHmm mumz muumwn c023 coapom anacouoo m.mcwmocmwm mucmmmum m>uso cmxoun was .mv.> mm um nmmsmumm umufim muuwmn a“ om.h mm mo mummsmnmm m um 30H“ anacouoo co mcflmocmcm mo muowmmm mnu w3onm m>uso UHHOm was .0 new m mmusmflm Eoum amxmu mumc mmumuumsaafl musmwm mass .m madmam 56 0 050.. 2252.8 234 20.0. #0. -0. u N u“ m 0. 000003 ow N In 0.878.000 .ll 0. W \ 2005 CNN:0 NOU o\oO_ lNOo\ooml In I @- Mw/uw) MOI-J Aavuouoo 57 Mean heart weight in the two groups was 1.2 :0.005 and 1.1 :_0.06 gm respectively. Table 3 indicates that at both perfusate pH 7.20 and pH 7.43, adenosine (lo-6M) increased heart rate signifi— cantly'(P<<0.05). However, Table 2 shows that this same concentration of adenosine at pH 7.42 and pH 7.20 had no effect on heart rate when the perfusate sequence was not altered. Thus the increase in flow at pH 7.20 in the former (Table 3) might be partially attributable to the increased heart rate. C. The Effect of Increasing Perfusate pH on the Coronary Flow Response to Adenosine (10‘3-5x10‘7M) The results of experiments in which adenosine's action on the coronary vessels was investigated in the presence of perfusates of increased hydrogen ion activities (Table 2, Figures 4, 5 and 6) indicated that adenosine not only retained its capacity to dilate when the pH of perfusing fluid was lowered, but that its coronary actions were en- hanced by an interaction with hydrogen ion. Our next ap— proach was the obvious; to investigate the effect on adeno- sine coronary dilation of increasing the pH of the perfu- sate above 7.42. This was accomplished equilibrating perfusate with 98% 02-2% C02, yielding a perfusate pH of 7.69. Hearts were first perfused with solution of pH 7.42, and adenosine (5x10-7M), increased mean coronary flow from Table 3 . 58 fusates of two different [H+]. to pH 7.20 followed by pH 7.43. Coronary flow and heart rate in response to adenosine in per- Hearts were first subjected All values are expressed as means :_S.E.M. (*) denotes statistical significance (P< 0.05). N = 8) Coronary_flow (ml/min Heart rate pH 7.20 pH 7.43 pH 7.20 pH 7.43 Pre—exper. 7. 1:0. 5 5 . 7:0. 33 248:5 . 1 254:4 . 2 10-8M 7.2:p.5 5.5:p.38 248:§.l 254:4.2 g 10'7M 11.7_+_o.e7* 6.010.410 25216.0 25715.5 2 5x10'7M 15.6:0.77* 9.51-9.80“ 25516.7 25714.5 '2 lO-GM l6.6:0.83* 12.4:0.80* 263:7.3“ 269:6.0* Postexper. 5.719.34 256:5.0 59 6.2 3; 0.24 ml/min to 12.8 :_0.8.ml/min, an increase of 106 per cent (Table 4, Figure 10). When hearts were subsequent- ly perfused with solution at pH 7.69, control flow fell insignificantly to 5.8 i 0.29 ml/min, and this was increased only to 8.6 :_0.57 ml/min in the presence of 5x10-7M adeno- sine (Figure lO). This represents a change of only 2.8 ml/ min above control as compared to a change of 6.6 ml/min at pH 7.42 in response to the same concentration of adenosine. Examination of Table 5 reveals that similar results were produced when diastolic and systolic flow rates were com- pared to their respective controls at both levels of per- fusate pH. The only statistically significant (P< 0.05) increases in flow at pH 7.69 occurred in response to 5x10-7M adenosine. Heart rate was not affected by adenosine (5x10-7M) at perfusate of pH 7.69. III. Coronary Flow Response to Adenosine (8x10-7M) at Perfusate pH 7.20 for 30 Minutes This experiment was designed to verify the findings of subsequent experiments in which theophylline was used to attenuate coronary dilation produced by adenosine. If the adenosine response does not want with time, then it is unlikely that attenuation by theophylline would be partially due to waning of the adenosine response. Further, since 7 the flow responses to leO‘ and 10-6M adenosine in hearts 60 0.011.000 0.0 0.00 0.0 80980000 0.0.0000 0.0.0.000 .04. .0.0 .000 .000 .0.0 .000 20:03.00.” 00.0-H.000 v.0-wfimv0 0.0 m...m 0.00 0.00 0.0 0.0 20-00% 0.0.0000 0.0.0000 0.0 0.0 0.3 0.: 0.0 0.0 20-0000 m. 0.0.0.000 0.0.H000 0.0 0.0 0.0 0.00 0....- 0.0 20:00 0.0.0000 0.0.0.000 0.0 0.0 0.0 0.00 0.0 0.0 508.0000 00.0 00.0 00.0 00.0 00.0 00.0 00.0 00.0 0000 00000 00000000 000000000 coauaaom-mdflmamuwm mo mm m u z .z.m.m.H G008 on» 000 005H0> 0000: oaflss c005 0:0 000 mmsH0> 30Hm Had 30.0 v00 000000000000 00000000000 0000000 E .05H0> Hmuaosfiummxmloum 00 uomQamu 5003 .00.0 mm 00 coamamuom 0005000250 an vasoaaom «v.0 am no 00000H00 £003 ommsmuom adamauaaa 0003 00000: so can .c08\dav 300m >u0couoo oaaoumhm 0:0 00H000000 .smma so 0000mm0 0.00000smuz .v 00009 .0000 0000: unoocmucomm 61 0uz .80 0000000 00 00000800 0023 Amo.ouvmv 0:000m00000 >000000000000 000 .z.m.m 0>00000000.H 0:008 00 00000000m 000 000000 000 .3000 000000>0 000000000 00>05o 00300 030 0:0 000 .3000 00000000 0008 000000 030 000008 020 .3000 000000000 000000 00>u5o 03» 000 000 .00.0 000 00.0 00 000000000 00 020:00xmn0so00 000000000 00 0:08\08v 300m m00couoo :0 0000mm0 0:» 00000800 005000 0009 .00 000000 62 5.0 :6 1‘ o. 93$ :2. mz.mozmo< buO. mb_xm 010— 0 2.23% 1.... was In 233:8 III N¢N In 232.3 (WW/IN) MO'IJ A8VNOHOO 63 in which the perfusate sequence was reversed (perfused at pH 7.20 immediately after stabilization at pH 7.42) appeared to be plateauing, it was of interest to see if the coronary vasculature would remain dilated for an extended period of time. To study this possibility, hearts were perfused with solution of pH 7.20 and adenosine (8x10-7M) was added to the perfusate. Coronary flow was monitored for approximately 30 minutes and flow was collected each 3-5 minutes with a graduated cylinder. Flow regularly reached a statistical maximum 5 minutes after addition of adenosine to the per- fusate (flow at this time was not significantly different from that measured at 10 minutes; Figures 11 and 12), and was found to have increased two and a half times above control. Twenty-five minutes later (30 minutes following adenosine administration) coronary flow was still two and a half times greater than control. Table 5 shows that coronary flow in these experiments was calculated and ex- pressed as both the per cent increase above control flow, and as the per cent of maximum flow. As can be seen (Table 5, Figure 12) 30 minutes following addition of adenosine, flow was still equal to 99 per cent of the re- sponse seen at minute 5. Figure 11 is a representative tracing taken from one experiment. 64 l2rnin1l6.0ml/min) 3 minfl4£ ml/min) 30mhll68mI/min) changed perfusate Adeno;ine (8x IO‘ M) lamin. (I66 ml/min) (no adenosine) pH 720. Figure ll: Adenosine (8xl0'7M) oncoronory flow for 30min. 01 perfusate 65 m.mumm~ n.mnmmm n.mnmm~ m.mHnm~ «.mummm v.mHmm~ n.mumm~ m.mHmm~ mama unmmm mm mm HOH NOH MOH OOH Nm 30am .xME M0 w mma mmH NOH moH mmH 00H hm HOHUCOU m>onm w ~.dum.oa q.¢um.ofl «.mum.oa H.&Hn.ma m.qum.oa m.QHv.oH m.QHm.m av.oum.m inflaxasv 30am om mm ON ma 0H m m o Ass oaxmv mcflmocmnm mo coaumuumacweoa may mcfizoaaom x.cHsV mafia m u z .30Hu Edstms no ucmo mom 0:» mm was Houucoo m>onm ucoo umm on» on such ommmmumxonmu can 09Hm> was» on vmummsoo Amo.ouvmv aaamoau umfiumum «H03 moumu 3on ucmcvomncm Ham aaucoavmmcoo ..cw8 m um adaaxma m monomou mafia Iocmom an voodooum 3oam humcouou .om.h am no coauaaom a nua3 cumsmumm mmz undo: may nows3 mcfiuso powwow .cfiE on m um>o much ammo: can 3on mumcouoo co mcwmocmom mo uomwum . m manna 66 muz .ONN Ia 203:8. 6 .EEOm .6 voice a .83 55.019 239.28 3 omaomto mo 30: 328.50 "2 950m 33%:me mz_mozua< 02.30.30u m2; .8 .mN .ON .0.— 1! a 1 q .0. e T! .m .m .0 mcuocmoq e U o O. 1m. ("lull I01) MO'L-l AUVNOHOD 67 IV. The Effects on Coronary Flow of Changing the pH of the Perfusate in the Absence and Presence of a Single Concentration of Adenosine (5x10‘1M) In this series of experiments we examined the effect of changing perfusate pH, in the absence and presence of adeno— sine, on coronary flow. To alter the pH, perfusate was selectively equilibrated with four experimental gas mix- tures: l) 95% 02-5% C02, 2) 98% 02-2% C02, 3) 90% 02-10% C02, and 4) 80% 02-20% C02. To determine if progressively lowering or raising the pH of the perfusate produced an additive or inhibitory action on the observed coronary flow response, the first three hearts in a group of seven hearts were exposed to the same sequence of perfusion: hearts were initially perfused at pH 7.43, followed by 6.89, 7.20 and 7.69 (Table 6, middle panel). A subsequent group of four hearts was perfused randomly following perfusion at pH 7.43 (Table 6, bottom panel). Note that the results are similar in both groups; coronary flow progressively increased as perfusate pH was lowered. The top panel in Table 6 presents means : S.E.M. from grouping the results in the other two panels and emphasizes the fact that re- gardless of the order of perfusing solution at different levels of pH, the results are similar. That coronary flow in the absence of adenosine is linearly regressed on per- fusate pH is illustrated in Figure 14 which is a negative Table 6. Effect of perfusates of differing pH (6.89-7.69) on coronary flow (ml/min) and heart rate in the absence and presence of adenosine (5x10-7M). The top panel presents mean values :_ S.E.M. from 7 hearts. Of these hearts, three were perfused in the same order: perfusate pH 7.42, 7.20, 6.89. Data from these three appear separately in the middle panel. The bottom panel shows data from the other four which were perfused randomly following perfusion at pH 7.42. In each case, the response to adenosine was compared to its respec- tive control (*), and to the adenosine response at pH 7.43 (#). Control flow rates were compared to the flow rate at pH 7.43 (o). P< 0.05 pH Control Adenosine Heart Rate 7.69:9.06 5.3:p.35 6.9:p.48*# 257:6.85 7.43:9.01 6.3:p.32 8.6:9.38* 255:7.37 7.20:9.02 7.2:p.27' l3.Q:p.60*# 25717.00 6.89:9.06 9.119.50' 18.6:l.l*# 255:9.9 7.68:9.08 4.8:p.7. 6.1:p.81*# 256:}6.02 7.45:9.06 6.419.64 7.7:p.39* 255:l6.4 7.18:9.07 7.3:p.47' 12.0:p.87*# 259:;4.9 6.87:9.02 9.4:p.46. 16.3i}.43*# 256:?1.2 7.70:9.04 5.6:9.3 7.6:p.4l*# 258:6.0 7.41:9.06 6.3:p.39 9.2:9.29* 25917.5 7.21:9.04 7.219.38. 13.8:p.53*# 2551;.5 6.90:9.33 8.9ip.85. 20.2:}.05*# 255:7.5 69 regression curve (b=-4.80) constructed by the method of least squares §=§+b(x-§) and tested for significance of linearity by an analysis of variance (Appendix A, Table A7). A 95% confidence interval (b:F0.05(5) 0.62) accompanies the regression curve. Figure 13 and Table 6 illustrate that the apparent enhanced ability of adenosine to dilate the coronaries as the perfusate pH is lowered cannot be accounted for by add- ing to adenosine's coronary effect the increase in flow due to pH alone. An interesting comparison was made between the coro- nary flow response produced by the 250 ug bolus of adeno- sine used routinely at the beginning of all experiments and the adenosine (5x10-7M) response at pH 6.89 in the current experiments. Bunger et;al, (11) reported that the decrease in coronary resistance in a similar guinea pig heart prep- aration in response to 250 ug adenosine appeared to be maximal. In this series of experiments we found that at pH 6.89 adenosine (5x10-7M) produced a flow response quantita- tively similar to that produced by a 250 pg bolus (compare top panel, Table 6 with Table 1, column 6). Spontaneous heart rate was not affected by the pH of the perfusate or by adenosine (Table 6). 70 h n z .z.m.m + mmsHm> some mm mommammflo mum mumo .mv.b mm um omGHmem umuwm mos mnemocmpm ou mmcommmu humconoo map can .mv.h mm no woman Inmm %HHMHuHcH mnm3 muummz Had .mcwmocmpm z oaxm mo mocmmmum Ucm mocwmnm may CH 30Hm mnmcouoo co mm mumwzwmmm mo powmmm one .ma wusmwm 6 N \‘g _«5 s. - N \m ‘V b .5 < 1- El “‘fi‘: V :2 2 °° e W (um/IN) M013 Auvuoaoo pH PERFUSATE FIGURE 13 72 Figure 14. Linear regression of coronary flow on perfusate pH; method of least squares used for computa- tion, linearity tested with an analysis of variance. N = 7 (o = observed mean values for coronary flow at respective pHs.) CORONARY nomad/min) O i ‘ 0‘__L L l L J 6.89 7.20 7. 43 7.69 Psarusns pH noun E 14 74 v. The Effects of Theophylline (10’6-10‘3M) on Coronary Flow and Heart Rate Reports in the literature are controversial with respect to the effects of theophylline on reactive hyperemia, and on the increased coronary blood flow produced in re- sponse to hypoxia. The nonuniformity of these reports has cast doubt on the hypothesis that adenosine is the metabol- ic mediator of coronary blood flow. In the following experiments the objective was to see if the capacity of theophylline to attenuate adenosine coronary dilation was altered by changing the hydrogen ion activity of the per- fusate. Table 7 and Figure 15 present results from preliminary experiments in which we studied the direct action of theo- phylline on coronary flow and heart rate to find concentra- tions which could be used to attenuate adenosine's coronary action but which by themselves were without a measurable effect on the heart. Concentrations of theophylline 6 to 10-4M were investigated at two levels 4 and 10-3M ranging from 10— of perfusate pH (7.43 and 7.20), while 5x10- theophylline were later investigated in a perfusate of pH 7.43 (Figure 17). Generally, coronary flow was not 6 4 affected by theophylline (10- ~10- 4 M); however, when hearts 3 were exposed to 5x10- or 10- M theophylline coronary flow was significantly (P‘<0.05) increased to 7.3:0.61 and A.Bo .Umv m.m m.v ®.m 0:50 H0663 602 lows .omm o.H~ .omm m.mH .oom m.na coflumudo .I I I Ediaé mo.o+a.ma hm.o+~.ma mm.o+v.ma 30am xmwm .nmmxwumom Asmuoaxmv.ummxm .uwmxmswum o u z coflumaao m>fluommm co AZm oaxmv unflamxmmOmna um uommwm '75 «m.mumn~ m¢.ouo.m .uwmxmumom .m.oeuqmm .mo.qwm.0H muoa «0.5mnmmm «Ho.~um.n vnods m.m¢Hvom mv.qua.w .uwaxmuwum Noo wm-~o wmm Noo amumo amm m u z ovum uummm ACflE\HEv 30am huwcouou 6.6Hmmm m~.QHH.m .ummxmumom n.muvm~ v.mH¢mm .mm.qum.m -.ouo.m vuofl o.o¢H~m~ «.mumvm o~.oum.m .m~.quh.v muoa o.o&nmm~ m.mumem m~.qwo.m m~.qum.v muoa n.amumem ~.6Hmv~ mm.qno.m om.QHo.m .ummxmumum Noo soa:~o mom Noo wm-~o «mm N8 aoa-~o «om Noo ammumo «mm m a z mama uumwm Asu\cfis\asv 30Hm mumcouoo .mwSHm> Hmucosflucmxcumum 0>wuoommou ou Umucmsoo con3 Amo.ouvmv cocoowuwcmwm Hmowumwumum monocmv Ac. .z.m.m .H memos mm pmucmmwum mum sumo .ocaaaanmomnu mo mocomnd can mocomoum 0:» ca seduca no consHooo .oom cm a mcw3oHHom 3oHu xuucouou macro Hocum aouuom .Amamcnm canvas pad mono muummn mo mucoum 039 ca 30Hm anacouoo oumumaxomoum co RSV mcwaa>£moonu mo vacuum .5 wands 76 .o~.s mm 00 soap Inflow nuHB ommsmumm maucmsvmmnsm mHmB paw «v.5 mm mo coHuSHom Suez >HHMfluHcfl pomsmnmm muck muummm .mm muMmsmumm mo mam>ma 03» um mum“ unmos mnomcmucomm paw 30am wnmconoo co sz mafiaawnmomsu mo uomwmm .mH musmflm 77 m. .59... mz_._.._>zn.ow1._. ¢a0_ 0.033.. 0-0. $-0- O.N ONN 0mm OVN 0mm owm 33.13 «8.»... -No.\.nm £2 .53 283:8 I 6.2 ENNIS N830. - «3.8 5? :8. 282.8 D 6.2 w A...%.........: 63.13 «8% - «038 5? .58 283.8 88.13 N8.6. - N oo\oom 5:5 :33 283.3 III .. m. (mm/um) M01 3 31.118 iBVBH SflOBNVlNOdS AHVNOH 03 78 10.3:0.66 ml/min (Figure 17, Table 8). Subsequent perfusion with fresh solution containing no theophylline quickly re- turned flow to its pre-experimental state. While perfusing hearts in the presence of leO-SM theo— phylline, the coronary response to an inflow occlusion of 30 seconds was observed and the peak rate of coronary flow was compared with pre- and postexperimental responses to occlusion observed in the absence of theophylline (Table 7, bottom panel). Figure 16 shows representative responses to inflow occlusion in the absence (pre- and postexperimental) and in the presence of theophylline and shows no significant difference in peak flow as measured. Although the shapes of the curves do appear to be different subsequent compari- son indicated that the time for excess flow to return half way to control was not significantly affected by theophyl- line. In quantitating the area under the diastolic portion of the curve by planimetry, it was found that theophylline decreased this area by only 8 per cent. 6 Heart rate was unaffected by 10- —lO_4M theophylline at either pH 7.43 or pH 7.20 (Figure 15). When the second group of hearts was subsequently exposed to 5x10"4 and lO-3M theophylline, heart rate was significantly (P<<0.05) increased from a pre-experimental value of 264:13.5 to 289:17.0 and 334:l0.5 beats/min respectively (Figure 17). Table 8 shows that after 10-15 minutes of perfusing the Figure 16. 79 The effect of theophylline (5x10 5M) on reactive dilation. The top and bottom panels show typical responses in the pre- and post- experimental states. During perfusion of hearts with solution of pH 7.42 (middle panel) theophylline was added to the reservoir prior ' to occluding inflow. N = 6 80 Peak Flow l3.0mI/min. 20 I20 ml/min. Theophylline (5x IO'5M) III III! ‘IIIIIIIIIIIIIIIIIIIIIIII‘IIIIIII' IIIIIIIII IIIIIIIIIIIIIIIII III III IIIIIIIIIIIII IIIIIIIIIII mIIII IIIIIII IIIIIIII'I'S'I IzIII IIII IIIIIIII IIIIIIIIIIIIIII III. I'IIIIIIIIIII'” III IIsIIIIII!IIIIIIIIIIIIIIIIIIII 20 flu: iIIIIIII'gggI: _: II IIIIIIIIIIIIIII II'IIIIIIIIIII :IIIII 'IIIIIIIIIII IIQIIIIIIIIIIIII '20 mein lo __ ._ ' I::.I|I. Ixmim IIIIIIIIIIII 30sec. Postexperlmemal Figure I6 81 m n z .on Houuaoo m>Huommmmn nuw3 cmummfioo cm£3 Amo.ouvmv mocmowmacmflm Hmowumwumum mmuoamo A«v .z.m.m_H mamms mm nmucmmmum mum mama .mv.h mm mo coausaom SUHB cwwSMMmm mumz muumwn c033 mumu pummn msomcmucomm cam 30am mumcouoo co mafiaamzmomnu mo pommmm .FH musmflm 82 m-o_ 2 89w; Zmzfidwmmommh Toma 0 TS 72$ 0 a V H I. H v _ I. 3 w coo-{rote G . v-I _ (UIHI‘IIH) M013 AUV ouoo 83 Table 8. Effect of theophylline on the coronary vasodilation produced by adenosine in perfusates with two different [H+]. Hearts were initially perfused with solution of pH 7.43 followed by a perfusate of pH 7.20. (*) indicates statistical signifi- cance (PI&OuI.PI. 2.70. 20.0. 5.0.0— 0 «ll-Ill _ + $3.13 Novoxom .. «ooxomm 5:3 8.29.53 2823a 83:3 N8.90. -mo.\.om 5.; 8.29.38 288.8 .. .... (WW/It”) MO'IJ AHVNOUOO 86 4M) attenuated the response to 8xlO-7M adenosine. The (10‘ maximum coronary flow was 8.2:O.5 ml/min in perfusate of pH 7.43. When hearts were subsequently switched to a per- fusate of pH 7.20, adenosine (8x10—7M) produced an increase in flow similar to that seen at pH 7.43 in response to the same concentration of adenosine. Theophylline (lo—4M) again produced a significant (P 30Hm mucmmmum m>uso um3oq .mv.n mm as oo3oaaom om.h mm um umuflm ommsmumm muumms aw 30am mumcouoo mzoSm a>uso momma .mH pcm ma mousmflm Eouw mum mama .Az loaxmv mcflmocmpm an omosoonm coflumaap humcouoo as» so mcwaawcmomnu mo uommmm .om musmam 91 ON 83E 2.: mz.._._>In_ouI._. «.9 0.0.6 ob ab. 0 0 d - q u d Nvu. In 8 cofiatoo BEE iil CNN In so :23th 655'» r lilllll 4.2%.me mz_mozmo< N_ m. (WW/ImiMO'Id AHVNOHOO 92 Hydrogen ion is also involved in controlling local blood flow and attempts have been made to evaluate its contribu- tion to reactive hyperemia. However, no one to date has examined the effects on reactive hyperemia of trying to simultaneously attenuate the possible contributions of potassium, hydrogen ion and adenosine to this response. We have made an attempt to do this and our results are as follows. Reactive dilation: Following release of a 30 second occlusion of the aortic inflow cannula, peak flow, volume of coronary flow for 20 seconds (VCF and the time re- 20)' quired for excess flow to return 50 per cent toward control (T50) were examined in the absence and presence of concur- rent theophylline, ouabain and alkalosis (perfusate pH 7.69). Control flow was not changed by these conditions. Results are presented in Table 10. Peak coronary flow, in the presence of theophylline, ouabain and alkalosis was slightly, but significantly reduced from 15.719.64 to 14.71 0.55 ml/min. VCF20 and T50 were also reduced during experi- mental conditions, but failed to return to control values in the postexperimental state. Figure 21 shows a repre- sentative tracing of reactive dilation before (pre—experi- mental), during (experimental) and after (postexperimental) treatment with test agents, and illustrates that the changes seen in the presence of test agents were slight. Table 10. 93 Effect of concurrent theophylline (5x10-5M), ouabain (1.4x10 M) and alkalosis (perfusate pH 7.69) on coronary flow following release of a 30 sec. inflow occlusion. VCFZO = volume of flow in first 20 seconds following release of occlusion. T50 = time required from release of occlusion for the flow rate to return to 50% of peak flow. Values are presented as the mean 1 S.E.M. (*) denotes statistical (P‘<0.05) significance when compared to pre-experimental values. N = 8 Peak flow (ml/min) VCF20(ml/min) T50(sec.) Pre-experimental 15.710.64 3.710.15 13.910.40 Experimental l4.710.55* 3.210.l9* 12.310.48* Postexperimental 14.910.58 3.310.23* 11.910.72* Figure 21. 94 Effect of concurrent theophylline (5x10 5M), ouabain (1.4x10 M) and alkalosis (perfusate pH 7.69) on reactive dilation following release of 30 sec. occlusion. Top and bottom panels show reactive dilation in the absence of test agents. N = 8 CORONARY FLOW(ml/n'dn) 95 PRE- EXPERIMENTAL " . “’ -w1 p... . .Ii’1 "7*: 7 ' w-ji ~44 a-‘ _. I , .~ .1 l t. . i -. . I I i" i—s‘ l ‘i J l' . HA -. 'I' 2 w J 2' " Peak Elow-ISOml/lnin Adjust mechanical o vet-'20 . 3.6ml Tso' l2.5 sec. EXPERIMENTAL . '. H‘ . , .i ; - i . l "W" I l " z 3‘ 1 ‘ ' ' i ' 'p-N “ , . ‘ ' . . ‘ . I i A o . . . l I on-..— ».._p..——... -_.-_+—,—-a—.-- A .9. o : o .y 20 l0 | l ‘ v t . ' l Peak F low . I4.0ml/rrh vcr20 - 3.3ml T50 ' '2. 3 sec. Pea‘k Flaw-Isomvmin VC I"20 - 35ml Figure 2| 96 Autoregulation: In the same group of hearts, the effects of theophylline, ouabain and alkalosis on autoregu- lation were investigated. Increasing perfuson pressure from 65 cm H20 to 95 cm H20 in the absence of test agents transiently increased flow from 6.310.41 to 11.410.61 ml/ min; it then quickly fell to 8.510.57 ml/min. Calculated resistance to flow increased nonsignificantly from 10.319.68 to 11.210.76 cm H20/ml/min. The reservoir was then quickly dropped to a point which produced a perfusion pressure of 35 cm H20 and flow decreased transiently to 1.410.37 ml/min, stabilizing at 4.410.13 ml/min. Calculated resistance decreased following the reduction of perfusion pressure to 8.0 cm HZO/ml/min. Subsequently, the reservoir was raised to return the perfusion pressure to 65 cm H20. This pro- duced a transient flow response typical of that seen upon release of a 30 second inflow occlusion. The perfusate containing theohyplline (5x10—5 M). ouabain (1.4x10-7M) and made alkalotic by equilibrating with 98% 02-2% C02 (pH 7.69) was then perfused through the system (with no observable effect on coronary flow or heart rate) and the pressure—flow responses were again observed (Table 11, Figure 22). Coronary flow was increased from 6.110.44 to 9.710.53 ml/min and calculated resistance increased from 9.812.0 to 13.310.64 cm HZO/ml/min when increasing perfusion pressure from 65 cm H O to 95 cm H 0. Subsequent lowering 2 2 97 m.m o.m «h.m v.v m.H ¢.H m o m o m U m m 2 0mm Eu mm o.mH ~.HH em.h m.m «h.m ¢.HH m.oa n.0H H.@ m.@ m o m o m U m . U u U m m m . . m m 0mm 50 mm. 0mm Eu mm Anamoamxam can cwmnaso , . .acwaawnmoanu ..m.Hv Hmucaefiuamxa u m ousmmaum ooum>maa um 30am xmam u m . - . \ Houucoo u U Aswa\aev 30am aumumimpmaum u m aucmmaum coumsoa um 30am Edeficws u 2 0mm 80\cws\aev oocmumflmou u m “max m a z .usHm> Houucoo a>wuoammou saws paumeoo cmn3 Amo.o my aucaowmwcmwm Honeymfiumum maumoflocw e .ousmmanm cowmcmuam new Immunoac 6cm mcwmmouoafi an uncommon ca aocaumflmou pmumasoamo 0cm Acfls\aav 30am anacouou co memoamxaa can Ash oaX¢.HV camnaso eAz oaxmv acaaaanmoonu ucmuusocou mo uoammm .HH MHQNB m 98 Figure 22. Effect of concurrent theophylline (5x10 5M), ouabain (1.4x10’7M) and alkalosis (perfusate pH 7.69) on coronary autoregulation. Left side shows responses in absence of test agents and right side shows responses in presence of test agents. N = 8 CO RONARY FLOW (mil min ) 99 Increasing perfusion pressure from 65cm H20 to 95cm H20. Control Conditions Experimental Condtions ..- .4 . v ’-o%‘- Increasing perfusion pressure from 35cmH20 to 65cm H20. Figure 22 100 of perfusion pressure to 35 cm H20 produced a transient reduction in flow, but flow quickly stabilized at 3.710.19 ml/min. Calculated resistance fell to 9.510.48 cm HZO/ml/ min which was significantly (P‘<0.05) higher than that seen at the same pressure in the absence of test agents. IX. Effect of Concurrent Theophylline (5x10-5M), Ouabain (1.4xlO'IM) and Alkalosis_(Perfusate pH 7.69) on Hypoxic Coronary Flow In a group of seven hearts the coronary flow response to hypoxia was investigated in the absence of theophylline, ouabain and alkalosis. When coronary flow was stable under control conditions, hypoxia was induced by switching hearts to a previously prepared reservoir equilibrated with 20% 02-5% C02 balance N2. Mean flow rate was increased from 6.1 1 0.52 ml/min to 14.110.83 ml/min. Diastolic flow and systolic flow were increased in a similar fashion (Figure 23). Hearts were then switched to fresh perfusate (pH 7.43), control data recorded (Figure 23, C2) and subsequent perfusion with test solution containing theophylline (5x10-SM), ouabain (1.4x10'7M) and equilibrated with 20% 02-2.5% C02 - balance N2 was begun. Mean flow under these conditions increased from 6.010.54 ml/min to a maximum of .14.010.62 ml/min. There was no difference in the maximum nnean flow achieved by hypoxia in the absence or presence of élrugs. Diastolic flow in the presence of test agents 101 n u z .z.m.m H mamme mum mumn mac .mucomm umou mo mocomnm n 4 .mucomm ummu mo oocomoum u m .A z mocmHmnl oo wmi o mom aufl3 pmumnnflaadqo anomSMHomv maxed»: on omcommou cfl 30Hm mumcouoo so Amm mummSMMomv mwmoaoxam 0cm Ask oaxs.ae aaaaaao .izmtoaxme aaaaasaaoaau uaaauaaaoa ma aoaumm .mm masons 102 §\\\\\\\\\\\§ \\ 0' \ .x. 1 r2 i l DIASTOLIC _] 1 Cl v ‘ . \‘:«:»::;~X»» ‘~:~?:i\\?:\ ‘-\R\>\\~‘\*—‘ ~ KS» n r \\\ \\\\\\\“‘ A c2 P SYSTOLIC - A" ///'l /97//, /'/,/ ‘,/ 1' _‘//’ . ‘, / // f V/ // 7/4 ,/ / z "/ C , // .- . ,/ A/V '/’-/" /./ / ,,// ,’ . A I'~/.’/x' / z r . //) CI 5 E] I Hypoxia \ \ \ \ §\\§Q\\\\\ ‘ .\\ \\ \KV . x ‘9; ‘ \ _\ ‘ \ “‘\\ ‘ ‘x‘\ g ‘ . \f‘ ~.\ ~.\\ W\ C (“NJ/IN) MO'H AHVNOHOD Figure 23 103 reached 21.110.74 ml/min as compared to 22.511.0 ml/min in their absence. Again statistical analysis revealed no sig- nificant difference. DISCUSSION Among the locally produced metabolites known to dilate the coronary vasculature, perhaps adenosine is the strong- est contender for the role of metabolic mediator of coro- nary flow. However, for some time following the introduc- tion of the adenosine hypothesis, investigators failed to find adenosine in coronary venous blood during ischemia and hypoxia. This was a source of criticism for the hypothesis because a physiologic mediator of blood flow must be present in the effluent during those conditions which evoke changes in blood flow. In addition, the proposed mediator must be present in quantities sufficient to produce the observed change in flow. With the improvement of experi— mental techniques it was soon shown that adenosine does appear in coronary venous perfusate during ischemia/hypoxia in quantities sufficient to produce the observed vasodila- tion. Currently the adenosine hypothesis faces another source of opposition. Skepticism regarding the hypothesis exists since the finding (2,27,28,56,72) that aminophylline and theophylline fail to attenuate coronary reactive hyperemia and hypoxic coronary dilation, while successfully attenuating 104 105 coronary dilation by exogenous adenosine. Work reported herein was aimed directly or indirectly at studying the possibility of an effect of perfusate hydrogen ion activity on adenosine's ability to dilate the coronaries, and/or on theophylline's ability to attenuate adenosine's coronary action. We investigated two major questions. The first being: Does altering the perfusate hydrogen ion activity affect the coronary adenosine dose- response relationship seen at perfusate pH 7.42? The second question was: Is the ability of theophylline to attenuate adenosine-induced coronary vasodilation pH sensi- tive? These questions are relevant to the observation that theophylline has little effect on coronary reactive hyperemia or coronary hypoxic dilation. I. Adenosine Coronary Dilation as Affected by Perfusate Hydrogen Ion Concentration Approaching the first question we compared the coronary dose-response curves for the effect of adenosine on the coronary vasculature in hearts perfused with solutions of different hydrogen ion concentrations. Several findings were of interest. We found that progressively lowering perfusate pH from 7.43 to 6.89 (in the absence of adenosine) produced an increase in flow which was significantly greater than the stable flow at pH 7.43. In none of these 106 experiments did altering the perfusate pH have an effect on spontaneous heart rate; therefore, the decrease in flow was attributed to a direct vasodilating action by hydrogen ion on the coronary vasculature. It is possible, however, that the increased flow seen when lowering the perfusate pH was not due to a direct action by hydrogen ion on the coro- nary vessels but rather was due to an indirect increase in transmural distending pressure subsequent to weakened cardiac contractile strength. No attempt was made to assess cardiac contractile activity so that the possible contribution to the increased flow produced by this mechanism is uncertain. At pH 7.43, 7.36 and 7.20, 10-6M adenosine produced rates of coronary flow which were not different from each other. However, when lowering the pH to 6.89, 5x10_7M adenosine produced an increase in coronary flow markedly greater than that achieved by lO-GM adenosine at higher pHs. Statistical treatment of the data at pH 6.89 revealed an interaction between adenosine and perfusate pH in producing the observed response. That this enhanced response at pH 6.89 must be related to potentiation of either adenosine's or hydrogen ions actions is demonstrated by the fact that the coronary flow increase cannot be attributed to summa— tion of the effects of hydrogen ion (pH 6.89) on coronary flow with the effects of adenosine (5x10-7M; pH 7.42) on coronary flow. In other experiments the perfusate pH was 107 increased from 7.43 to 7.69, but coronary flow was not affected. If decreasing pH from 7.42 to 7.20 produced an acidosis which weakened contractile strength, yielding a passive increase in flow, one might argue that alkalosis could improve contractile strength, thus reducing coronary flow. When adenosine (5x10-7M) was added to the perfusate at pH 7.69, both diastolic and mean flow increased to a much lesser extent than previously seen at pH 7.43 when the same adenosine concentration was used. Statistical analysis indicated a significant interaction between adeno- sine and hydrogen ion to produce this response. Thus, it is possible that the decreased hydrogen ion activity pro- duced a direct reduction in adenosine's dilating action, or perhaps myocardial contractile strength was increased thereby decreasing the transmural pressure gradient and minimizing the adenosine-flow response. How does one explain the fact that the perfusate hydrogen ion activity seems to potentiate adenosine's coro- nary action at a pH of 6.89 while lowering the hydrogen ion activity of the perfusate to a pH of 7.69 retards adeno- sine's ability to dilate the coronary vasculature? Recently Raberger, Weissel and Kraupp (73) reported from studies per- formed on an anesthetized dog, that adenosine's (intracoro- nary injection, l-2 ug/kg) coronary dilator action corre- lated directly with the hydrogen ion concentration of the 108 blood. They found that this effect of adenosine was sig- nificantly dependent on arterial blood pH only if changes in systemic blood pH were accompanied by comcomitant changes in extracellular buffer capacity. A decrease in buffer capacity and pH (i.v. infusions of 0.1N HCl, 2 ml/min) led to an increase in adenosine's dilating action, while a rise in pH and extracellular buffer capacity (i.v. infusions of either 5% NaHC03, 5 ml/min, or tris-hydroxyamino- methane, THAM, 1M, 2 ml/min) resulted in a decrease. Eberlein (23) also reported that adenosine's coronary dilating action increased as arterial PCO2 increased. Moreover, the effects of dipyridamole and hexobendline- coronary dilators with a potentiating effect on adenosine's coronary action (27,28,62) were found by Wolner and Kraupp (94) to be significantly dependent on the extracellular pH. In the isolated perfused guinea pig heart we found that varying the pH of perfusing solution over the range of 7M adenosine resulted 7.69 to 6.89 in the presence of 5x10— in changes in coronary flow not unlike those reported by Raberger §1_31, (73). As perfusate pH was decreased from 7.43 (control) to 6.89 a marked increase in coronary flow was produced which could not be accounted for by simply adding the individual effects on coronary flow regularly produced by pH 6.89 and by leO-7M adenosine. Subsequent statistical treatment of these findings revealed that 109 indeed lowering the pH to 6.89 produced a potentiation of adenosine's ability to dilate the coronaries. Moreover, increasing perfusate pH from 7.43 to 7.69 resulted in a reduction of adenosine's coronary dilating ability. Thus, although altering the pH was accomplished differently in the two studies, the results are similar and might be suggestive of a common mechanism of "interaction" by adenosine and hydrogen ion on the coronary vessels. Further, Raberger §£_31, (73) reported other observa- tions indicating that concurrent with adenosine's coronary action was an increase in intracellular lipid and carbo- hydrate metabolism accompanied by increased release of hydrogen ions and total CO from the myocardial cells. 2 Increased total C0 release from the myocardium would have 2 an effect on extracellular buffer capacity similar to the infusion of acid in Raberger's study; both would reduce buffering capacity. Raberger postulated that the effect of adenosine on myocardial lipid and carbohydrate metabolism created a metabolic acidosis which was transferred to the coronary vasculature. When these investigators infused THAM (tris-hydroxyaminomethane) prior to HCl administration, they found that even though arterial pH did decrease, five times more HCl was required to drop the pH by 0.1 unit than was required when THAM was not administered prior to HCl. Also, in the THAM-pretreated experiments, arterial pH 110 shifted from 7.7 to 7.1 but adenosine's coronary dilator action was not enhanced. This was attributed to the fact that THAM entered the myocardial cells and buffered much of the hydrogen ion before it could be released from the cell. From this study they concluded that adenosine's coronary dilating action was mediated by an effect on myocardial metabolism as well as an effect on the coronary vasculature. It seems reasonable to speculate that during myocardial ischemia and/or hypoxia there is an increased release of hydrogen ion from the tissues concomitant with an increased release of adenosine. The increase in hydrogen ion activ- ity conceivably could affect appropriate coronary adenosine receptors thus increasing the vascular response to adeno- sine. Studies dealing with hydrogen ion activity in exer- cising skeletal muscle and those concerning adenosine's vascular action in this bed might be relevant to the specu- lation that concomitant release of adenosine and hydrogen ion from the myocardial cell during ischemia interact to dilate the coronary vessels. Rudke eE_21. (81) and Goll- witzer and co—workers (37) reported that associated with exercise hyperemia in active skeletal muscle is an increase in venous blood hydrogen ion activity. Although no attempt was made in these studies to assay for adenosine in the venous effluent, Berne and his co-workers (8) reported from constant—flow experiments on exercising skeletal muscle the 111 appearance of adenosine in venous blood coming from the exercising limb. They proposed that adenosine could be the mediator of the hyperemia associated with exercise. Additionally, Rubio gE_§1. (78) were able to find adenosine in coronary venous blood during reactive hyperemia, but made no attempt to measure hydrogen ion activity. Thus, from such reports as those made by Rudko (81), Gollwitzer (37), Berne (8) and Rubio (78) it seems possible that a con- comitant release of adenosine and hydrogen ion from exercis- ing skeletal muscle and/or from stressed cardiac muscle could produce an increased blood flow that is due to potenti- ation of adenosine's actions by hydrogen ion. Other possibilities could account for the apparent enhanced coronary action of adenosine at a low pH. Evidence indicates that cardiac contractile strength is weakened by an acidotic perfusate (59). Decreased extravascular com- pression of the coronaries resulting from weakened contrac- tile strength would result in a net increase in coronary transmural pressure (passive dilation). An increase in coronary blood flow could accompany such an action. Fuchs §E_§1. (33) recently found that hydrogen ion inhibits the binding of Ca2+ by troponin in cardiac muscle, and Katz (59) reported evidence that a fall in intramyocardial pH, as in ischemic myocardium, might be directly responsible for a reduction in contractile strength. 112 More speculative mechanisms for the increased response to adenosine at a low pH could include an inhibitory action by the increased hydrogen ion activity on enzymes responsi- ble for the degradation and/or rephosphorylation of adeno- sine. Such an action would possibly result in greater tissue concentrations of adenosine. In keeping with such a possibility is the report by Jacob and Berne (49) that adenosine deaminase (the enzyme responsible for the deamina- tion of adenosine to inosine) and nucleoside phosphorylase (an enzyme capable of rephosphorylating adenosine to yield AMP) are leached out of the isolated, perfused cat heart during recirculation of perfusate. Based on the assumption that during myocardial ischemia and/or hypoxia a concomitant release of adenosine and hydro- gen ion occurs, our finding that hydrogen ion potentiates adenosine's coronary dilation could possibly explain, in part, theophylline's inability to block reactive hyperemia. It is conceivable that increased hydrogen ion activity in the vicinity of the coronary adenosine "receptor" helps adenosine more effectively dilate the coronaries thus pre- cluding the0phylline attenuation. In the experiments discussed thus far, the coronary action of adenosine was always initially examined in a per— fusate of pH 7.43 followed by subsequent perfusion at a pH of either 7.36, 7.20 or 6.89. We therefore deemed it 113 necessary to "reverse the sequence of perfusion," i.e., assess adenosine's coronary effects first at pH 7.20 then switch to a perfusate of pH 7.43 and again treat with adeno- sine. This seemed important for the sake of detecting a patterned adenosine coronary response which might have resulted from the experimental bias of always perfusing hearts at pH 7.43 initially. Figure 8 presents data from experiments in which coronary vessels were perfused first with solution of pH 7.20 followed by perfusion at a pH of 7.43. Comparison of the results shown in this figure with results presented in Figure 5, in which coronary vessels were perfused initially at pH 7.43 followed by perfusion at pH 7.20, reveals some marked differences. We discovered that lO—GM adenosine in a perfusate of pH 7.20 produced a much greater response in hearts perfused first at pH 7.20 than in those hearts perfused first at pH 7.43 followed by perfusion at pH 7.20. Note that the portion of the curve (Figure 8) at pH 7.20 from lO-BM adenosine and beyond has been shifted up and to the left. This indicates that at a low pH less adenosine is required to produce a given coro- nary response, or alternatively, increasing perfusate pH to 7.42 competitively antagonized the ability of adenosine to dilate the coronary vasculature. At pH 7.42, ten times as much adenosine was needed to produce a coronary response similar to that produced by lO-7M adenosine at pH 7.20. 114 Hence, this finding suggests that adenosine might be acting at a coronary receptor site which is sensitive to hydrogen ion. Statistical analysis of the coronary response to adenosine in hearts perfused first at pH 7.20 suggests that the difference in adenosine's action from that at pH 7.20 in hearts first perfused with solution of pH 7.43 results from potentiation by hydrogen of the adenosine action in the former (Appendix A, Table A5). The only difference in protocols of experiments presented in Figures 5 and 8 is that of reversing the order of perfusion heretofore used, i.e., we perfused initially with a perfusate of a pH other than 7.43. In our early experiments in which hearts were first perfused with solution of pH 7.43, a new perfusate with a lower pH was not introduced for some 30 to 45 minutes following initial stabilization of coronary flow. During this period of time, the hearts probably achieved a steady state between the metabolic production of hydrogen ions and CO2 and the ability of the NaHCO3 in the perfusate to buffer. Conversely, hearts first treated with solution of pH 7.20 were not allowed the additional 30 to 45 minutes to achieve stable buffering conditions. Consequently, the abil- ity of NaHCO to buffer the increased hydrogen ion of the 3 perfusate in addition to that released from the myocardium might have been temporarily overwhelmed, allowing hydrogen 115 ion concentration to increase and resulting in significant potentiation of adenosine's coronary action. II. Adenosine's Coronar Action at a Low pH for an'Extende" eriod of Time Examination of the results from experiments in which hearts were initially perfused with solution of pH 7.20 followed by perfusion at pH 7.43, revealed that the coronary 7 6 response to 5x10“ M and 10- M adenosine at pH 7.20 were not statistically different and that at 10-6 M, the increase in coronary flow was reaching a plateau. A steady level of coronary flow at pH 7.20 in response 7-1076M adenosine raised the question of whether or to 5x10" not adenosine can maintain an increased coronary flow over an extended period of time at a low pH. An answer to this question seemed pertinent to the adenosine hypothesis for if adenosine is to be assigned a significant role in producing and maintaining the increase in coronary flow accompanying prolonged exercise, it must be shown that adenosine's coro- nary dilating action does not diminish during periods of heavy myocardial work. Additionally, this experiment served as a means of testing if attenuation of the adenosine coro- nary response by lO-4M theophylline (from other experiments in this study) was partially due to a waning of adenosine dilation with time. The finding that adenosine dilation at 116 a low pH is maintained for 25-30 minutes is interesting. Adenosine has been postulated not only as the mediator of coronary blood flow, but of skeletal muscle blood flow dur- ing exercise (8). Additionally, it is known that the pH of venous effluent blood from active skeletal muscle decreases (37,81) during exercise. With periods of increased myo- cardial oxygen demand, coronary flow increases and adeno- sine has been found in the coronary venous effluent (78,79). If myocardial tissue pH and/or coronary venous effluent pH decrease under conditions of increased oxygen demand, it would be tempting to speculate that the low pH coupled with simultaneous adenosine release interact to produce the in- creased flow in both of these vascular beds. To my knowl- edge, the ability of adenosine to maintain an increase in skeletal muscle blood flow has not been studied over an extended period of time. Again, statistical evaluation of this response suggested that adenosine's action was being enhanced by the hydrogen ion concentration of the perfusing fluid and that under these conditions of low pH adenosine is capable of maintaining elevated coronary flow for at least 25 minutes. 117 III. Effect of Variable Perfusate pH on the Coronary Action of a Single Concen- tiSn of Adenosine Varying the pH of perfusate stepwise between 7.69 and 6.89 as well as randomizing the order of perfusion at dif- ferent pHs produced a marked increase in coronary flow. When adenosine (5x10-7M) was subsequently added to the per- fusate, we found that its dilating action when compared to that at pH 7.43, was markedly enhanced by perfusates with a low pH and noticeably diminished at pH 7.69. ~Thus, regard- less of whether our studies dealt with a range of adenosine concentrations at a constant perfusate pH, or if they assessed the action of a single concentration of adenosine over a range of perfusate pHs, the coronary dilating capa- city of adenosine was affected similarly by the hydrogen ion activity of the perfusing fluid; decreasing the hydro- gen ion activity inhibited adenosine's coronary actions and increasing the hydrogen ion activity enhanced adenosine's coronary action. An interesting sidelight of the experiments in which the coronary action of a single concentration of adenosine was investigated over a range of pHs is the finding that upon reducing the perfusate pH to 6.89, the extent of coro— nary dilation produced by adenosine (5x10-7M) was virtually equal to that produced by a 250 ug bolus of adenosine at pH 7.43. Others (11) have reported that in the isolated 118 perfused guinea pig heart maximum coronary dilation is pro- duced by a bolus of 250 pg of adenosine. Another report (78) indicates that infusion of 56 nmoles/lOO ml adenosine produces maximum coronary dilation in the dog. Both of these concentrations are much greater than 5x10-7M used in our experiments. Thus, several of our findings concerning adenosine coronary dilation as affected by changes in the perfusate pH, have shown that increasing the hydrogen ion activity in the perfusing fluid enhances adenosine's dilating ability. It therefore seems reasonable that if the tissue pH de- creased during reactive hyperemia and/or hypoxia, theophyl- line's failure to block these responses might be partially explained on the basis that the increased hydrogen ion activity enhances adenosine dilation of the coronary vascu- lature thereby precluding theophylline attenuation. IV. Theophylline Attenuation of Adenosine Coronary Dilation as Affected’by Perfusateng Using concentrations of theophylline that produced no observable change in coronary flow, we sequentially attenu- ated the increase in coronary flow produced by 8xlO-7M adenosine and found that in hearts perfused initially with solution of pH 7.43 followed by perfusion at pH 7.20, theo- 6 4 phylline's (10- -10- M) ability to attenuate adenosine's 119 coronary dilation was virtually unaffected by the hydrogen ion concentration of the perfusate. When we subsequently conducted experiments in which hearts were initially exposed to perfusate of pH 7.20 and were later perfused at pH 7.42 we found that the total absolute reduction in flow produced by theophylline at pH 7.20 appeared to be greater than that seen in previous experiments at pH 7.20 or 7.42 by the same concentration of theophylline. However, this apparent dif- ference in effects by theophylline is not interpreted as being a “potentiation," by the increased hydrogen ion con- centration, of theophylline's capacity to block adenosine at a low pH, but rather can be explained on the basis that at pH 7.20 adenosine was more effectively dilating the coro- nary vessels, thus providing theophylline with a larger flow to attenuate. When hearts were perfused at pH 7.42 first and then switched to pH 7.20, theophylline (lo-4M) was successful at both pHs in attenuating the adenosine response. However, in hearts which were immediately switched to per- fusate of pH 7.20 following stabilization, theophylline (lo—4M) at pH 7.42 failed to significantly attenuate the adenosine response (Results, Section VII, Figures 18 and 19). Tables 8 and 9 indicate that adenosine's dilating action at pH 7.43 under these two sets of conditions was different. Table 9 shows that adenosine (3x10'7M) failed to dilate as well at pH 7.43 when hearts were first exposed to perfusate 120 of 7.20 as compared to its dilator action at pH 7.43 in hearts first perfused at pH 7.43 (Table 8). This might suggest that under the former conditions, less adenosine was binding to respective receptors, thus providing a reduced amount of flow for theophylline to attenuate. In the latter condition perhaps more receptors were activated by adenosine thus providing a greater amount of adenosine- induced flow for theophylline to competitively antagonize. we thus concluded from these results that the inability of theophylline to block reactive hyperemia is probably not affected by an increased hydrogen ion activity in the blood. Had theophylline's ability to attenuate adenosine coronary dilation been diminished at the low pHs, then the failure of theophylline to attenuate reactive hyperemia might have been explained. V. Coronary Reactive Hyperemia as Affected by Theophylline and Perfusate pH Any contribution by adenosine to coronary reactive hyperemia must be reconciled with reports that theophylline, a competitive inhibitor of adenosine (10,11), fails to ».block, consistently, reactive hyperemia (9,27,28,55,56). Several investigators (15,78,91) have suggested that some of the conflict concerning theophylline's inability to regularly block reactive hyperemia is attributable to the ways in which investigators variously characterize reactive 121 hyperemia (9,15,56,78). Others have proposed (15,84) that tissue concentrations of adenosine during occlusion of the coronary vessels reach levels which can not be successfully blocked by theophylline. Still there are those (84) who argue that usable concentrations of theophylline (those which do not exert cardiotonic actions) are too weak to block coronary reactive hyperemia. We have found that in the presence of 5x10—5 M theophyl- line and at pH 7.43, peak reactive dilation resulting from a 30 second inflow occlusion was not reduced when compared to a control response in the absence of theophylline. However, peak flow may not be a good index of reactive hyperemia as evidenced by the study of Curnish and co- workers (15). They found that aminophylline, while without a measurable effect on peak reactive hyperemia, reduced the volume of reactive hyperemic flow, and the duration of reactive hyperemia by 42 and 31 per cent respectively. In our study we found that in the presence of leO-SM theo- phylline, the total area under the diastolic hyperemic curve (quantitated by planimetry in sq. cm) was only re- duced by 8 per cent as compared to control, and that T50 was not affected. Also studying reactive hyperemia, wadsworth (91) found that in the presence of aminophylline (10 mg/kg i.v.) the duration of reactive hyperemia was noticeably reduced when compared to control responses in the 122 absence of aminophylline. Conversely, Eikens and Wilcken (27.28) reported that in their studies, aminophylline (10 mg/kg, slow i.v. injection) failed to affect either the duration of reactive hyperemia or the volume of excess flow following release of 4-, 8- and 60 second occlusions. Nadsworth's experiments were performed on anesthetised cats, and reactive hyperemia was studied by occluding the left anterior descending coronary artery for 30 seconds. Eikens and Wilcken studied unanesthetised greyhounds when occluding for 4 and 8 seconds, but used anesthetised mongrels when studying responses to 60 seconds of occlusion. In Eikens and Wilckens studies they found no qualitative dif- ferences in reactive hyperemic responses in the presence and absence of aminophylline whether animals were anesthetised or unanesthetised. Perhaps in our experiments and in those of Curnish §1_§1, (15), peak flow was not affected because it is more strongly influenced by the vascular distending force produced by the sudden surge of perfusate as the in— flow occlusion is released, while the duration and volume of flow might be more strongly affected by vasodilator metabolites, and are therefore more susceptible to blockade by theophylline/aminophylline. Later, when attempting to attenuate the possible contribution of adenosine, potassium and hydrogen ions to reactive dilation, we found that the concurrent presence of 123 theophylline (5x10-5M), ouabain (1.4x10-7M) and alkalosis (perfusate pH 7.69) produced a small but significant effect on peak flow following release of the occlusion, and was responsible for a slight but significant reduction in the volume of coronary flow and in the time required for flow to return half way toward control. Several minutes after perfusing with fresh solution, coronary inflow was similarly occluded and upon release it was found that the volume of coronary flow and time for flow to return toward control were still reduced. Hence it was concluded that the reduc- tion in responses during the postexperimental state was due to the fact that test agents were still present or con- versely, that the slight reduction in volume of flow and in time for flow to return toward control in the experimental state was due to something other than an effect by theo- phylline, ouabain and alkalosis. No attempt was made to study, individually, the possible contributions of potas- sium and hydrogen ions to the reactive hyperemic response. VI. Effect of Concurrent Theophylline, Ouabain and Alkalosis on Hypoxic Coronary Dilation Others (2,91) have been unsuccessful in blocking, with theophylline/aminophylline, coronary dilation produced by hypoxia. In an effort to minimize possible contributions by adenosine, potassium ions and hydrogen ions to the coronary 124 response to hypoxia, we added theophylline and ouabain to an hypoxic perfusate and made it alkalotic by reducing the con- centration of carbon dioxide. The response of the coronary bed under these conditions was compared to a similar coro- nary response produced by hypoxia in the absence of test agents and no difference was found. It appears that with the particular blocking agents used in this experiment, in conjunction with the degree of hypoxia produced, our results fail to support but do not rule out a role by adenosine, potassium ion and hydrogen ion in producing the coronary dilation accompanying hypoxia. It is possible that tissue levels of adenosine during hypoxia were not blockable by the concentration of theophylline used in this experiment. Bunger eE_§1, (11) have recently reported that theophylline is a competitive antagonist of adenosine and that theophyl- line attenuation of adenosine coronary dilation can be greatly overcome by increasing the concentration of adeno- sine in the perfusate. Failure by Afonso and co-workers (2) in the dog, and by Wadsworth (91) in the anesthetized cat to block hypoxic coronary dilation with amin0phylline led these investigators to conclude that adenosine is prob- ably not involved in producing the observed coronary hyperemia. Conversely, Scott gE_a1. (84) have found that lO-3M theophylline, a concentration much greater than can be used to block coronary hypoxic/reactive hyperemias in 125 the isolated perfused heart, is effective in abolishing renal vasoconstriction produced by perfusing an isolated, denervated bioassay kidney with hypoxic coronary sinus blood from a donor dog. Additionally, theophylline (10’3m) blocked renal vasoconstriction produced by injection of adenosine (lo—20 ug) into the renal artery of the bioassay kidney. VII. Effect of Concurrgnt Theophylline, Ouabain and Alkalosis on Coronary Autoregulation Ono and co-workers (71) reported that pretreatment of the renal vascular bed with theophylline-ethylenediamine (aminophylline) blocked the kidney's ability to autoregu- late. We compared the ability of the coronary bed to auto- regulate in the presence of concurrent theophylline (5x10—5M), ouabain (1.4x10-7M) and alkalosis (perfusate pH 7.69) with its ability to autoregulate in the absence of these test agents. Upon raising perfusion pressure from 65 cm H20 to 95 cm H20 we found that in the presence of test agents both peak flow and steady state flow at the elevated pressure were significantly reduced while calcu- lated resistance was increased. In explaining these re- sults two factors must be considered: as pressure is elevated, the increased transmural distending pressure should elicit myogenic constriction of the coronary 126 vasculature. Additionally, the increase in pressure pro“ duced an increase in coronary flow which should enhance washout of adenosine, potassium ions and hydrogen ions, thus favoring a return of flow towards control. However, the coronary dilating action of the unblocked portion of these chemicals would tend to dilate the coronary vessels. The effect of test agents, both during the transient in- crease in flow accompanying sudden elevation of perfusion pressure and during the new steady state flow, appears to have reduced the relaxing effects of adenosine, potassium ion and hydrogen ion on coronary vessels. Subsequent lowering of hydrostatic pressure from 95 cm H20 to 35 cm H20, produced, in the presence of theophylline, ouabain and alkalosis, a significant reduction in the abil- ity of the coronary vessels to readjust flow toward control. It is reasonable to assume that at 35 cm H O perfusion 2 pressure, coronary flow is still related to a composite interaction of myogenic smooth muscle activity and the op— posing actions of vasodilator metabolites. Thus in the presence of test agents, the relaxing effect of adenosine, potassium ion and hydrogen ion on smooth muscle is effec- tively reduced and the coronary vasculature is less capable of autoregulating flow (Table 11). It is difficult to attribute the effects seen at a high or at a low perfusion pressure (in the presence of test agents) to an individual agent, nor can a proportionate contribution be assigned to each. SUMMARY The possibility that adenosine, a vasodilator, is a significant contributor to coronary reactive and hypoxic hyperemias was central to work reported in this study. Theophylline, a competitive antagonist of adenosine, attenu- ates the coronary response to exogenous adenosine but does not greatly affect the magnitudes of reactive and hypoxic hyperemias. Since in states of increased cardiac metabol- ism the tissue hydrogen ion activity might be increased, this study deals with the possibility that theophylline's ineffectiveness in attenuating reactive hyperemia and hy- poxic dilation might be related to an interaction of hydro- gen ion with adenosine and/or theophylline. We found that lowering perfusate pH by increasing the PC02 had a statistically significant potentiating effect on adenosine dilation of the coronary vessels at a pH below 7.0 as compared to adenosine's action at pH 7.43. Conversely, it was found that increasing the pH of the per- fusing fluid to 7.69 inhibited adenosine's ability to dilate the coronary vasculature. Also in hearts switched to a perfusate of pH 7.20 following stabilization, the coronary response to adenosine appeared to be enhanced. To account 127 128 for the increased coronary flow produced by adenosine at a low perfusate pH several possible mechanisms are considered, including 1) an interaction of hydrogen ion with adenosine to enhance coronary dilation, 2) weakening of contractile strength by the acidotic perfusate with a subsequent in- crease in coronary transmural distending pressure, and 3) the possible interference by acidosis of the enzymatic degradation and/or rephosphorylation of adenosine. We also investigated the coronary response to adenosine at a low pH for an extended period of time (30 min). This experiment warranted attention for two reasons. First, it served as a 'check' on experiments in which theophylline was used to attenuate the coronary dilation produced by adenosine. we wanted to ensure that theophylline attenua- tion was not partially due to waning of the adenosine response. Secondly, if adenosine is to be assigned an important role in the regulation of coronary flow during prolonged exercise, it must be shown that adenosine's dilat- ing action does not diminish with time. We found that 25 minutes after maximal dilation was achieved by 8xlO-7M adenosine, coronary flow was still 99 per cent of the maxi- mal response and 159 per cent greater than control. Thus, adenosine is capable, at a low pH, of sustaining an in- creased coronary flow for an extended period of time. 129 Theophylline was without effect on coronary flow and spontaneous heart rate in concentrations up to lO-4M, although at higher concentrations (5x10.4 and 10-3M) coro- nary flow and heart rate were both increased significantly. Other hearts were used to see if theophylline attenuation of the adenosine response is pH sensitive. Upon reducing perfusate pH to 7.20 it was found that vasoinactive concen- trations of theophylline attenuated the adenosine response as effectively as seen at pH 7.42. Further, in hearts initially perfused at pH 7.20 (following stabilization at pH 7.42), adenosine increased coronary flow to a greater degree than was normally seen but theophylline was still able to effectively attenuate this response. Theophylline, 5x10-5M, was essentially without effect on the reactive hyperemia seen following 30 seconds of coronary inflow occlusion. Since the potassium and hydro- gen ions have also been suggested as mediators of hypoxic and ischemic dilation and of autoregulation, ouabain 7 (1.4x10— M), a blocker of potassium vasodilation, and alkalosis (perfusate pH 7.69) were combined with theophyl- line (5x10.-5 M) in an attempt to attenuate these manifesta- tions of local regulation by minimizing the contribution of potassium and hydrogen ions and adenosine. Hypoxic dilation was unaffected. Both the volume and duration of hyperemic flow following release of a 20 second inflow 130 occlusion were reduced but failed to return to control after normalizing the perfusate. Peak reactive dilation was not affected. It is hard to determine if the reduced responses in the presence of test agents were effected by these agents. In previous experiments any effect by theo- phylline or hydrogen ion disappeared within a few minutes of perfusate normalization. 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C.: Autoregulation of renal blood flow and glomerular filtration rate, including data on tubular and peritubular capillary pressures and vessel wall tension. Circ. Res., Suppl. 15, no. l-2:I-132, 1964. wadsworth, R. M.: The effects of aminophylline on the increased myocardial blood flow produced by sys- temic hypoxia or by coronary artery occlusion. Eur. J. Pharmac. 20:130, 1972. 139 92. Wedd, A. M.: The action of adenosine and certain re- lated compounds on the coronary flow of the per- fused heart of the rabbit. J. Pharmacol. Exp. Therap. 41:355, 1931. 93. Winton, F. R.: Arterial, venous, intrarenal, and extra- renal pressure effects on renal blood flow. Circ. Res., Suppl. 15, no. 1-2:I-103, 1964. 94. Wolner, E., and O. Kraupp: Die abhangig keit pharmako- dynamisch nervorgerufener Anderugen der AVD-O des herzens vom extracellularen pH. Naunyn-Schmied. Arch. exp. Path. Pharmak., 253:298, 1966. APPENDICES APPENDIX A STATISTICS AND ANOVA TABLES 140 STATISTICS USED The nature of the adenosine and theophylline dose— response studies made their analysis applicable to the use of a RCB analysis of variance (two-way ANOVA) using factori— als. Factorial analysis of variance is the test of choice when dealing with studies in which two or more variables (such as adenosine and perfusate pH) might interact in producing a common response (83). The majority of the studies presented herein are based upon an interaction of the variables involved. Analysis of variance, however, is merely the initial means of testing for data variance. In order to subsequently recognize differences in control and experimental means, several tests were used. If in the initial analysis of variance the treatment effect was sig- nificant (calculated F greater than tabular F), Tukey's procedure, i.e., LSR (least significant range test) was used to determine statistical difference between/among means (R. R. Sokal and F. J. Rohlf, Biometry) (83). If, however, the initial variance ratio test was not significant (calculated F less than tabular F), then an a priori test, LSD (least significant difference) was used to test for mean differences. If the design of the experiment was not 141 142 concerned with interaction of main effects, the Student t test modified for paired replicates was used to answer the question: Is the mean difference (5) amongst pairs sig- nificantly different from zero? In other experiments a regression analysis (method of least squares), was applied to the data to determine if the dependent variable was significantly regressed on the inde- pendent variable.~ Using the method of least squares, linearity of the regression curve is assumed; however, an analysis of variance was always applied to test for linear- ity. Appendix A presents ANOVA tables from appropriate experiments. 143 Analysis of Variance (ANOVA) Tables computed from experi- ments in which the effect of adenosine on coronary flow was examined at perfusate pH 7.42 vs 7.36 (Table A1), 7.42 vs 7.20 (Table A2), 7.43 vs 6.89 (Table A3), 7.43 vs 7.69 (Table A4), and 7.43 vs 7.20 when hearts were initially treated with perfusate of pH 7.20 (Table A5). Key: df = degrees of freedom SS = sum of squares MS = mean square * = significance at a 0.05 ** a = 0.001 Table A1. ANOVA (2x4 Factorial) Source df SS MS Cal.F Tab.F Blocks 8 33 4.13 *3.72 1.97 Treatment 7 219 31.3 *28.2 2.51 perfusate pH 1 17 17 *15.3 5.29 adenosine 3 196 65.3 *58.8 3.34 interaction 3 6 2 1.80 3.34 Error 56 62 1.11 Total 71 Table A2. ANOVA (2x4 Factorial) Source df SS MS Cal.F Tab.F Blocks 8 41.2 5.15 *3.06 1.97 Treatment 7 376 53.7 *32.0 2.51 perfusate pH 1 76.6 76.6 *45.6 5.29 adenosine 3 284 94.7 *56.4 3.34 interaction 3 15 5 2.98 3.34 Error 56 94 1.9 Total 71 continued 144 Table A3. ANOVA (2x5 Factorial) Source df SS MS Cal.F Tab.F Blocks 5 27 5.4 *4.86 2.85 Treatment 9 414 46 *41.4 2.41 perfusate pH 1 130 130 **117 11.4 adenosine 4 210 52.5 *47.3 3.07 interaction 4 74 18.5 *16.7 3.07 Error 45 50 1.11 Total 59 Table A4. ANOVA (2x5 Factorial) Source df 83 MS Cal.F Tab.F Blocks 7 429 61.3 *39.8 2.51 Treatment 9 322 35.8 *23.2 2.33 perfusate pH 1 34.5 34.5 *22.4 5.29 adenosine 4 255 63.8 *41.4 3.01 interaction 4 42.5 10.6 *6.9 3.01 Error 63 97 1.54 Total 79 Table A5. ANOVA (2x5 Factorial) Source df SS Ms Cal.F Tab.F Blocks 7 134 19.1 *5.62 2.51 Treatment 9 1234 137 *40.3 2.33 perfusate pH 1 292 292 *85.9 5.29 adenosine 4 864 216 *63.5 3.01 interaction 4 78 19.5 *5.74 3.01 Error 63 214 3.4 Total 79 145 Analysis of Variance (ANOVA) Tables computed to determine if linearity occurs when regressing coronary flow on perfusate adenosine concentration (Table A6); when regressing coronary flow on perfusate H+ activity (Table A7), and when regress- ing coronary flow on perfusate adenosine concentration in hearts perfused first at pH 7.20 (Table A8). Table A6. ANOVA Source df SS MS Cal.F Tab.F Treatment 2 75 37.5 *20.2 3.40 Regression 1 62.6 62.6 *33.6 4.26 Remainder 1 12.4 12.4 * 6.7 4.26 Error 24 45 1.86 Total 26 b = 0.0013 8b = 0.0045 Table A7 . ANOVA Source df SS Ms Cal.F Tab.F Treatment 3 56.8 18.9 *19.7 3.72 Regression 1 56.1 56.1 *58.4 5.72 Remainder 2 0.78 0.39 0.41 4.32 Error 24 23.1 0.96 Total 27 b = -4.80 Sb = 0.62 continued Table A8. ANOVA 146 Source df SS MS Cal.F Tab.F Treatment 3 438 146 *37.0 2.95 Regression 1 337 337 *85.4 4.20 Remainder 2 101 50.6 *12.8 3.34 Error 28 110 3.94 Total 31 b = 0.031 Sb = 0.01 147 Analysis of Variance (ANOVA) Tables computed from experi- ments in which the effects of theophylline on adenosine dilation of the coronaries was studied. In Table A9 hearts were perfused first with solution of pH 7.42. In Table A10 perfusate of pH 7.20 was first used to perfuse the coro— naries. Table A9. ANOVA (2x4 Factorial) Source df SS MS Cal.F Tab.F Blocks 8 146 18.3 *6.2 2.41 Treatment 7 132 18.9 *6.4 2.51 perfusate pH 1 10 10 3.4 5.29 theophylline 3 114 38 *12.9 3.34 interaction 3 8 2.66 0.9 3.34 Error 56 165 2.95 Total 71 * Significance (P<<0.05) Table A10. ANOVA (2x5 Factorial) Source df SS MS Cal.F Tab.F Blocks 8 176 22 *5.5 2.41 Treatment 9 530 58.9 *14.7 2.33 perfusate pH 1 357 357 *84.3 5.29 theophylline 4 161 40.3 *10.1 3.01 interaction 4 12 3 0.75 , 3.01 Error 72 288 4.0 Total 89 * Significance (P‘<0.05) APPENDIX B RAW DATA 148 149 omncflucoo 0.0 0.0m 0.0..” m.m 0.3 0.5 0.~ 023.. 5.0 5.m 0.0a .70 0.~ 0.0a «.0m 0.0a 0.0m 0.0a 0.0 0.0a 0.0 0.0 0.NH 0.5 0.0 0.0a 0.5 0.0 0.0a 0.5 0.0 0.- 0.NH 0.H 0.MH 0.5 0.H 0.ma 0.0 0.H 0.~H 0.0 0.H 0.NH 0.0 0.~ 0.5H 0.0 0.0 0.0a 0.0 0.0 0.0a 0.0 0.H 0.0 0.0 0.H 0.0a 0.0 0.~H 0.0m 0.0H 0.5 0.HH 0.0 0.0 0.0a 0.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.H~ m.ma 0.0 0.0a m.m 0.0 0.~H 0.5 0.0 0.HH 0.5 0.0 0.HH 0.5 0.0 0.5a m.0H 0.~ 0.~H 0.0 0.~ 0.HH 0.0 0.m 0.HH 0.0 0.~ 0.~H 0.0 0.0 0.0a 0.~H 0.N 0.NH 5.0 0.N ~.~H m.0 0.~ 0.HH 0.0 0.N 0.HH 0.0 0.5 0.0a 0.0a 0.m 0.0a 0.5 0.~ 0.5 0.0 0.m 0.0a 0.0 0.0 0.0a 0.0 m o z m o s .01 o z m o z m b s z5ioax0 z5ioa amuoaxm zmIoH o Amv.5 mmv owaoumMm u m .owaoumawo n o .cmofi u 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SE 7.43 .4 8. 288 7.66 . . 288 7.97 .2 7. 240 7.72 . . 240 7.46 . . 238 7.66 . . 240 7.48 .0 . 240 7.67 . . 252 7.40 .4 . 252 7.74 . . 252 7.40 .8 . 252 9.69 . . 252 7.42 .8 . 276 7.70 . . 276 163 Umdfiflflflou m.m o.m m.m m.v m.m h.¢ m.m m.¢ m.m m.v m m.m H.m m.m o.m w.m o.m v.m m.v v.m m.m m.m o.m m.m o.m m.m o.m m.m m.m m.m (m.m m.m m.m H.m m.m m.m v.m m.m m.m m.o m.m ¢.m o.m h.¢ m.m m.v m.v v.m ~.m m.m o.m o.m m.m m.m «.4 m.m v.m v.m m.v v.m m.v H.m 0.4 m.v o.v >.v m.m h.v m.m v.¢ m.m o~.h mv.b om.h «v.5 on.» ~m.m o~.h mw.b o~.b «war mm wuoa muoaxm mica mica o 30Hm Houusoo u 0 Ah magma .ma ousmwm .muasmmmv om.h mm an omBOHHOM mv.w mm muMmsmumm um 30Hm anacouoo so msflaamnmomnu mo uommmm .mm magma 164 vmw smw omw omw wmw mew wmw mew wvw mew m www www www www www mww mmw www www wmw wmw wmw wmw wmw wmw wmw ovw wmw wmw oww mww ovw www mww www www www www www mww emw wmw wmw sew sew wmw vmw wmw sow sow vmw sew wmw wmw sow wmw sew ovw wmw ovw www www www www www www www www www www ow.w we.» ow.» ww.» on.“ mm.“ mm.» mm.» .bwhhlllmwub .mm wuow muowxm muow muow , o wfldm ”~ng mpowgwfifiomm omscwucoolumm GHQMB 165 Table 310. Effect of theophylline (5x10-4 and 10'3M) on coronary flow and spontaneous heart rate at perfusate pH 7.42. (Results, Figure 17, Table 7) C - control Flow Rate c 5x10‘4 10‘3 c 5x10”4 10‘3 5.1 6.3 8.0 252 264 312 6.0 8.0 11.4 252 276 324 5.8 6.8 9.6 216 228 312 7.6 9.4 12.6 312 342 360 7.0 8.3 10.8 264 300 324 6.0 5.2 9.4 288 324 372 X 6.1 7.3 10.3 264 289 334 166 omscwucoo w.m w.m e.ee w.oe w.we m.ee w.ww o.we m.w w.w m w.m o.w m.oe w.m w.we w.we w.we o.wH w.m e.m w.w m.o m.m m.oe o.ee m.ee o.ee m.we m.m o.w w.w w.w w.oe m.oe o.HH m.HH w.wH m.ee m.m o.m m.oe m.w o.we o.oe m.we e.ow w.we o.ee m.m m.m w.m w.w w.ow w.m w.ee m.oe o.ee o.oe m.w w.w e.ww m.oe m.ew. m.me m.ee w.mw o.me e.ew o.oe m.w e.wH m.ow o.eH e.mw e.ee w.me w.ee e.me w.oe o.m o.m w.w w.ow e.m m.oe m.m w.oe o.oH w.w w.m o.ow w.w m.ww e.ee w.ww w.we w.we e.ww e.w w.m bmmw, we.w ow.w we.w ow.w wen» bhqw we.w owablw we.» mm .omge zeuoe .omna s nee. .omza zmuo mnemocmca Am menus .mw magmas .muesmmme ow.w an an omzoeeom we.w mm mummsmumm um Azwnowxwv mnemocmom an couscoum 30am mnmcouoo co mafiaamcmomnu mo uomwmm .Ham magma 167 Hum th Hum Ohm mmm mmm hwm 0mm mmN 0mm m ovm NmN ovm mmm mvm mmN ovm mNN ovm vmm mhm mmm ohm mum ohm mvm mhm mvm ohm mvm mmm mmm mmm mmm vwm mmN vow vmm wow wow wow mmN vwm mmm vow mmm mmm emm mmN vmm ohm mum mum ohm chm ohm chN ohm vow ohm vwm vwm «mm vmm chm cum ohm onm emm vmm mmm mmm mmm mmm mmm mmm mmm mmm mmm mmm mmm Ohm mmm mmm Nmm NmN mmm mmm mmm mmm vmm oom vmm oom «mm mmm mmm mum mmm ‘ mum cm.h N¢.h om.n «v.5 om.b‘ mw.h om.h mm.» om.b mm.b. mm .0059 2 Ioa .omca 2 tea .0659 2 led mnemocmod U ovum unmwm noncaucooulaam manna 168 H.h mym .meh...HeHH. mvm meNH _.meme..H9¢H m-m N.va w.m m.w m m.v 0.0H m.v N.HH o.m N.vH N.m v.mH N.m m.mH m.m m.m v.0H m.HH. ¢.oa o.HH w.oa w.MH N.HH N.mH o.HH N.mH v.0 v.5 o.v «.5 o.m v.m m.m v.HH m.n «.ma v.5 v.NH m.m v.m m.m N.m 0.5 0.0H m.m ¢.NH m.m o.ma m.m o.ma v.m o.m 0.5 v.oH o.m N.HH v.m o.NH o.oa o.ma N.OH o.ma m.m v.m m.h m.oa m.h m.HH N.m o.NH m.m m.MH v.m m.ma o.m m.h m.h v.0H N.m m.NH m.m N.MH m.OH w.MH m.OH m.ma N.v N.m v.w o.m m.o H.HH o.h ¢.HH w.m w.HH m.m w.HH N.m m.m N.m m.HH m.m o.ma N.HH o.vH N.NH o.mH N.NH o.hH m.h o.m mv.h om.h .Nv.b. om.b, _6N¢sh..omebfi...mw.h .oN.h .mv .w om. b mm. 50 om. n ma .omnfi Svloa .Om£9,2mloaxm .0059 Smloa .Omse Zmloa wcdmocmcd 1m manna .me munmem .muesmmmo .Nv.h mm um comemumm we owBoHHOM o~.h mm um umuwm ommnmnmm muumms GA A: noaxmv mnemocmom an omu¢ooum.30am Xumsouoo so mcaaaanmomnu mo uommwm .mam manna 169 Effect of concurrent theophylline (5x10-5M), ouabain (1.4x10‘7M) and alkalosis (perfusate pH 769) on reactive dilation. (Results, Figure 21, Table 313. Table 10) C = absence of test agents, E = presence of test agents Peak Flow (ml/min) VCF20 T50 C E C C E C C E C 15.0 14.0 15.0 3.6 3.3 3.5 12.5 12.3 14.5 14.0 13.5 13.0 3.6 8.1 3.1 16.0 14.2 14.5 17.5 15.0 17.0 3.7 3.2 3.3 14.0 12.4 11.5 13.5 13.0 13.0 3.1 2.4 2 2 13.5 10.5 10.0 14.0 13.0 15.0 3.5 3.2 3.3 13.5 14.0 11.0 16.0 16.5 14.0 3.9 3.8 3.6 13.5 12.0 9.2 18.0 17.0 17.5 4.6 4.0 4.3 15.0 12.2 13.5 17.5 15.5 15.0 3.6 2.8 2.8 13.0 10.5 11.0 X 15.7 14.7 14.9 3.7 3.2 3.3 13.9 12.3 11.9 170 Table 814. Effect of concurrent theophylline (5x10-8M), ouabain (1.4x10 M) and alkalosis (perfusate pH 7.69) on hypoxic coronary flow. C = control, A = absence of test agents, P = presence of test agents Coronary Flow Mean Systolic Diastolic C A C P C A C P C A C P 6.6 12.2 6.6 12.6 0.5 4.0 0.5 5.5 12.0 22.0 12.0 21.0 4.4 14.2 4.0 14.2 5.0 10.0 3.0 10.5 6.0 20.5 6.0 20.0 7.4 13.0 7.4 12.8 1.5 5.5 2.5 7.0 15.5 24.0 18.0 21.0 6.6 14.2 6.6 14.0 2.0 3.5 1.5 2.54 13.0 25.0 11.0 21.0 4.0 11.2 4.0 18.2 3.0 7.5 2.0 6.5 5.5 17.5 5.0 18.0 6.2 17.2 6.3 16.4 1.0 12.5 1.0 11.0 11.5 23.0 11.0 21.0 7.6 16.6 7.2 16.0 4.0 9.5 3.5 10.0 11.5 25.5 10.5 23.0 NI 0‘ o H H .5 o H 6.0 14.0 1.0 7.5 1.0 7.6 10.7 27.5 10.5 21.5 171 m.m o.w w.m e.e m.me m.HH m.w m.m m.m o.oe H.m m.m m w.m m.w m.w w.e o.ee m.m m.m o.He H.ee w.ee o.e m.m m.w m.w o.e m.e m.oe H.m o.m e.oe w.w m.w e.m m.w m.m m.w o.e m.e m.ew m.oe e.m o.m m.m m.m o.w e.w m.ow m.m w.m o.e w.ee m.we w.m e.w m.ew m.oe m.m m.o m.m o.m w.m m.e m.me m.ae m.w m.m e.ow o.oe H.o m.m v.5 o.w m.e o.m w.we o.HH e.w m.m o.oe m.m m.m w.m w.ew m.m o.m o.e e.me w.ee m.m e.o m.ee m.we m.we w.m m.oe m.w w.m w.e o.ee m.me m.m 0.5 o.mw m.me o.m m.e m a m m m a m 4 m a m a mocmumMMmm 30am magnum mocmumMmmm 30am mflmmum mocmumwmmm 30am magnum Aowm so mmv Aowm Eu may Aowm 80 mos whommmum omuwzoq mummmmnm owum>oam mocmmmum Honucou mucmmm ummu mo mocmmmum u m mucmmm ummu mo monomnw u d Awe menus .ww musmem .muwsmmmv .mnnume Isomm> hnmcouoo mnu ca coaumasmmMOpsm co Amm.n mm ovumsmummv mflmoamxam can .12 noexe.eo cemamso .12 noexmo mceeemnmomap ucmunsocoo mo uomumm .mwm menus