ABSTRACT ROLES OF ADENINE NUCLEOTIDES IN CAUSING CORONARY REACTIVE DILATION IN GUINEA PIG HEARTS BY Philip Kuocherng Liu In guinea pig hearts, perfused by a modified Ringer‘s solution, no ATP was found in the coronary venous effluent unless inflow concen- tration of ATP was greater than 1.5 uM during perfusion of exogenous ATP. ATP seems to be degraded during a transit through the coronary circulation. This thesis was supported by the finding that more than 802 of adenine base of ATP, ADP or AMP, or 86% of 14C of 14C-ATP per- fused into the coronary circulation was 'recoverable in the venous effluents. Chromatographic analysis of the venous effluents revealed that two-thirds of the adenine compounds in the effluent were AMP during perfusion of ATP, ADP or AMP. No ATP, ADP, IMP, or cyclic AMP was found in the effluent. Thus, if adenine nucleotides are released during coronary reactive dilation, they will appear in venous effluent as AMP. ATP and AMP concentrations of coronary venous effluent were measured by the firefly bioluminescence and enzymatic (myokinase and 14C—ATP) assays, respectively, before, during and after reactive dilation eli- cited by 30 seconds arterial occlusion in three types of guinea pig heart preparation. ATP concentration did not change during reactive Philip Kuocherng Liu dilation in all three preparations (N = 6, N = 4 and N = 11 for the first, second and third preparations). AMP concentration increased significantly during reactive dilation in two preparations (N = 11 and N = 6 for the first and second preparations) but not in the third preparation (N = 6). The magnitudes of control coronary flow and reac- tive dilation were the same in all three preparations. The third preparation differed from the remaining two preparations in that it had the least surgical damage to the cardiac tissues and the venous effluents collected were immediately centrifuged to avoid contamination with cell debris from the heart. The rise in AMP during reactive dilation in the first two preparations, therefore, seems to result from contaminated cell debris. It is concluded, that AMP is not released from the guinea pig heart during reactive dilation, and adenine nucleotides seem not to be directly involved in the coronary reactive dilation. ROLES OF ADENINE NUCLEOTIDES IN CAUSING CORONARY REACTIVE DILATION IN GUINEA PIG HEARTS BY Philip Kuocherng Liu A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Physiology 1977 DEDICATION This thesis is dedicated To: To: To: Formosa for the strength she gives Phil the Formosan Aborigines for their encouragement from afar and Phil 0......0.0000COOOOOOOOOOOO000......O... WY not? ii ACKNOWLEDGEMENTS I wish to express appreciation to the members of my guidance committee: Drs. C. C. Chou, J. B. Scott and B. H. Selleck for their invaluable guidance, encouragement, and support throughout the course of this study. I have benefited greatly from the perception and per- sistence shown by each of them. I thank Dr. Chou for providing me the opportunity to come to Michigan State University. I also thank Dr. Scott for his generosity and realism, Dr. Selleck for suggesting ideas and assistance. Finally, special thanks to Timothy Diller, Steve McAlpine, Gary Merrill, Keith Tait, Bruce Talsma and Mrs. Yanee Thoonsuwan for their technical assistance. iii TABLE OF CONTENTS Page LIST OF TABLESOOOOOOO0.00.00.00.0000.00.000.00.0IOOOOOOOOOOOOOOCO Vii LIST OF FIGURESOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO0.... Viii LIST OF ABBREVIATIONS.O...OOOOOOOOOOQOOOOOOOOOOOOOOOOOOOCOOOOOOOO ix INTRODUCTIONOOCOO0.0...O...OOOOOOOOOOOOOOOOO...OOOOOOOOOOOOOOOOOO 1 LITERATURE REVIEWOOOOOO...0.......0...OOOOOOOOOOOOOOOOOOOOO00.... 3 I. Definition and Major Forms of Local Regulation of BlOOd FIOWOOOOOOOOOO...0.00......IOOOOOOCOOOOOOOOOOOOO0. 3 II. History of Coronary Reactive Hyperemia.................. 4 III. Effects of Duration of Arterial Occlusion on Coronary ReaCtive HyperemiaOOOOOOOOOOOIOOOO...OOOOOOOOOOOOOOOOOOO 5 IV. Proposed Mechanism of Coronary Reactive Hyperemia....... 6 V. Metabolic Hypothesis of Coronary Reactive Hyperemia..... 8 A. Oxygen Tension, Hydrogen Ion, and Carbon Dioxide..... 9 B. Adenosine Hypothesis................................. 10 C. Adenine Nucleotides.................................. 14 VI. Disputes of Adenine Compound‘s Participation in Coronary Reactive HyperemiaOOOOOOOOO0.00.0.0.0...OOOOOOOOOOOOOOOI 15 urn-ODS...O0.0...I.OOOOOOOOOOOOOOOOOOOOOOOO0.000COOOOOOOOOOOOOOOO 19 I. Experimental Preparations.O...OOOOOOOOOOOOOIOCOOOOOOOOO. 19 A. Surgical Preparations of Isolated Guinea Pig Hearts .1—1'1-Vitro.‘0....OOOOOOOIOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 19 1) Preparation I. O O O O O O O O O O O O O O O O O O O O O O O O O O I O O O O O O O O O 20 2) Preparation II 0 O O I O O O O O O O O I O O O I O O O O O I O O O O O O O O O I O O O 20 3) Preparation 1110.00.00.00...OOOOOOOOOOOOOOOO00.... 23 iv TABLE OF CONTENTS-continued B. The Coronary Flow Measurement........................ C. Criteria of an Acceptable Surgical Preparation....... II. Experimental Procedures................................. E. Survival of ATP during a Passage through the Coronary Circulation.......................................... Coronary Venous Recoveries of the Adenine Base during Intraarterial Perfusions of ATP ADP, or AMP......... Coronary Venous Recoveries of lac during Intra- arterial Perfusion of 14C-ATP........................ Identifications of Products Formed from Hydrolysis of Adenine Nucleotides in a Transit through the Coronary Circulation.......................................... 1) ATP, ADP or AMP................................... 2) 14c-AIP........................................... Endogenous Release of ATP and AMP from the Heart during Reactive Dilation............................. III. Statistical Analyses Of ResultSOOOOOOOOOOOOCOOOOOOOOOOOO RESULTSOOOOOOOOI...OI.00....OOOOOOOOOOOCOOCOOOOOOOOO0.0.0.0000... I. Survival of ATP during a Passage through the Coronary CitCUlat-ionooooooooooooooooooooooooo0.000000000000000... II. Coronary Venous Recoveries of the Adenine Base and of 14C during Intraarterial Perfusions of Adenine Nucleo- tideBOOOOOOOOOOOOOOOOOOOOOOOOOOO...OOOOOOOOOIOOOOOOOOOOO III. Identifications of Products Formed from Hydrolysis of Adenine Nucleotides in a Transit through the Coronary CirCUIationOOOO.I0.0.0000...OOOOOOOOOIOOOOOOOOO0.00....0 IV. Endogenous Release of ATP and AMP during Reactive DilationOOOOOOOC.OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO V. Comparison of Reactive Dilation Elicited by an Occlusion of Perfusion for 30 Seconds in Preps. I, II and III Hearts.0..0..C0.0I.0..00.00.000.000.000000000000000COOOO DISCUSSIONOOOOOOODO0.0.C.0..OCOOOOOOOOOOOOOOOOOOOOOOOOOO0.0.0.... I. Release of ATP during Coronary Reactive Dilation........ II. Metabolism of Exogenous Adenine Nucleotides in a Transit through the Coronary Circulation of Guinea Pig Hearts... III. Release of AMP during Coronary Reactive Dilation........ Page 24 24 25 25 26 27 27 27 28 31 31 32 32 37 37 43 46 50 51 52 58 TABLE OF CONTENTS--continued Page SUMMARY AND CONCLUSIONS................... ...... ................. 62 APPENDICES A. REAGENTS.................................................. 65 I. Krebs—Ringer-Bicarbonate Perfusate................... 66 II. Standard Solution Containing Carrier Nucleotides and Nucleosides for Chromatographic Separation........... 66 III. l4C-ATP.............................................. 68 IV. Ammonium Formate..................................... 68 V. Formic Acid.......................................... 69 VI. Myokinase-TEAr-Mg++ Buffer Solution................... 69 VII. Anion-exchange Resin................................. 70 B. CORONARY REACTIVITIES TO ARTERIAL OCCLUSIONS AND HYPOXIC PERFUSION OF PREPAMTION I’ll WTS.OOOOCOOOOOOOOOOOOCOOOO 71 C. CWICAL ANAIJYSES.OICOCOOOOOOOOOOOO0.0IOOCOOOOOOOOOOOOOOO. 77 I. Firefly Bioluminescence Assay of ATP................. 78 II. Anion Exchange Chromatographic Separations of Nucleosides and Nucleotides.......................... 80 1) Rapid Separation of ATP, ADP and AMP.............. 83 2) Specific Separation of ATP, ADP, IMP, cAMP, AMP and NUCIeosides O O O O O O O O O O O O I O C O 0 O O O O O O O O O O O O O O O O 0 O 83 3) Modified Specific Separation of Inosine from ' Nuc18081de8 O O O O O O O O O O O O O O O O O O O O I O O O O O O O O O O O O O I O O O O 86 III. Quantitative Assay of AMP by Myokinase and 140(U)—ATPOOOOOOOOOOOOOOOOOOOOOO.OOOOOOOOOOOOOOOOOOOO 87 BIBLImRAPHYOOO0.00000000000000000000000000000000000000000000COOO 90 vi LIST OF TABLES TABLE 5. 3-1 0 Venous Recoveries of Adenine Base and 14C................... The Percentage of AMP,Nuc1eosides Appearing on the Chroma— tograms of Venous Effluent during Perfusions of Adenine NUCleotides0.0.0.0.0000...OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO Concentrations of Venous ATP (ng/ml) before (C ), during (R) and after (C2) Coronary Reactive Dilation............... Concentrations of Venous AMP (ng/ml) before (C ), during (R1 and R2) and after (C2) Coronary Reactive D lation....... Magnitudes of Reactive Dilation and Heart Rates in the Beginning (Pre-Expt) and at the End (Post-Expt) of Experi- ments in Preps. I, II and III Hearts........................ ATP Concentrations in the Effluents from Pulmonary Artery (PA) and from the Heart Chamber Reservoir (HC) of Prep. III Hearta...’OOOOOOOOOOIOCOOOIOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO. vii Page 38 42 44 45 47 76 LIST OF FIGURES FIGURE Page I. The non-recirculating perfusion system...................... 22 2. Survival of exogenous ATP during a passage through the coronary circulation of Prep. I hearts...................... 34 3. Survival of exogenous ATP during a passage through the coronary circulation of Prep. II hearts..................... 36 4. Typical Chromatograms of arterial and coronary venous per- fusate during perfusions of adenine nucleotides............. 41 5. Typical recordings of reactive dilation in Preps. I, II and III heartBOO0.0..0....0.0.0.0...OOIOOOOOOOOOOO00.0.0000....0 49 B-l. Coronary vascular reactivities to different durations of arterial occlusions in Prep. III hearts..................... 73 B-2. A typical recording of coronary flow during hypoxic perfu- SioninaPrep. III heart.OOO...0......OOOOOOOOOOOOOOOOOOOOO 75 C-1. The apparatus for anion exchange chromatographic separa- tionSooocooooooooooooooooooooooooooooooo00000000000000.0000. 82 C-2. Chromatograms of standard samples and inflow perfusate analyzed by the specific separation method.................. 85 viii ATP bpm cAMP 14C—ATP CCF Expt. No. gm IMP I.U. uCi U8 LIST OF ABBREVIATIONS Adenosine 5'-diphosphate, AMW = 419.3 Absorbance at wavelength 258 nm Adenosine 5'-mon0phosphste, AMW = 347.4 Anhydrous molecular weight, gm/mole Adenosine 5'-triphosphate, AMW a 487.2 Beats per minute of heart rate Cyclic adenosine 3':5‘-monophosphate, AMW = 351.2 Uniformly labeled 14C ATP, or 14C(U)-ATP Control coronary inflow, inflow rate immediately before occlusion Experimental number Gram Inosine 5'-monophosphate, AMW = 392.2 International unit of enzymatic activity Molar, mole per liter Microcurie Microgram, x 10-6gm Micromolar, x 10-6 M Milligram, x 10-.3 gm Milliliter, x 10'3 liter Millimolar, x 10"3 M Number of experiments ix LIST OF ABBREVIATIONS--continued ng/ml nM : PA : Prep. Samples Wt : Nanogram per m1 (ng = x 10-9 gm) Nanomolar, x 10-9 M Pulmonary artery Heart preparation Cl: First control sample (before arterial occlusion) Rl(or R): First postocclusion sample (during increase of coronary flow. Collection started from the onset of increased flow till 5 ml of sample were collected). R2: Sample collected immediately after R1 C2: Second control sample, collected 5 minutes after Rl Guinea pig heart mass (wet weight), gm Times gravity INTRODUCTION Physiologically, the myocardium is underperfused relative to its metabolic rate. In fact, the heart has the lowest flow to metabolism ratio compared to other organs in the human body. However, the ratio is kept relatively constant, i.e., when the metabolism is increased, the flow also increases. Therefore, the heart exhibits an intrinsic regulatory mechanism for its blood flow. The local regulatory mechan- ism of coronary blood flow is believed to be mediated through numerous factors, such as myogenic, tissue pressure, and metabolic factors. Among the metabolic factors are interstitial contents of oxygen, hydro- gen ions, potassium, magnesium, prostaglandins and adenine compounds. In actuality, all of these factors may function in concert. However, the participation of adenine compounds are still controversial. Chemical and bioassay studies have suggested that adenosine and adenine nucleotides (ATP, ADP, AMP) may play a role in local regulation of coronary blood flow. It has been shown that ATP concentration in coronary effluent of the Ringer's perfused guinea pig heart increases during reactive hyperemia (91). Using the kidney and forelimb to assay the vasoactivity of coronary sinus blood, Scott §£_§l, (84) have shown that during coronary reactive hyperemia, coronary sinus blood raises renal vascular resistance while it lowers forelimb resistance. Intra- arterial injection of AMP or adenosine to the kidney and forelimb produces similar effect. Thus these studies suggest that coronary reactive hyperemia is in part due to endogenous release of these adenine compounds. In contrast to the above observations, no adenine nucleotides (8), but only adenosine (ll, 78, 81) and its breakdown products, i.e., inosine and hypoxanthine (75) have been found in coronary effluent during myocardial ischemia, hypoxia, or coronary reactive hyperemia. The absence of adenine nucleotides in coronary effluent in these studies, however, does not completely exclude the possibility of the release of adenine nucleotides by the heart in these conditions. ATP has been shown to be very unstable in the blood and plasma (25), and during transit through the coronary circulation (4, 91). The nucleotides released might have been rapidly hydrolyzed to nucleosides and appeared in coronary effluent as adenosine, inosine or hypoxanthine. The present studies were designed 1) to study the metabolism of adenine nucleotides in a transit through the coronary circulation, and 2) to determine concentrations of ATP and AMP in the coronary effluent collected before and during coronary reactive dilation to see if ATP and AMP concentra- tions increase during this vasodilatory phase. LITERATURE REVIEW I. Definition and Major Forms of Local Regulation of Blood Flow According to Haddy and Scott (42), local blood flow regulation is defined as that regulation of flow which is intrinsic to an organ. Specifically excluded in this definition is regulation directly result- ing from remote influences such as vasomotor nerve (constrictor or dilator) activity or the concentrations of vasoactive substances in the inflowing blood. Local regulation can be observed in numerous organ preparations. It occurs not only 1.21272 and _i_rl £1.52, but also _i_n__v_i§_r_g, even when the organ under question is isolated and perfused with cell- free solutions. The four major forms of local regulation are: 1) autoregulation, i.e., the ability of an organ to adjust its own vascular resistance and thereby keep its flow rate nearly constant in response to alterations of arterial pressure within physiological range, 2) autoregulatory escape, i.e., the tendency for blood flow to return to normal during continuous administration of an extrinsic vasoactive stimulus, 3) active hyperemia, i.e., increase in an organ blood flow induced by increased metabolic activity within the organ, for example, exercise hyperemia in skeletal muscle, and 4) reactive hyperemia, i.e., the transient increase of organ blood flow following relief of a period of ischemia. Knowledge concerning local regulation of blood flow in the heart may be of utmost practical significance. According to an article in Newsweek (67), coronary heart disease accounts for 54 percent of all deaths in the United States of America. By understanding the physio— logical mechanism whereby coronary flow is regulated we may be able to devise better techniques for maintaining cardiac function in individuals with heart disease and lower the death rate. In this Literature Review, our knowledge concerning reactive hyperemia will be described in detail. II. History of Coronarngeactive Hyperemia The history of reactive hyperemia originated with studies of organs other than the heart. Roy and Brown (76) ascribe the first recorded observation of reactive hyperemia to Cohnheim (24) who studied blood flow in frog's tongue in 1872, though reactive hyperemia may have been known to physiologists prior to 1872. Olsson (73) gives credit to Bier (12) for naming this transient increase of blood flow "reactive hyperemia". Since Cohnheim's observation in 1872, others have confirmed the occurrence of reactive hyperemia in the intact extremities of mammals, for example, the observations of reactive hyperemia in the forearm and the leg of unanesthetized men (58), in innervated extremi- ties of anesthetized and unanesthetized dogs (64), and in sympathecto- mized human hands at different temperatures (35)- These early studies have been further extended to other organs, especially to the heart. Hilton and Eichholtz (44) observed reactive hyperemia in the coronary system of dog hearts with the heart—lung preparation in 1925. A decade later, Katz g£_al, (54, 55) also demon- strated coronary reactive hyperemia by occluding coronary flow in iso- lated fibrillating dog hearts perfused with defibrinated blood at constant pressure and temperature. Working with in gizg_dog hearts, coronary reactive hyperemia was demonstrated in anesthetized open-chest dogs by Chenugt_al.(18), Coffman and Gregg (19), Moir and Downs (63), Rubio g£_§l, (81), Scott g£_§l, (84), and in unanesthetized dogs by Olsson and Gregg (74). More recently other investigators (16, 62, 91) also observed reactive hyperemia in the coronary system of isolated guinea pig hearts. In these latter studies, the guinea pig hearts were perfused with Krebs-Ringer—bicarbonate solution containing pyruvate and glucose. III. Effects of Duration of Arterial Occlusion on Coronarngeactive Hyperemia Several investigators have observed a rather definite relationship between duration of occlusion and the magnitude of reactive hyperemia. Roy and Brown (76) suggested that "intensity" and duration of reactive hyperemia in frog's web are both directly proportional to the duration of occlusion. Observations of Freeman (35) on sympathectomized hands of human beings indicate that the "amount" of reactive hyperemia is influenced by temperature of the organ and by duration of the occlusion. Montgomery g£_§1, (64) have found that the "intensity" and duration of reactive hyperemia in animal extremities are causally and quantitatively related to events occurring during arterial occlusion, and suggested that the reactive hyperemic phase can be described as a repayment of its flow debt which occurs during arterial occlusion. However, working with _i_r_1_ 23:32 dog hearts, Coffman and Gregg (l9) , and Olsson and Gregg (74) found that flow debt1 is overpaid during coronary reactive hyperemia in dogs. These investigators (19, 74) as well as Olsson (73) have reported that when the preocclusion flow is constant, the reactive hyperemic flow2 is predictably determined by the duration of coronary occlusion, because reactive hyperemic flow and its duration increase in proportion to the duration of coronary occlusion. Furthermore, they observed that peak flow during reactive hyperemia increases with increasing duration of occlusion up to 15—30 seconds. Lastly, in the in yi££2_Ringer's per— fused guinea pig coronary system, it has been reported that diastolic hyperemic flow (excess diastolic flow during hyperemia) increases linearly with duration of occlusion, and peak diastolic flow responds similarly with duration of occlusion but in less linear fashion (16). IV. Prgpgsed Mechanism of Coronary Reactive Hyperemia Several factors have been proposed to account for reactive hyper- emia. Among these are metabolic, myogenic, viscosity, and tissue lCoffman and Gregg (l9), Olsson ££.§l: (73, 74) define: 1) flow debt: the flow volume that the heart is deprived Of during an arterial occlusion period, and thus is the product of control flow rate and dura- tion of the occlusion; 2) reactive hyperemic flow: the excess flow dur- ing reactive hyperemia, is the difference between total volume of hyperemic flow and volume of basal flow which is the product of the pre- occlusion flow and duration of reactive hyperemia; 3) duration of reac— tive hyperemia: the time taken from the onset of reactive hyperemia to the point where the flow returns to the preocclusion level; 4) repayment of flow debt: the ratio of reactive hyperemic flow to the flow debt. 2Ibid. pressure. In actuality, all of these factors may function in concert as the mechanism(s) through which reactive hyperemia operates. When arterial inflow is reduced or stopped, the perfusion pressure decreases. Intra- and extracellular pH and oxygen tension of the organ will de- crease due to decreasing blood flow. In conjunction with these changes, the vascular transmural pressure and capillary hydrostatic pressure also decrease. Decreased pH and oxygen tension (metabolic factors) are vasodilatory (44, 61). Reduction in vascular transmural pressure (myogenic factor) deactivates vascular smooth muscle and causes dilation of the resistant vessels (5). Decreased capillary hydrostatic pressure occurring with arterial occlusion causes influx of tissue fluid into the lumen of the capillary. This flux of tissue fluid decreases the viscos- ity of the capillary blood, and causes a gradual decrease in tissue fluid pressure. Thus, arterial occlusion changes blood viscosity and tissue pressure in a manner which tends to decrease vascular resist- ance (42). However, while the myogenic hypothesis may account for initiation of reactive hyperemia, it fails to explain the increased peak flow and the extended duration of reactive hyperemia when arterial occlusion and organ ischemia are prolonged. Likewise, the viscosity hypothesis fails to explain the occurrence of reactive hyperemia in hearts perfused with cell-free perfusate (16, 62, 91). V. Metabolic Hypothesis of Coronary Reactive Hyperemia The metabolic hypothesis was first suggested by Roy and Brown (76). According to this hypothesis, local regulation of blood flow is mediated through changes in the concentration of vasoactive metabolites in the tissue that lead to adjustment of flow to a level more suitable to the tissue metabolic rate. Evidence for the involvement of vasoactive metabolites in causing reactive hyperemia is from several studies. Crawford g£_§l, (27), Nelemans (66), and Scott 32 31. (84) reported that venous blood from nor- mally perfused organs, including the heart,is vasodilatory when compared to arterial blood if the venous effluent is tested immediately after collection. However, earlier studies of Jelliffe £5 31, (50) have indi- cated that coronary sinus blood tested 2-10 minutes after collection does not produce vasodilation. Thus the vasoactive metabolites in the coronary sinus blood seem to be unstable. The vasoactive metabolic factors usually considered are oxygen, hydrogen ion, carbon dioxide, potassium ion, magnesium ion, prostaglandins, osmolality, adenosine, and adenine nucleotides. Bacaner g£_gl, (3), Gregg (39) and Scott g£_§l, (84) have demon- strated that coronary metabolism is quite flow sensitive. According to Haddy and Scott (41), a decrease in flow to metabolism ratio, by de— creasing flow and/or increasing metabolism, will cause local active vasodilation. For example, exercise (an increase in metabolism of the organ), or arterial constrictions and occlusions (a decrease in flow to the organ) will cause local vasodilation. Conversely, increasing the ratio by an increased flow causes vasoconstriction. For example, vasoconstriction is observed when arterial pressure is suddenly in- creased (an increase in flow to the organ while the metabolism of the organ is constant). Therefore, flow to metabolism ratio, which affects the tissue fluid concentration of oxygen and vasoactive metabolites, should also be considered in searching for local mechanisms which regu- late blood flow. A. Oxygen Tension, Hydrogen Ion, and Carbon Dioxide Hilton and Eichholtz (44), proposed oxygen tension to be involved in reactive hyperemia because they observed an increase in coronary blood flow when the heart was made hypoxic. More evidence for oxygen being a factor in the metabolic hypothesis is from Katz and Lindner (55), who proposed that the cause of reactive hyperemia was a diffusable dilator substance which was eliminated by the presence of oxygen. Furthermore, Eckenhoff e£_§l, (31) reported that cardiac oxygen consump- tion was found to have highly significant correlations with coronary flow and coronary resistance. Eckenhoff e£_§1, further proposed that coronary blood flow is adjusted in such a way to meet demand of the heart for oxygen, or for supply of arterial blood. Coffman and Gregg (20) demonstrated that myocardial ischemia stimulates coronary blood flow, and thus increases oxygen availability. On the other hand, Haddy and Scott (41) indicated that the fall in coronary vascular resistance during myocardial hypoxia may result in part from passive vasodilation, because there is an associated fall in left ventricular contractile 10 force which lowers intraventricular and tissue pressure (29, 65, 68). Therefore, vasodilation that occurs during myocardial hypoxia is mediated by both chemical and physical factors. Others have suggested that the hydrogen ion and carbon dioxide are also involved. Daugherty g£_§l, (29), and McElroy g£_al, (61) observed a reduction in coronary vascular resistance when local blood pH is decreased or pCO is raised. An opposite response was observed 2 when local blood pH was raised or pCO was lowered. Hilton and Eichholtz 2 (44), McElroy g£_al, (61), Ng e£_§l, (69), and Need and Little (94) indicated that the action of carbon dioxide is mediated through the hydrogen ion. However, Berne (9) suggested that carbon dioxide has little contribution to regulation of coronary blood flow under physio- logical conditions such as during physical exercise, and likewise be suggested that hydrogen ion probably does not have an important role in local control of coronary blood flow. B. Adenosine Hypothesis It has been known since 1929 that injection of adenosine produces vasodilation in most systemic vascular beds excepting the kidney where it produces vasoconstriction (6, 30). The formation of purine nucleo- sides in the hypoxic or anoxic myocardium of dog (7), rabbit (48), and rat (36-38) hearts has been reported. Furthermore, Berne g£_§l, (8, 11), Jacob and Berne (49), Richman and Wyborny (75), and Stoner g£_§l, (90) reported that, while no detectable adenine nucleotides were present in the venous effluent of normal and hypoxic hearts, significant amounts of inosine and hypoxanthine, i.e., adenosine breakdown products, 11 were found in the venous effluent from the hearts undergoing vasodila- tion. The adenosine hypothesis of Berne (8) for metabolic regulation of coronary flow was based on: 1) The amount of adenosine increases in hypoxic myocardium. 2) Adenosine can move across cell membrane. 3) Adenosine is a coronary vasodilator. 4) Inosine and hypoxanthine, i.e., the degradation products of adenosine, are present in coronary effluent during myocardial anoxia, hypoxia, or coronary reactive hyper- emia. In this hypothesis, Berne postulates that the reduced myocardial oxygen tension, which is caused by either 1) hypoxemia, 2) decreased coronary blood flow, or 3) increased myocardial oxygen utilization, results in a net hydrolysis (dephosphorylation) of cellular ATP to AMP. Because nucleotides, including AMP, do not diffuse readily through the myocardial cell membrane (45), he proposes that AMP is further dephos- phorylated to adenosine by 5'-nucleotidase present on the membranes' lining compartments open to the extracellular space (80). Adenosine then rapidly diffuses out of the myocardial cell and produces vasodila— tion upon reaching the coronary arterioles by diffusion through the interstitial fluid. Subsequent studies tend to support this hypothesis. Richman and wyborny (75) reported that adenosine was found in venous effluent of anoxic Krebs-Henseleit perfused rabbit hearts when the cardiac adenosine deaminase activity was inhibited by 8-azaguanine. Other investigators also reported that a highly significant level of adenosine was found in the venous effluent in hypoxic or anoxic hearts. For example, Katori and Berne (53) found that adenosine appears in the artificial perfusates 12 of anoxic cat and guinea pig hearts treated with 8-azaguanine. Probably, the most important finding in support of this hypothesis, is that of Rubio‘ggngl. (81) who found that adenosine, without 8-azaguanine, appears in coronary sinus blood during the hyperemic phase of reactive hyperemia. This study was performed in anesthetized dog hearts perfused by their own blood. No adenosine was found in either arterial or coro- nary sinus plasma before occlusion. But, 130 nM adenosine was found in coronary sinus plasma collected during reactive hyperemia produced by occlusions for 40 seconds. Since 600-800 ml of blood were required for their adenosine analysis, they made four to six blood collections in their study. From these data, they further estimated that there must have been a minimum.concentration of 750 an adenosine in the extracellu- lar fluid of the heart during reactive hyperemia. In their study, intraarterial infusion of 560 nM adenosine produced a maximum coronary dilation. Further support for the adenosine hypothesis follows. Reduction of myocardial oxygen tension was found to cause a decrease in the myo- cardial ATP (96) and creatine phosphate (CP) (7) and an increase in the myocardial ADP, AMP and adenosine of dog (40, 57) and rabbit (48) hearts. Schsuer and Stezoski (83) suggested that a causal relationship exists between alterations of myocardial adenine compounds and adjustments of the coronary blood flow, because changes in ATP and CP levels occur as fast as the changes in blood flow. Similarly, Olsson (72), and Rubia ggugl. (79, 82) suggested a regulatory role of adenosine in controlling blood flow during coronary hypoxia, because coronary vascular resistance 13 decreases when tissue adenosine levels and the rate of adenosine release from the heart are increased. Hoffman and Okita (45) reported that, in guinea pig hearts (Langendorff preparation) perfused with recirculated Krebs-Henseleit solution containing 8-14C-ATP and beta, gamma 32P-ATP, the ATP was degraded to ADP, AMP, adenosine, inosine, and hypoxanthine. In addition, results of these studies indicated that ATP and ADP could not enter myocardial cells without chemical modification. They, however, suggested that nucleosides could pass through the membrane. Adenosine is a nucleoside, its concentration is increased during reduction of oxygen tension in the heart, and it passes freely through the myocardial cell membrane. These findings support the hypothesis that adenosine is re— leased by the heart to regulate the blood flow during reactive hyperemia and myocardial hypoxia. Another convincing support to the adenosine hypothesis is from the experiments of Scott 35 El: (84). When local vasodilation was induced in a donor organ (heart) by decreasing the flow-metabolism ratio (i.e., arterial constriction, or arterial occlusion), the venous blood from this donor organ produced vasodilation in the assay forelimb and vaso- constriction in the assay kidney. Although the chemical nature of the vasoactive substance(s) was not clear, they suggested that the chemical was not oxygen, hydrogen ion, sodium, potassium, osmolality, acetyl- choline, or histamine, because none of these substances produces directly opposite effects on kidney and limb. They, however, suggested that the chemical may be AMP and/or adenosine because the local injection of 14 either chemical' produced vasodilation in the assay forelimb and vaso- constriction in the assay kidney. C. Adenine Nucleotides ATP, an adenine nucleotide, is the fuel for myocardial contraction. ATP and its derivatives (ADP, AMP, adenosine) are present in the cells of cardiac tissue and are coronary vasodilators. Several studies, however, show that ATP is rapidly hydrolyzed in blood and in plasma (25) and in a single transit through the coronary circulation (4, 91). Baer and Drummond (4) demonstrated that exogenous ATP injected (4.9 pg) into the vasculature of the Ringer's perfused rat hearts is hydrolyzed to AMP, adenosine and inosine within 10 seconds. They also found a rapid hydrolysis of 5'AMP to adenosine and inosine during a single pass through the heart. They therefore suggested that ATPase and 5'-nucleotidase are present in coronary vascular cells, and that these enzymes are accessible to ATP and S'AMP in the extracellular fluid of rat hearts. Because ATP can be hydrolyzed to adenosine, the presence of adenosine in coronary venous blood during reactive hyperemia as described in the previous section (Adenosine Hypothesis) can also be interpreted as an evidence for the release of adenine nucleotides, such as ATP, during reactive hyperemia. Collingsworth (25) demonstrated that the half-life of exogenous 14C-ATP in canine whole blood (pH = 7.5-7.7) at 37°C is 18 minutes, while it is less than 3 minutes in the plasma (pH = 7.8) at the same temperature. Collingsworth further reported that the hydrolysis of ATP in the whole blood or the plasma can be reduced by lowering the tempera- ture. 15 For example, while 17% of the exogenous ATP added to the blood was hydrolyzed in 1% minutes at 3°C (half-life - 6 min.), 55% was hydrolyzed in 1% minutes at 37°C. Chen £3.2l, (18) reported, in blood perfused dog hearts, that there was no increase in plasma ATP and AMP during coronary reactive hyperemia, but there was an increase in plasma ATP and AMP in the coro- nary venous effluent during active hyperemia. Stowe 25 2;, (91) reported that ATP concentration in venous effluent increased during coronary reactive hyperemia in the Ringer‘s perfused guinea pig hearts. They, however, found that if arterial ATP was raised to the level attained during reactive hyperemia in their experiments, no coronary vasodilation occurred. Thus, the increased venous ATP during reactive hyperemia appears to be not large enough to produce vasodilation. However, Stowe 35 El! suggested that the venous ATP concentration found during coronary reactive hyperemia was a small fraction of the ATP released by the myocardium, because they observed a rapid disappearance of exogenous ATP in a transit through the coronary circulation of the guinea pig heart. VI. Disputes of Adenine Compound's Participation in Coronary Reactive Hyperemia Some investigators found evidence to dispute that adenine com— pounds (ATP, ADP, AMP, adenosine) are the only metabolites which regu- late coronary blood flow. For example, in the open-chest dog, Meir and Downs (63) found that individual infusions of high ATP, ADP (approxi- mately 10 MM each), AMP or adenosine (approximately 45 MM each) do not 16 produce coronary vasodilation as large as that occurring after a 20- second arterial occlusion in the same dog heart. They concluded that adenine compounds were not the only mediators through which the myocar- dium regulated its blood flow. _Pharmacological studies using dipyridamole (Persantin), lidoflazine and xanthine derivatives (aminophylline and theophylline) reveal more controversial aspects concerning the role of adenine compounds in coronary reactive hyperemia. Dipyridamole and lidoflazine are coronary vasodilators and are believed to potentiate adenosine vasodilatory effect. Because they inhibit adenosine uptakes by unlysed red blood cells (56), by platelets (77), and by the lung and myocardium.(2), these drugs prevent adenosine from reaching the site of adenosine deamination in the tissue. By such inhibition, adenosine can produce greater vasodilation on the vessels. If one can demonstrate that either one of these two drugs can potentiate reactive hyperemia, such findings will significantly substantiate the role of adenosine during reactive hyperemia. Eikens and Wilcken (33-34) reported that dipyridamole did not alter coronary reactive hyperemia in dog hearts elicited by occlusion for 4, 8, or 60 seconds. Bitter and Pauly (13) did not observe an in- crease in total volume of hyperemic flow during reactive hyperemia in dog hearts elicited by stopping perfusion for 30, 60 or 120 seconds after lidoflazine administration. Juhran‘g£_§l, (52) reported that dipyridamole had no effect on peak coronary reactive hyperemic flow of 17 the dog hearts but did increase the duration and volume ratio3 of the response if the arterial occlusion exceeded 30 seconds. Xanthine derivatives block coronary dilation produced by exogenous ATP or adenosine. For example, Bunger g£_§l, (15) reported that, in the Ringer's perfused guinea pig heart, additions of theophylline (10-6 - 10-4 M) into the perfusate will inhibit the coronary vasodilatory effects produced by ATP (8 x 10-7 M), or by adenosine (5 x 10-7 M) previously added to the inflow perfusate. Thus, these drugs should inhibit or attenuate reactive hyperemia if adenine compounds are involved in the hyperemia. Afonso gt-gl, (1), Curnish g£_§l, (28), and Juhran and Dietmann (51) all observed that peak coronary reactive hyperemic flow was not altered after administration of aminophylline or theophylline. Eikens and Wilcken (33, 34) also reported that aminophylline did not affect the coronary reactive hyperemia elicited by an arterial occlusion for 4, 8, or 60 seconds in dogs. Bittar and Pauly (13) did not observe changes in total volume of reactive hyperemic flow elicited by the occlu- sion for 30, 60 or 120 seconds after injection of aminophylline. On the other hand, Curnish £2 31. (28), and Wadsworth (93) found reductions of the duration4 and the volume of coronary blood flow during reactive 3The ratio of the total volume of hyperemic flow to the flow debt. Volume of hyperemic flow is that collected from the onset of reactive hyperemia to the point where the flow returns to the control level. Because reactive hyperemia is characterized by a very gradual return to control flow, it is difficult to determine the duration of reactive hyperemia. Some investigators prefer to use the period from the onset of reactive hyperemia to the point where the flow has returned 50% toward control level as the duration and the flow volume measured by planimetry during the duration as the volume of coronary blood flow during reactive hyperemia (28). 18 hyperemia after aminophylline injection. To summarize the experiments cited in this review, it appears that the issue of adenine compounds in the regulation of coronary blood flow during reactive hyperemia is not completely settled. Berne and his co- investigators have produced strong evidence for their adenosine hypothe- sis. However, as described in this literature review, adenine nucleo- tides may be released from the cardiac tissue and produce vasodilation during reactive hyperemia but they are quickly hydrolyzed such that no significant amount of nucleotides can be detected in the venous effluent. This present study, therefore, was designed to investigate l) the stability of ATP, ADP and AMP during a transit through the coronary circulation, and 2) if ATP and AMP concentrations would increase during reactive hyperemia in a heart perfused with artificial perfusate. METHODS I. Experimental Preparations A. Surgical Preparations of Isolated Guinea Pig Hearts in linza Guinea pigs (Hartley strain, Cannaught Laboratories, Willowspring, Ontario) of either sex,fed §g_libitum and weighing 300-500 grams, were sacrificed by a blow on the neck and then secured on a dissecting tray by two hemostats on the hindlimbs. After bilateral thoracetomy, the heart was exposed through an incision on the pericardium. The ascending aorta was isolated and freed from surrounding tissues. Two silk threads (size 000) were passed beneath the aorta and a tie was made on the aortic arch so that there was a space about 1.5 cm in length between the tie and the heart for cannulation. An incision was made on the ventral wall of the aorta which was immediately cannulated through the incision with a polyethylene tubing (PE 240, i.d. 0.066 inch). Care was taken to avoid damaging the aortic valve. The heart was immediately perfused via this cannula with a modified Krebs-Ringer—bicarbonate solution (293 i 2 mOSm/kg, pH 7.5, and in mmoles/L NaCl 119.7, KCl 4.7, CaCl 2.5, 2 KHZPO4 1.2, NaHCO3 24.9, glucose 5.5, pyruvate 2.0) at 65 cm H20. The perfusate was equilibrated with 95% 02 and 5% C02 at 37°C throughout the experiments. The procedure for the preparation of the perfusate is described in APPENDIX A (page 65). 19 20 After the cannulation of the aorta, the heart was transferred to the heart chamber reservoir as shown in Figure l. The time taken from sacrifice to completion of cannulation was about 3 minutes. The heart was weighed at the end of all experiments for calculation of coronary flow per gram of heart tissue. Three types of Langendorff heart preparation were used in the study and they varied as follows: 1) Preparation I, the standard Langendorff preparation (Prep. I) The preparation which prevents accumulation of perfusate within the heart chambers as described by Bunger g£_al, (l6) and Merrill (62) was used. The left atrial appendages were excised and the mitral valve was cut. The apex of the right ventricular wall was punctured (avoiding visible coronary vessels). The anteriomedian area of the right atrial appendages was cut open. All surgical maneuvers were performed with great care so that damage to the sinoatrial node was minimized. Outflow samples from the heart were collected from the outlet of the heart chamber (Figure l). The sample, therefore, contained the perfusate which was passed through the intact as well as incised coronary vessels, coronary sinus and thebesian veins. The samples were not centrifuged before chemical analyses. It has been shown that adenine compounds (ATP, ADP, AMP, cyclic AMP, adenosine) and hypoxia produce coronary vasodilation in this preparation (15, 16). 2) Preparation II (Prep. 11) Because the Prep.I heart does not allow collections of true coro- nary venous effluent, a second heart preparation was designed. In this 21 Figpre 1. The non—recirculating perfusion system. The diagram shows that PA is cannulated with an inverted L—shape PE tubing, while VC and PV are tied. The coronary venous effluent is collected from PA catheter in Preps. II and III (or from the outlet of the reservoir as shown in (b) in Prep. 1). The height between the outlet of PA catheter and the pulmonic valve is 3—5 cm. The non-recirculating perfusion system is: Three reservoirs (f, g, h) are situated above the heart to give 65 cm hydrostatic perfusion pres- sure. Three 3~way stopcocks (d) are in the perfusion circuit (e) to direct specific perfusate to the heart. The normal (f), the hypoxic (h), and the heart chamber (b) reservoirs and the glass distillation column (c) are jacketed by circulating 37 C water so that the heart is kept at the same temperature throughout the experiment. Arrows indicate the direction of circulating water from or to the thermo- regulator. All the reservoirs (b, f, g, h) are covered with a plastic sheet to retard evaporation. Long rubber latex tubings (approximately 100 cm) connect the reservoirs (f, g, h) to the distil- lation column, and these tubings allow adjustments of the height of the reservoirs. Reactive dilation is produced by occluding these tubings. The whole circuit is washed by 95% ethanol and rinsed with a large volume of distilled water at the end of each experiment. LA: Left Atrium PA: Pulmonary Artery RA: Right Atrium VC: Venae Cavae LV: Left Ventricle PV: Pulmonary Veins RV: Right Ventricle FM: Flowmeter WT: Water (37°C) from or to FT: Flow Transducer the thermoregulator REC: Recorder PREM: Preamplifier a: Collection tubes, or graduated cylinder. b: Heart chamber reservoir. f: Reservoir for normal perfusate (equilibrated with 95% oxygen and 5% carbon dioxide gas mixture throughout the experiment). The reservoir' is also calibrated and marked so that the volume of the perfusate inside of the reservoir can be estimated. g: Reservoir for radioactive material is not jacketed. h: Reservoir for hypoxic perfusate (equilibrated with 95% air and 5% carbon dioxide gas mixture). This reservoir is also cali- brated and marked so that the volume of the perfusate can he estimated. 1: Rubber latex tubing (i.d. 0.25 inch). th 22 ‘fi O) 01 / > d 0 2 Z i2 . ”<1: WT «E I c 4-WT [ii Id!) m e lifii VC Pv PA fl ’ \ CATHETER _L.. WT ‘1 ‘ ' 2 RA ' V .0 "0 ""1 i m RV / I// a "WM/fl b I Le grRAOTMER 3 7° C THERMO- REGULATOR 23 preparation, the atria and ventricles were left intact and the pulmonary veins were tied. The pulmonary artery was cannulated with an inverted L-shape polyethylene catheter (PE 280, i.d. 0.085 inch), and the vena cava was tied so that the coronary venous effluent was directed into the pulmonary arterial catheter (Figure l). The pulmonary arterial catheter was deeply inserted into the right ventricle and therefore the pulmonic valve and the right ventricular endocardium might have been injured. Coronary venous samples collected from the pulmonary arterial catheter were also not centrifuged before chemical analyses. The efflu- ent collected from the catheter might have been contaminated by the in- jured cell debris. 3) Preparation III (Prep. III) In order to minimize cardiac damage and sample contaminations, a third heart preparation was designed. This preparation was essentially the same as the Prep II except: a) care was taken to minimize damage to the pulmonic valve and the right Ventricular endocardium.by minimal insertion of the catheter into the pulmonary artery, b) the pulmonary arterial catheter and collection tubes were siliconized,5 and c) the venous effluent samples collected from the pulmonary arterial catheter were immediately centrifuged at 22,500 x g at 4°C for 20 minutes. We have demonstrated that this preparation exhibits reactive and hypoxic vasodilation (Figures B-l, B-2, APPENDIX B, pages 73 and 75). 5The catheter was thoroughly washed and rinsed in distilled water, and was then immersed in a solution of one part Siliclad (contains soluble concentrated silicone, Clay Adams, Becton, Dickson and Company, Parsippany, N. J.) to 100 parts of distilled water by volume, rinsed and then dried at room temperature for 24 hours. 24 we have also found that during a resting steady state the effluent from the pulmonary artery contains less than 0.1 ng/ml ATP, but that from the heart chamber reservoir contains 1 to 5 ng/ml ATP (Table B-1, APPENDIX B, page 76). This indicates that in this preparation the effluent collected from the heart chamber may be contaminated by ATP originating probably from tissue debris but the effluent from.the pulmonary artery seems to be free of the contamination. B. The Coronary Flow Measurement An extracorporeal flow transducer (BLC-2024-F17, i.d. 3/32 inch) was used to measure the rate of coronary inflow in all three prepara- tions. The transducer was placed about 8 cm below the glass distillation column and 6 cm above the aortic valve of the heart (Figure l). The transducer was connected to a Biotronex BL—610 Pulsed-Logic flowmeter (Biotronex Laboratory, Inc., Silver Spring, Md.), which was connected to a Hewlett Packard 350-1000B DC preamplifier. The coronary flow, either phasic or mean flow, was continuously recorded on a Sanborn (model 296) direct-writing oscillograph. The preamplifier and the flow- meter were calibrated at the beginning of each experiment and mechanical zero was checked periodically during the experiments. Throughout this thesis, mean coronary inflow was used to express the results unless otherwise indicated. C. Criteria of an Acceptable Surgical Preparation The heart was allowed to stablize for 20—30 minutes following surgery. After the stablization period, the heart was discarded if it 25 did not satisfy the following criteria: 1) the flow rate was stable (within 0.5 ml/min) in two consecutive measurements within six minutes, 2) the spontaneous heart rate (the intrinsic heart rate) was at least 200 bpm (beats per minute, less than 200 bpm indicates possible damage to the sinoatrial node), and 3) the magnitude of the peak coronary flow rate, following the release of an occlusion of inflow for 30 seconds, was at least twice greater than control flow. The measurement of this third criterion was called the pre-experimental reactive dilation. The data from an experiment were discarded if the following criteria were not met: 1) the magnitude of reactive dilation performed at the end of the experiment had to be at least 85% of the magnitude of the pre-experimental reactive dilation, and 2) the control coronary flow had to be less than 7 ml/min/gm (more than 7 md/min/gm generally indicates incompetence of the aortic valve or a punctured aorta which allows leakage of perfusate). Furthermore, all experiments were per- formed within 90 minutes after the stablization period. II. Experimental Procedures A. Survival of ATP duripg a Passage through the Coronary Circulatiqp While measuring coronary flow, various amounts of carrier ATP were added into the perfusate (zero to 0.1 mg of ATP per ml of perfusate) perfusing the coronary circulation. Four minutes later (to allow dead- space perfusate to pass through).0r when coronary flow became steady after dilation, 2 m1 of the venous effluent were collected for the 26 determination of ATP. The samples, without being centrifuged, were analyzed for ATP by using the firefly bioluminescence assay (see APPENDIX C—I, page 78) within 3 minutes. Surgical preparations I and II were used for this ATP survival study. In order to see if hydrolysis of ATP will occur in the inflow perfusate during the 90~minute period of the experiment, ATP was added into a test tube containing the arterial perfusate (20 ng/ml) and incu- bated at 37°C. ATP concentrations in the tube were determined every 5 minutes for 80 minutes of incubation. The ATP concentration of the per- fusate remained unchanged for 80 minutes in the test tube. The stability of ATP in the uncentrifuged effluent samples was also studied by adding various amounts of ATP to test tubes containing venous effluent which had been assayed to be free of ATP. The ATP concentration, immediately following additions of ATP, ranged from 4 to 12 ng/ml. The concentra- tion remained essentially unchanged at room temperature (25°C) for 45 minutes. These studies indicate that ATP present in the inflow perfusate, or venous effluent remains stable for at least 45 minutes. B. Coronagy Venous Recoveries of the Adenine Base during. Intraarterial Perfusions of ATP, ADP, or AMP The study was performed in Preps. II and III hearts. Various amounts of individual ATP, ADP or AMP were added into the perfusate to make concentrations of the nucleotide in the perfusate range from 2 to 121 uM. When coronary flow reached a steady state, samples were obtained from both the effluent and the perfusate for the measurement of the 27 adenine absorbance at 258 nm (A258).6 The perfusate without adenine compounds was used as a blank when A258 was measured. The venous recovery of the base was expressed as percent of the outflow to the inflow A258. Venous recovery of less than 100% indicates either extraction or destruction of the adenine base in the perfusate during the passage through the coronary circulation of the heart. C. Coronary Venous Recoveries of 14C dur Intraarterial Perfusion of 17*C-ATP 14 14 Four to five p01 of C—ATP (uniformly labeled C-ATP, specific activity - 482 mCi/mM, Lot No. 893-209, New England Nuclear) were added to 20 ml perfusate (0.5-0.6 uM ATP) and the whole was perfused through the coronary circulation. 14C radioactivities (cpm/ml, counts per minute per ml) of the effluent were compared to cpm/ml of the arterial perfusate. A decrease in the cpm/ml of the perfusate after passing through the heart would indicate uptakes of the ATP carbon skeleton by the heart. The venous recovery of 140 was expressed as percent of the outflow to the inflow 140 radioactivities. Preparations II and III were used in the study. D. Identifications of Products Formed from Hydrolysis of Adenine Nucleotides i; a Transit through the Coronary Circulation 1) ATP, ADP or AMP Different amounts of ATP, ADP or AMP were added to the arterial perfusate to make the nucleotide concentration ranging from 14 to 130 uM. 6The wavelength of maximal absorbance of ATP and AMP was experi- mentally found to be 258 nm on a spectrophotometer (Beckman DB). 28 When venous A258 and coronary flow became steady, 30 to 50 ml of coro- nary venous effluent and arterial perfusate were collected. Both samples were immediately analyzed, without being centrifuged, by chroma- tography for specific biochemical intermediates. The chromatogram of arterial perfusate shows the purity of the adenine nucleotide entering the heart, while the chromatogram of venous effluent shows the inter— mediates formed during the transit. The chromatographic procedure, i.e., the rapid separation, to identify these intermediates is described in APPENDIX C-II, page 80. These studies were performed in Preps. II and III hearts. Since the concentration of ATP, ADP or AMP perfused in these studies was high (14—130 uM) enough to induce heart block, additional experiments were performed in which 14C—ATP was used instead of carrier ATP. In this way the concentration of ATP was reduced to 0.5-1.1 uM. 2) léC-ATP In two experiments, 5 or 10 uCi of l4C-ATP (uniformly labeled 14C—ATP, specific activity - 482 mCi/mM; Lot No. 893-209, New England Nuclear) was added to 25 ml perfusate (0.5 or 1.1 uM.ATP) which had been equilibrated with a gas mixture containing 95% 02-52 C02 (normal gas). The radioactive perfusate was left in the radioactive reservoir (Figure 1) without further equilibration with the normal gas mixture. Prep. II was used in these two experiments. Immediately following the perfusion of radioactive perfusion of the radioactive perfusate, thirty m1 (including the fluid from the dead-space) venous effluent were col— lected from the pulmonary artery. The sample was chromatographically analyzed by the specific separation method (APPENDIX C—II). 29 In three other experiments, surgical preparations II and III were used. Approximately 4-5 uCi of 1l'C-ATP were added to 20 ml freshly pre- pared perfusate; ATP concentration was approximately 0.5-0.6 uM. Fifty microliters of this solution were withdrawn and added to 2.0 ml dis- tilled water (1 to 41 dilution). This diluted sample, Sample T, was used for calculation of total radioactivities perfused. The rest of the 14C-ATP solution (approximately 19.9 ml) was poured into the small reservoir (g) shown in Figure l, and was continuously bubbled with the normal gas mixture. When coronary flow became steady, the perfusate was switched from that containing no 14C-ATP to that containing 14C-ATP. Immediately following the switch, venous effluents were simultaneously and continuously collected from two sources, one from the pulmonary artery (called Sample P) and the other from the heart chamber reservoir (called Sample H) as shown in Figure 1. When 10 to 15 ml of the radio- active perfusate had passed through the heart, approximately 0.3 ml sample (called Sample A) was withdrawn from the inflow perfusate tubing at 7 to 10 cm above the heart. Simultaneously, 0.5 m1 of the venous effluent (called Sggple V) was withdrawn from.the PA catheter. To 0.05 ml of Sample A and Sgpple V were added 2.0 ml distilled water to make 1 to 41 dilution of the two samples. These diluted samples were used for the measurement of venous recoveries of 14C from the heart. Just before the radioactive reservoir was empty, two fivedml aliquots of non-radioactive perfusate were added to the reservoir tO'wash off the residue of lac-ATP. The volumes of Sample P and Sample H collected over the entire perfusion period were recorded. A half ml of 30 Sample P was also diluted with 5.0 ml distilled water (1 to 11 dilution). The diluted Sample P was used for calculations of 14C recoveries from the anion exchange resin. Carrier ATP, ADP, IMP, AMP, cAMP, inosine, and adenosine, l to 4 mg each, were added into the remaining undiluted Sample P. These carrier chemicals served as indicators of specific nucleotide position on the chromatogram. The whole was then chromatographed either by the specific or the modified specific method (APPENDIX C-II). One ml of each eluate collected on the fraction collector was plated onto a planchet and dried to infinitive thinness. The 14C radio— activities (cpm/ml, counts per minute per ml) of each of these planchets were counted by a Gas—Flow Detector (model 470) in an Automatic Planchet Changer (model 1042) for one minute by decade Scaler (8703 series) and the data were listed by a Printing Lister (stock No. 000-008437) (Chicago Nuclear Corp., Chicago, 111.). Also the absorbance of the eluate in each fraction collector tube was measured with a spectrophotom- eter (Beckman DB) at wavelength 258 on. One ml of each diluted sample A, l, _11, _I_I_ and 1 was plated separately and its cpm/ml were measured. The 14C venous recovery, i.e., the recovery of 14C in the perfusate after one transit through the coronary circulation, was expressed by the ratio of cpm/ml of diluted Sample V to cpm/ml of diluted Sample A. The recovery of 14C after passing through the resin was determined by dividing total cpm in the eluate collected on the fraction collector tubes (the sum cpm/ml of the chromatogram multiplied by 9.8 ml) by the total cpm contained in Sample P added to the resin (cpm/m1 of diluted 31 Sample P multiplied by the dilution of 11 and then by the volume of undiluted Sample P added into the resin). E. Endogenous Release of ATP and AMP from the Heart during Reactive Dilation Before each experiment, venous effluents were sampled successively to measure endogenous ATP by the firefly bioluminescence assay (FBA, see APPENDIX C-I). These measurements were performed because surgical trauma may cause release of endogenous ATP. When no detectable ATP was present in the effluent, coronary reactive dilation was elicited by stopping the perfusion for 30 seconds. Three or four venous samples, five ml each, were collected, i.e., one before the occlusion (C1), one or two during reactive dilation (R, or R1 and R2), and one 5 minutes after resumption of perfusion (C2). The venous samples were discarded if the magnitude (peak flow) of the coronary reactive dilation was not at least twice greater than that of the flow before this occlusion. One quarter m1 of each venous sample was used for the measurement of ATP by the FBA.within 5 minutes. Three ml of each remaining venous sample were analyzed for AMP using myokinase and 14C-ATP (see APPENDIX C-III, page 87). Surgical preparations I, II and III were used in these studies. III. Statistical Analyses of Results Student's t-test modified for paired comparisons, the least sig- nificant difference test, or randomized complete block of analysis of variance (89) was used in the statistical analyses of results. The statistical significance was set at p values less than 0.05. RESULTS I. Survival of ATP duripgra Passage through the Coronary Circulation The survival of exogenous ATP in a transit through the coronary circulation was studied by adding various known amounts of ATP to the arterial perfusate perfusing Preps. I and II hearts and assaying the ATP in the effluent. The arterial ATP concentration above which venous ATP becomes detectable (greater than 0.1 ng/ml) is called the threshold concentration. As shown in Figure 2, ATP was not found in the effluent from the Prep. I when inflow ATP concentration was less than 60 ng/ml (0.12 uM). The threshold concentration of ATP was 75 ng/ml (0.15 pH) in the Prep.I5 Figure 3 shows that in Prep. II, no ATP was found in the venous effluent collected from the PA catheter when inflow ATP concentration was less than 500 ng/ml (1.1 MM). The threshold concentration of ATP in the Prep. II was between 750 and 900 ng/ml (1.5-2.0 uM). 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Coronary Venous Recoveries of the Adenine Base and of 140 during Intraarterial Perfusions of Adenine Nucleotides Table 1 shows that venous recoveries of the adenine absorbance (A258) of carrier ATP were 84 i 2% (N - 3); that of ADP, 96 i 5% (N - 2); and that of AMP, 81 i_3% (N = 4). When l4C-ATP was perfused into the coronary vasculature, venous recoveries of 14C radioactivities were 86 :;7% (N = 3). This value was statistically the same as that of carrier ATP (84 :;2%) (analyzed by randomized complete block of analysis of variance and the least significant difference test) in spite of the fact that the concentration of carrier ATP (2-26.5 uM) used was four to fifty times greater than that of 14 C-ATP (0.5—0.6 uM). The result in these studies indicates that more than 80% of the inflow adenine absorbance can be recovered in the effluent during per- fusions of ATP, ADP or AMP. This suggests that disappearance of ATP during transit through the coronary circulation (Figure 3) results from hydrolysis of ATP. The exact chemical nature of the adenine absorbance in the effluent, however, is not clear. For example, it is not known if the adenine absorbance is due to the presence of adenine, adenosine, AMP or ADP. Therefore, attempts were made to identify the chemical nature of the effluent adenine absorbance. III. Identifications of Products Fogmed from Hydrolysis of Adenine Nucleotides in a Transit through the Coronary Circulation Various amounts of ATP, ADP or AMP were added to the perfusate (14 to 130 HM) perfusing the heart and samples were obtained from the 38 Venous Recoveries of Adenine Base and 14C Table 1. Average Surgical Percent Prep. Venous Wt (Expt. No.) Percent Venous Recovery* (UM perfused) Recovery (gm) Carrier ATP (A258) II (53) 82(5.5); 86(9.0); 88(13.5); 86(26.5) 86 2.5 III (54) 79(2.0); 93(4.3); 86(7.l) 86 2.3 III (62) 81(13.9) 81 1.6 _(mean 3; S.E.) - 84 i 2 Carrier ADP (A258) II (55) 100(110) 100 1.6 III (66) 97(3.3); 94(6.8); 88(15.l); 90(25.4) 86(82.5) 91 2.7 (mean i S.E.) - 96 i 5 Carrier AMP (A258) II (56) 86; 88; 87; 85(14.2) 87 2.4 III (61) 77; 73(5.8) 75 2.5 III (65) 73(4.0); 65(6.l); 86(8.4) 75 2.0 III (83) 82(6.9); 83(19.9); 86(38.6); 91(121) 86 1.8 (mean j; S.E.) - 81 i 3 14C-A'I‘P (14C) II (57) 79(0.5) 79 1.9 III (59) 78(0.6) 78 2.1 III (60) 100(0.5) 100 2.0 (mean : S.E.) - 86 i 7 * Venous Recovery = Venous A 258 °r 14 Arterial A or 258 C 14 C x 100% 39 inflow tubing and effluent. Both were analyzed by anion exchange chromatography. Similar experiments were performed by using 14C-ATP (0.5-1.1 MM) and only the effluent was analyzed. The typical chromatograms from these studies are shown in Figure 4. The left column of the figure shows the chromatograms of the arterial perfusates. During perfusion of ATP, AMP or 14C-ATP, the only adenine compound identifiable in the arterial perfusate was the particular compound added to the perfusate. However, during perfusion of ADP, there were AMP and nucleosides on the chromatogram of arterial perfusate in addition to ADP. The amounts of AMP and nucleosides present were small (Figure 4). As shown in the right column of Figure 4, only AMP and nucleosides appeared on the chromatograms of the effluent during the perfusion of ATP, ADP or AMP. In addition to AMP and nucleosides, a small fraction of inosine and three unknown chemicals were present in the venous effluent during 14C—ATP perfusions. However, in all of these experi- ments, no ATP, ADP, IMP or CAMP was present in the effluent. AMP ap— peared to be the main products in the venous effluent because AMP frac- tion occupied the largest area under the curve on all venous chromato- grams. Table 2 shows the percentage of AMP and nucleosides found on the chromatograms of venous effluent during these perfusion experiments. The table indicates that more than 68% of the chemicals identified on the chromatograms were AMP. In these experiments, high concentrations, i.e., 14.2-130 HM, of carrier ATP, ADP, or AMP were used so that A258 could be detected. 40 Figure 4. Typical chromatograms of arterial and coronary venous perfusate during perfusions of adenine nucleotides. Equal volumes of arterial perfusate and coronary venous effluent were sampled during the perfusion of ATP, ADP or AMP. The samples were chromatographically analyzed (with gradient elution) on two separate anion exchange resin columns. Arterial chromatograms (left column) show purity and stability of adenine nucleotides entering the coronary. The venous chromatograms (right column) show the products formed during the passage through the coronary vasculature. ' Similar experiments were performed by perfusing uniformly labeled l(‘C-ATP. In these experiments, only the venous effluent from the pulmonary artery was analyzed by the specific (or the modified specific) separation of anion chromatography. The purity of 14C-ATP in the inflow perfusate was checked as described in APPENDIX A, page 65. The products in the venous effluent on the chromatogram were identified by the indicator nucleotides and nucleosides which had been added into the venous effluent before the chromatographic separation. The ordinate is the absorbance or 14C radioactivities of the eluate. A - Absorbance at wavelength 258 nm (mp). cpm - Counts per minute of 1 md eluate. The abscissa shows the test tube number of a fraction collector. Each test tube collects approximately 9.8 ml of eluate. N - Number of experiments. 41 ANION EXCHANGE CHROMATOGRAPHY ARTERIAL PERFUSATE CORONARY VENOUS DURING ATP INFUSION N-l losuucoou a: a} , , 4.0» scoou “via nu‘ooou W‘LOH m‘cooriInum‘coon 3 (Purlty Chock) f j t .. ' s. s. " '. ‘ 1 s z 3 - . - a t ...................................... . 1w. ....-- _I—fl—fl—Tz‘I—III—‘fi _‘I—fi—t—‘Ifl DURING ADP INFUSION «.2 A Wtouu Inn coon m I W: "I g I ‘. .8 E I i I: i E ‘3 l ...... ..-\- - . .. .°'.-......' _T—fl—ffi—Ts We DURING AMP INFUSION III-2 A s. 25"” 3 8 5‘: .o i s i T .4 z j E E .4 . é E O ........... ' . ”IR-m...” . . . . . I ' -' "-. —:—-;.—-a—a—-a—.:. ‘3—1—I‘I—1—1—fu DURING Mc-ATP INFUSION u-s r230." HCOOH an ”on HCOOH v.75“ NH‘COOH ma 5 -‘ I 3 £5 m a i: Z — D G O 0 0 ATP ‘¥&% 8 '2' 40 60 m m m Froctlon collector tube no. 40 I20 FroctIon collector tube no. Figure 4 42 Table 2. The Percentage of AMP,Nuc1eosides Appearing on the Chromato- grams of Venous Effluent during Perfusions of Adenine Nucleotides Percent of Total Venous A or Venous cpml 258 2 Heart Nucleotide Coronary Flow Prep. Perfused (uM) AMP Nucleosides Unknowns (ml/min/gm) II ATP (130) 84 t 16 none II ADP (110) 92 8 none III ADP (82.5) 75 25 none II AMP (14.2) 79 21 none III AMP (103) 76 23 none 113 izc-ATP (0.5) 81 10 9 3.96/5.94 II l4C-ATP (1.1) 75 12 13 2.27/6.2 III 14C-ATP (0.6) 72 10 18 3.81/5.24 1(A258 or cpm of the particular component/total A258 or cpm of the eluate on the chromatogram) x 100%. 14 2Coronary flows are expressed as the flow before/during C—ATP perfusion. l4 3Perfusate was not gassed with 95% O -5% CO after adding C-ATP. 2 2 43 But with the high concentration of adenine nucleotides, the heart rate was decreased, perfusate accumulated in the heart chamber and impeded the coronary flow. Consequently, the coronary flow during the perfusions could not be accurately measured. Concentration of 14C-ATP used was much lower than that of the carrier nucleotides. During the perfusion, coronary flow increased in all five experiments (Table 2). This indi- l4 cates that C-ATP perfused had reached the coronary arterioles and produced vasodilation. IV. Endogenous Release of ATP and AMP during_ Reactive Dilation Table 3 shows the concentration of ATP in the effluent before, during and after coronary reactive dilation in three preparations. Concentrations of ATP during reactive dilation were not significantly altered in all hearts. Table 4 shows the AMP concentrations before, during and after coronary reactive dilation. The concentrations of venous AMP during reactive dilation (R1) were significantly greater than before reactive dilation (C1) in Preps. I and II hearts. The results from Prep. III hearts were different from those of Preps. I and II hearts. The concen- tration of AMP in all samples from Prep. III, C1 as well as R1, was approximately one—tenth of those from Preps. I and II hearts. Furthermore, the concentration of AMP was not significantly increased during reactive dilation in Prep. III. 44 Table 3. Concentrations of Venous ATP (ng/ml) before (C 1), during (R) and after (C2 ) Coronary Reactive Dilation Expt. No. C R C Wt (gm) Prep. I Hearts (N = 6) 0.3 16 19 20 22 29 30 Mean 1 S.E. 0.3 0" 2 0.3 0.1 0.1 0.3 l+ C>C>C>C>PJC> l+ c>c>c>c>c> l+- C>C>C>C>P4C> Prep. II Hearts (N - 4) 53 54 55 57 0 0 0 0 Mean i;S.E. 0.4 <0 0.1 1.2 l+ CDF‘CDCD .1 2.0-1:200 Prep. IIIa Hearts (N = 5)@ 58 59 62 64 65 Mean : S.E. All samples had less than 0.1 ng/ml ATP. 2.0 i 0.1 00000 00000 00000 NI—‘I—‘NN coon-no Prep. III Hearts (H,'4§l 80 0 81 82 84 85 86 .4 Mean i S.E. 1.6 i 0.9 0.4 c>u~c>a~c>c> l+- F‘FJCDCDCD |+- c>c>c>uac>c> 2 0.2 0.5 c: :11 N L I+ c: 2.: A11 reactive dilations were elicited by a 30-second inflow occlusion. Wt(gm) - guinea pig heart wet weight. Since the lowest detectable ATP concentration in our ATP assay by firefly bioluminescence is 0.1 ng/ml, all zero values in this table represent ATP concentration less than 0.1 ng/ml. @: These samples were collected from nonsiliconized catheter in the pulmonary artery, and were not centrifuged before chemical analysis. 45 Table 4. Concentrations of Venous AMP (ng/ml) before (Cl), during (R1 and R2) and after (C2) Coronary Reactive Dilation Expt. No. C1 R1 R2 C2 Wt(gm) Prep. I Hearts (N - 11)} 20 26 88 61 15 1.7 22 60 150 78 48 2.7 24 130 200 160 140 1.5 25 78 124 108 82 2.8 26 64 118 76 73 2.6 27 42 130 88 61 2.4 28 46 130 79 110 2.3 16 68 80 not measured 49 1.2 19 88 137 not measured 79 1.2 29 66 195 not measured 164 2.3 30 96 156 not measured 92 1.6 Mean 1 S.E. 72 i 9 139 _-l_- 11* 93 i 12 84 i 12 2.0 i 0.3 Prep;_II Hearts (N - 6)1 44 62 210 120 94 4.3 45 70 116 92 52 3.2 46 112 156 128 108 2.6 47 40 172 124 22 2.4 48 116 210 140 90 2.2 49 94 210 140 70 2.4 Mean 1 S.E. 82 i 12 179 i 16* 124 i 7* 73 i: 13 2.9 i 0.3 Prep. III Hearts (N - 6)2 80 0 35 10 0 2.2 81 0 0 0 0 2.4 82 0 0 0 0 2.0 84 0 40 0 0 2.8 851 0 0 0 0 2.1 86 0 0 0 0 3.8 Mean 1: S.E. 0 12.5 i 4.5 1.7 i 0.1 0 2.6 1; 0.3 All reactive dilations were elicited by a 30—second inflow occlusion. * - significantly different from.Cl(randomized complete block of analysis of variance, p< 0.05). lAmmonium formate (0.4 M, 150 ml, hydrostatic pressure = 90 cm H 0) was used for the separation of ADP and AMP from labeled ATP in the esin. 2Distilled water (10 m1, drained by gravity) and formic acid (2.4 N, 50 m1 at 60 cm H20) were used to separate ADP and AMP from labeled ATP in the resin. 46 V. Comparison of Reactive Dilation Elicited by_g§ Occlusion of Perfusion for 30 Second§_ in Preps. I, II and III Hearts Table 5 shows the magnitude of reactive dilation and heart rates before and after using the heart for various experiments for 90 minutes. No exogenous adenyl compound was perfused into the heart during these 90~minute experiments. Control coronary flow and the magnitude of reactive dilation, did not change significantly after the 90-minute period in all of the three preparations. Also, the ratios of the peak coronary flow (PCF) to con— trol coronary flow (CCF) were the same among all preparations. The heart rate of Preps. II and III decreased significantly after the 90 minute period. The decrease in heart rate might be due to Bainbridge reflex. The pressure in the left atrium might have increased during this period in Prep. II and III because the pulmonary veins were tied. The elevated pressure might initiate Bainbridge reflex to lower the heart rate (10). This would not occur in Prep. I because its left atrium ‘was excised to prevent accumulation of perfusate in this chamber. Figure 5 shows the typical recordings of reactive dilation in three preparations. In Prep. I (heart weight - 2.6 gm), coronary flow increased from a control of 7 ml/min to a peak flow of 17 ml/min. The duration of dilation was 20 seconds. The weights of Preps. II and III hearts were 2.25 and 2.1 gm, control flows 8 and 6.5 ml/min, peak flows during reactive dilation 24 and 14 ml/min, and durations 18 and 20 sec- onds respectively. These indicate that the response of the three heart preparations to 30 seconds occlusion of arterial inflow was similar. 47 .Amo.ouve .oomfiumeaoo menace pom wowmfiooa amoulu m.ucoooumv umNMIoum Bonn uaoummmqo manomofiwwawwm « new .unwao3.um3 unmom mam modest I DB moaa\umon .oumw ammo: 1 mm .am\oH\E 3 mooooom om no Sea a mo GOfimoaooo as a woufioaao coaumawo m>fiuomou sense Beam usdouou soon I mom . c o o A w a maw\oaa\aa Ge .ooamoaooo ouomon eaoumwooaaa 30am Houuooo mumoouou I moo I o H 32.. do H m.~ to H og No H Ha $me38 to + ~.~ I I I I 3 H: o + ooN H.o + m.~ s.o + m.a ~.o +_e.m oomeoum I o+«%~ House moHNR flofimsufiTga m.o + m.~ I. I. I. I. o HH m + New ~.o + m.~ o.o + o.o «.0 + a.~ nomemum I oHafi HoHNN Towfio moumsofiTta «.0 + o.~ I. I. I. I. a H e + Hmm H.o + e.~ a.o + H.o ~.o + m.m nexMIoum us me Amoo\momv mom moo z .aoum oaumm Hmoawuom muumum HHH mom HH .H .memum ea muomawuoexm mo AuQMMIumomv mam meu us was Aumxmlmumv wofiooawmm sou ca moumm uumom mom oowumaan o>Huomom mo moooufiowmz .m manna 48 Figure 5. Typical recordings of reactive dilation in Preps. I, II and III Hearts. Coronary reactive dilation was elicited by an occlusion of inflow for 30 seconds in all hearts. C1, R1 and R2 represent the time during which 5-ml samples were collected. Chart speed of all recordings = l mm/second, 1 cm pen deflection - 11 ml/min coronary flow. 49 \7 ___ 4 c. Occlusion R. R2 0 30 PREPII . l\ C. Occlusion R. R; O 30 PREPZIII _. ./\ L a c. Occlusion R, R2 0 30 TIME (see) Figure 5 DISCUSSION The purpose of this study was to determine if adenine nucleotides participate as vasodilators in coronary reactive hyperemia. Specifically, the experiments were designed to study the metabolism of ATP, ADP and AMP during a transit through the coronary circulation; and to measure concen- trations of ATP and AMP in the coronary venous effluent during reactive vasodilation. The studies were performed on isolated guinea pig hearts which were perfused by non-recirculating, cell—free perfusate. Artificial perfusate was used because ATP has been shown to have a half-life less than 3 minutes in whole blood and plasma at 37°C (25). In addition to common electrolytes, the perfusate also contained glucose and pyruvate to en- sure a better cardiac performance and a more constant coronary flow (16). Three types of guinea pig hearts were prepared according to Langendorff techniques (16). In the first preparation, Prep. I, the wall of the atria and the right ventricule was cut open, the mitral valve was made in- competent, and the effluent was collected from drainage flowing over the heart‘s surface. The second preparation, Prep. II, was a modified Langendorff preparation in which the mitral valve, the atria and right ventricle were left intact, the venae cavae and pulmonary veins were tied, and the coronary venous effluent was collected from a catheter inserted into the right ventricle via the pulmonary artery. The third 50 51 preparation, Prep. III, was the same as the Prep. II except that the catheter tip remained in the pulmonary artery to avoid damaging the pulmonic valve. Furthermore, the catheter and collection tubes were siliconized, and the venous effluent collected was centrifuged immedi- ately at 22,500 x g for 20 minutes at 4°C. By these modifications, damages to the cardiac tissue were minimized and true coronary venous effluent could be obtained from the catheter in the pulmonary artery. Contaminations of cellular elements in the effluent were minimized by centrifugation. I. Release of ATP during Coronary Reactive Dilation Venous effluent ATP concentrations were measured before, during and after reactive dilation elicited by occluding the coronary inflow for 30 seconds. No significant change in ATP concentrations was ob- served during reactive dilation in all three heart preparations (Table 3). This finding is in agreement with that of Chen e£_el, (18) who reported that coronary venous plasma ATP did not rise significantly dur— ing reactive hyperemia in dog hearts. Berne e£_§1, were also unable to detect any adenine nucleotides in the effluent of hypoxic rabbit (75), and cat hearts (8). Stowe e£_el, (91), however, found an increase in coronary venous ATP during coronary reactive dilation in guinea pig hearts. In their experiments (91), ATP was undetectable in the venous effluent before arterial occlusion,but approximately 4 nmoles/L of ATP were detected in the effluent during reactive dilation. The preparation 52 used by Stowe e£_el, was similar to Prep. I of this present study. The duration of arterial occlusion and the method for ATP analysis were also similar. It is, therefore, difficult to speculate on the reason for the discrepancy in the results. It is possible that in our experiments, the heart might release ATP during reactive dilation but a part or all of the ATP released was rapidly hydrolyzed before samples were collected. In this regard, Beer and Drummond have shown a rapid hydrolysis of ATP in Ringer's perfused rat hearts (4). Stowe e£_el, (91) also reported that ATP added to the coronary circulation of guinea pig hearts did not appear in the effluent unless ATP concentration in the inflow perfusate was greater than 40 nmoles/L. Even above this concentration, only 11.5% of ATP added to the perfusate appeared in the venous effluent. It is, therefore, possible that ATP added to the coronary circulation is either degraded or re— tained by the heart during the transit through the circulation. We therefore decided to study the metabolism of exogenous ATP, ADP and AMP during a transit through the coronary circulation. II. Metabolism of Exogenous Adenine Nucleotides in a Transit Ehrough the Coronapy Circulation of Guinea PigfiHearts We first studied if exogenous ATP added to the perfusate can sur- vive in a transit through the coronary circulation. Various amounts of ATP were added into the perfusate perfusing the heart and the coronary venous ATP concentrations were determined by the firefly biolumines- cence assay. 53 It was found that no venous ATP was detected if inflow concentra- tion of ATP was less than 0.15 HM (Figure 2) in the standard Langendorff preparation (Prep. 1), and less than 1.5 uM (Figure 3) in the modified preparation (Prep. 11). However, ATP added to the test tube containing either inflow or outflow perfusate remained stable for at least 45 minutes (A, II, METHODS). These studies thus indicate that ATP was either rapidly hydrolyzed during a transit through the coronary circula- tion, or retained by the cardiac tissue. Baer and Drummond (4) have observed a rapid hydrolysis of l4C—ATP in a passage through isolated Ringer‘s perfused rat hearts. Our results from Prep. I are similar to those of Stowe egnel. (91) who conducted similar experiments in the same guinea pig heart preparation. The threshold concentration of ATP, i.e., the inflow ATP concen- tration above which.venous ATP becomes detectable, in Prep. II (1.5 uM) was about tenfoLd greater than that of Prep. I (0.15 pH). The differ- ence appears to be due to minimization of surgical damage to the Prep. II hearts. In Prep. 1, incisions were made on the atria, right ventri- cle and mitral valve. The incisions would surely incise the small vessels in these areas. From the ruptured small arteries, ATP perfused could leak out, survive hydrolysis and drain into the venous effluent intact. For the study of ATP, ADP and AMP metabolism during a transit through the coronary circulation, Prep. I therefore appeared to be not as adequate as Preps. II and III. The latter two preparations were there- fore used in the subsequent experiments. 54 The next series of experiments concerned the fate of exogenous ATP during a transit through the coronary circulation. In one series of experiments, l4C-ATP was added into the perfusate (0.5—0.6 uM) perfusing the heart and radioactivities of 14C in the inflow and outflow perfusates were compared. As shown in Table l, 86% of 140 in the inflow perfusate were recoverable in the venous effluent. This indicates that 86% of the carbon skeletons of ATP perfused survived the transit through the coro- nary circulation. Since the inflow concentration of 14C-ATP was below the threshold concentration, no ATP was expected to survive the transit. Presence of 14C radioactivities in the venous effluent, therefore, indi- cates hydrolysis of ATP during the transit and appearance of hydrolytic products of 14C-ATP in the venous effluent. Similar finding was obtained when carrier ATP was added to the inflow perfusate at concentratiousranged from 2 to 26.5 uM. In these experiments, the adenine absorbances of inflow and outflow perfusates were compared. As shown in Table 1, 84% of the adenine absorbance present in the inflow perfusate were recoverable in the venous effluent. This indicates that about 84% of the adenine base perfused survived a transit through the coronary circulation. The percentages of 14C and adenine baseIsurvivingduring a transit through the coronary circulation are statistically the same (Table l). Perfusions of carrier ADP at concentration ranged from 3.3 to 110 DM, or carrier AMP at 4.0 to 121 pH, were also made, and recoveries of adenine base in the venous effluent were determined. During ADP persu- sion, 96 i;5% of adenine base in the arterial perfusate were recovered 55 in the venous effluent, and during AMP perfusion, the recovery was 81;: 3% (Table 1). These studies, therefore, indicate that ATP, ADP or AMP added to the perfusate was not taken up by the heart, and more than 80% of the adenine base were recoverable in the effluent. The next series of experiments was, therefore, performed to identify the chemi- cals recovered in the venous effluent by anion exchange chromatography. As shown in Figure 4, only AMP and nucleosides were identified in the venous effluent during perfusion of carrier ATP. But during perfu- sions of 14C-ATP, small fractions of three unknown chemicals were also found, in addition to AMP and nucleosides (Figure 4). Nevertheless, lliC-ATP was used, the main chemical regardless of whether carrier ATP or presence on the chromatogram is AMP which comprises at least 68% of chemicals appearing on the venous chromatogram, as shown in Table 2. Furthermore, no ATP, ADP, cAMP or IMP could be identified in the venous effluent whether carrier ATP or l4C-ATP was used in the experiments. This indicates that the major hydrolytic product of ATP during a passage through the coronary circulation is AMP. Beer and Drummond (4) have found labeled ATP, AMP, adenosine and inosine in the venous effluent following injection of 0.1 ml of 0.1 mM (4.9 ug) 14C-ATP into the coronary circulation of Ringer's perfused isolated rat hearts. AMP was found to be the major chemical in the venous effluent: of the total Chemicalsidentified, 52% was AMP, 23% was adenosine, 11% was inosine and 10% was ATP. Thus, their findings are similar to ours except that no ATP was identified in our experiments. Since they injected 14C-ATP as a bolus, it is difficult to calculate 56 the concentration of ATP in the arterial perfusate. But, if the concen— tration was high, ATP could survive the transit and appear in the venous effluent. In guinea pig hearts, we have found that if inflow concentra— tions of ATP are greater than 0.15‘uM in Prep. I, and 1.5'uM in Prep. II, ATP perfused will appear in the venous effluent (Figures 2 and 3). In this regard, we expected to identify ATP in the venous effluent when 130 UM (64 ug/ml) carrier ATP were perfused in our experiment. ATP was not identified in the venous effluent even at this high concentration. At this high concentration, ATP regularly produces arrythmia which in turn produces irregular coronary flow in the guinea pig heart. Cardiac metabolism of ATP might be affected by arrythmia and irregular coronary flow. Another possible reason why we did not detect ATP in the venous effluent at the high concentration is dilution of carrier nucleotides in the eluate during chromatographic elutions. The spectrophotometer might not be sensitive enough to measure A of the diluted nucleotide 258 in the eluate. The problem of arrythmia and insensitivities of measure- ments have been avoided by using 14C-ATP in this present study. Hoffman and Okita (45) have found ADP, AMP, adenosine, inosine and hypoxanthine in the effluent of guinea pig hearts during perfusions with recirculating Ringers containing radioactive ATP. We, as well as Baer and Drummond (4),have not found ADP in the venous effluent during perfusions of 14C-ATP. The reason why Hoffman and Okita found ADP in their study may be due to the fact that they used a recirculating perfu- sion system. Collingsworth (25) reported that ADP, AMP and nucleosides 14 were found in the breakdown products of C-ATP incubating in dog's 57 whole blood and plasma at 37°C. The result of Hoffman and Okita's experiments may be similar to that of Collingsworth. ATP can be hydrolyzed to AMP by three common pathways (25): aden l c clas AMP(phosphodiesterase) ATP i_BT'OI‘ATPase) A_ADn(ADPase) g_ (5'-nuc1eotidase)Adenosine \\xa rase) '5 I ‘ifi @denylic (adenosine Ry - l deaminase) deaminase) '- IMP (5 nucleotidase;Inosine The first pathway is hydrolysis of ATP by adenyl cyclase to cAMP, which is then hydrolyzed by phosphodiesterase to AMP. The second pathway is hydrolysis of ATP by B~aaATPase to ADP, which is then hydrolyzed by ADPase to AMP. The third pathway is direct hydrolysis of ATP to AMP by apyrase. AMP could then be hydrolyzed to IMP, adenosine, or inosine. As shown in Figure 4, no IMP was present in the venous effluent during perfusions of 14C-ATP. This suggests that adenylic deaminase, which converts AMP to IMP, is probably not accessible to extracellular AMP in guinea pig hearts. During perfusions of carrier ADP (82 and 110 uM), AMP and nucleo- sides were identified in the venous effluent and AMP was the major degradation product of ADP hydrolysis (Figure 4 and Table 2). ADP added to the perfusate seems to be hydrolyzed to AMP by ADPase during a transit through the coronary circulation. It is, therefore, possible that con— version of ATP to AMP in the coronary circulation might, in part, have taken the second pathway, i.e., hydrolysis of ATP to ADP by Bir—ATPase, then to AMP by ADPase. 58 In contrast to ATP and ADP, most of the AMP added to the perfusate remained unchanged following a transit through the coronary circulation (Figure 4, Table 2). About 20% of the venous adenine compounds were nucleosides during AMP perfusions (Figure 4, Table 2). These studies thus show that ATP and ADP are degraded to AMP while most of AMP remains unchanged during a transit through the coronary circulation. Therefore, if adenine nucleotides are released during reactive hyperemia, the chemical best identified in the venous effluent is AMP. In the subsequent studies, venous effluent was sampled before, during and after reactive dilation for the measurement of AMP concentrations. An enzymatic assay using myokinase and uniformly labeled 14C—ATP was used to determine the AMP concentration. III. Release of AMP During Coronary Reactive Dilation As shown in Table 4, venous AMP concentrations during reactive dilation were significantly greater than control values in Preps. I and II hearts, but were not in Prep. III hearts. The result from Prep. I and Prep. II hearts indicates that during dilation AMP is released from the heart. This conclusion, however, is challenged by the result of Prep. III. The magnitude of coronary flow and the reactivity to arterial occlusion of Prep. III were essentially the same as those of Preps. I and II (Table 5 and Figure 5). We have also found, as Bunger epuel, (15, 16) did in the standard Langendorff preparation (Prep. 1), that the coronary circulation of Prep. III hearts exhibits: 1) reactive 59 dilation whose magnitude and duration are proportional to the duration of arterial occlusion (Figure B-l, APPENDIX B); 2) vasodilation under a hypoxic condition (Figure B-2, APPENDIX B); and 3) vasodilation in response to perfusion of ATP over the concentration range between 0.5 and 1.1 MM (Table 2). These studies, therefore, show that functionally Prep. III is essentially the same as Preps. I and II, and indicate that the reason why venous AMP did not rise during reactive dilation in Prep. III is not due to surgical preparation or reactivity of the Prep. III. Prep. III and Prep. II hearts were prepared in the same way except that the possible damage to the pulmonic valve during the catheterization was avoided in the Prep. III hearts. In addition, all catheters and sampling test tubes used in Prep. III experiments were siliconized and all samples collected were centrifuged immediately at 22,500 x g for 20 minutes at 4°C before AMP assay. All these efforts were made in order to prevent possible contaminations of venous effluent by cell debris and cellular elements. Indeed, as shown in Table 4, the AMP concentration during the control period was zero ng/ml in Prep. III: that is, markedly and significantly lower than those in Preps. I and II. If most of the AMP present in the venous effluent of Preps. I and II was derived from contaminated cellular elements, the rise in venous AMP during reactive dilation is not due to release of AMP from the functioning myocardial cells during arterial occlusion. Rather, it seems that during arterial occlusion cell debris were accumulated ip_§igp_as a result of no flow. Upon relief of the arterial occlusion, the AMP-rich cell debris enters 60 the sampling tubes, and gives higher AMP concentration in the sample than the control sample. It must be assumed, with this hypothesis, that cell debris containing AMP are continuously released from the heart and are contaminating the venous effluent. Since venous effluent of Prep. III was centrifuged, the procedure might have caused AMP degradation or sedimentation in the effluent. This possibility was ruled out by the following experiments. In three experi- ments, known amounts of AMPwere added into the venous effluent (20 ng/ml) collected during the control period. The concentration of AMP in these samples did not change after centrifugation. The centrifugation prob- ably eliminates only cell debris in the sample. It is believed that the venous effluent obtained in the experi— ments performed on Prep. III is the true and most dependable coronary effluent among those obtained in all experiments. Since venous AMP was not significantly altered in Prep. III, it is reasonable to conclude that AMP concentration in the coronary venous effluent is not signifi— cantly altered during coronary reactive dilation in guinea pig hearts. This conclusion is in agreement with the finding in dog hearts by Chen e£_el, (18). They found that AMP concentration in the coronary sinus plasma does not significantly change during reactive hyperemia in blood perfused dog hearts. The conclusion may imply that AMP as well as ATP and ADP are not released from the heart and do not act as a vasodilator in reactive hyperemia. This implication may not be true in the rat heart because it has been demonstrated that two—thirds of the injected 5‘AMP were rapidly degraded to adenosine and inosine during a single 61 transit through the rat heart perfused by Ringer's solution (4). In guinea pig hearts, however, about two-thirds of the AMP perfused through the heart were unchanged (Tables 1 and 2). Therefore, AMP, ADP and ATP may not be released from the guinea pig heart during reactive hyperemia to play a role in the dilation. SUMMARY AND CONCLUSIONS The aim of this present study was to determine if adenine nucleo- tides play a role in reactive hyperemia in guinea pig hearts. The standard Langendorff preparation (Prep. 1) was used in the beginning of this present study but the preparation was found to be inadequate for the study because venous effluent seemed to be contaminated by cellular elements derived from the heart. The standard Langendorff preparation was therefore modified, in that the atria and ventricles were left intact and the venous effluent was collected from a catheter in the pulmonary artery (Prep II). This preparation was also found to be inadequate for the study and was slightly modified, in that the catheter in the pulmonary artery had minimum insertion to avoid damag- ing the pulmonic valve; the catheter and the sampling tubes were sili- conized and venous effluents collected were centrifuged immediately. 1. In all three preparations, venous ATP, as measured by the fire- fly bioluminescence assay, did not significantly change during reactive dilation elicited by 30-second: arterial occlusion. When ATP was per- fused into Prep. I and Prep. II, no ATP was detected in the venous effluent unless ATP concentration of inflow perfusate was raised above 0.15 MM in Prep. I and 1.5 uM in Prep. II. This indicated that no ATP would appear in the venous effluent unless ATP released during reactive dilation waslarge enough to raise local concentration above 1.5 uM in Prep. 11. 62 63 2. To investigate if the ATP lost during a transit through the coronary circulation is due to its degradation, the adenine absorbances of the inflow and outflow perfusates were measured during perfusions of ATP (2.0-26.5 uM), ADP (3.3-110 uM) or AMP (4.0-121 pH) by a spectro- photometer at a wavelength of 258 nm. The study was performed on Preps. II and III hearts, and 84 j; 2%, 96 i 5%, and 81 j; 3% of the adenine base in the inflow perfusate were recovered in the venous effluent during perfusion of ATP, ADP or AMP respectively. In addition, we found that during perfusion of 14C-ATP at low concentration, i.e., 0.5-0.6 uM, 86 j;7% of 14C radioactivities in the inflow perfusate were recoverable in the venous effluent. These studies indicated that most of the ATP perfused were degraded during a transit through the coronary circula- tion. 3. In order to identify the degradation products of ATP during a transit through the coronary circulation, ATP, ADP or AMP was perfused through the circulation and the venous effluents were analyzed by anion exchange chromatography with gradient elution. The major hydrolytic product of ATP, ADP or 14C-ATP was AMP. AMP remained largely undegraded after a transit through the coronary circulation. No ATP, ADP, IMP or cyclic AMP was found in the effluent. These studies indicated that if nucleotides are released during coronary reactive dilation, the chemical which will appear in the venous effluent is AMP. 4. AMP concentrations in the coronary effluents before, during and after coronary dilation were then measured by an enzymatic assay using myokinase and 14C-ATP. The AMP concentration significantly increased 64 during reactive dilation in Preps. I and II hearts but did not change significantly in Prep. III hearts. Also, the AMP concentration of all samples obtained from Preps. I and 11 hearts were about tenfold greater than that from Prep. III hearts. The rate of coronary flow as well as the magnitude of reactive dilation of the three heart preparations were the same. Prep. III differed from Preps. I and II in that it had least surgical damage to the cardiac tissues and the venous effluents col- lected were immediately centrifuged to avoid contamination with cell debris from the heart. The increased AMP concentration during reactive dilation and high AMP in all samples of Preps. I and II, therefore, seems UJbe due to contamination of venous effluent by cell debris. It is therefore concluded, that venous AMP is not altered during reac— tive dilation in guinea pig hearts. Adenine nucleotides, therefore, may not be involved in coronary reactive hyperemia in guinea pigs. APPENDICES APPENDIX A REAGENTS 65 REAGENTS I. Krebs-Ringer-Bicarbonate Perfusate For each experiment, 2 liters of perfusate were prepared as follows: NaCl 13.8 gm, KCl 0.70 gm, KHZPO 0.326 gm, glucose 1.982 gm, 4 sodium pyruvate 0.44 gm, NaHCO3 4.18 gm were added and mixed in 2 liters of distilled water. The solution was then bubbled with 95% oxygen and 5% carbon dioxide gas and stirred continuously. After 5 to 10 minutes bubbling, 0.554 gm of anhydrous CaCl2 was added to the solution and the whole was bubbled with the gas throughout the experiments to keep constant p02 and pH. Reagent grade NaCl, KCl and anhydrous CaCl2 were obtained from Baker Chemical Co. (Phillipsburg, N. 3.), NaHCO D-glucose, and 3’ KB PO from Mallinckrodt Chemical Works (St. Louis, Mo.), and sodium 2 4 pyruvate.was from Sigma Chemical Co. (St. Louis, Mo.). II. Standard Solution Containlpglparrier Nucleotides and Nucleosides for Chromatoggephic Separation ATP and AMP were obtained from.Mutritional Biochemical Corp. (ATP disodium salt, control No. 2166; adenylic acid, muscle, control No. 6857, Cleveland, Ohio). From Sigma Chemical Co., adenosine (Lot No. 97B-0040, anhydrous mol. wt. 267.2), DMP (5'-inosine monophosphate, sodium.salt, Lot No. 678-7400), cAMP (adenosine cyclic monophosphate, sodium salt, Lot No. 1030-7010), Ba-ADP (ADP, barium salt, equine muscle, 66 67 Lot No. 109B-7200) were obtained. All the compounds were kept in a dessicator at -10°C. A typical standard solution of 20 ml containing (micromoles) adenosine 15, AMP 18, cAMP 7, IMP 10, ADP 10, ATP 30 was prepared for a standard chromatographic separation as follows: Sixty- three mg of AMP were added to 100 m1 freshly prepared perfusate and the whole was warmed in a hot water bath for at least 10 minutes to ensure complete dissolving of the AMP crystals and then allowed to cool. To 10 ml AMP solution, 4 mg of adenosine, 3 mg of cAMP, 5 mg of IMP and 14 mg of ATP were added. Conversion of Na-ADP from Ba-ADP was made according to the procedure described by Collingsworth (25). Seven mg of Ba-ADP were mixed with 5 m1 of 0.2 M sodium sulfate (1.4 mg of NaZSO4 per 20 mi of water) with agitation and the whole was then centrifuged at 600 x g for 7 minutes7 at room temperature. The supernatant (Na-ADP, approxi— mately 1.6 micromoles) was decanted and added to the previously pre- pared 10 ml nucleoside and nucleotide solution, and the whole was brought to 20 ml by adding perfusate. Purity checks on each carrier nucleotide were performed by either the specific separation method or the rapid separation method (APPENDIX C-II). Each carrier nucleotide being checked for purity was added to 30-40 m1 of freshly prepared perfusate and then dumped onto the resin column. Carrier ATP and AMP used in this thesis were tested for purities by specific separation method, and also along with studies of the degradation of ATP, AMP in the coronary vasculature (Figure 4). 7According to Dr. B. H. Selleck, two minutes may be sufficient to separate Na-ADP from barium sulfate. Longer centrifugation of the solu- tion at room temperature might generate heat which would cause degrada— tion of Na-ADP. 68 Since Na-ADP converted from Ba-ADP is notoriously unstable even at 10°C below zero, a simultaneous purity check of Na-ADP was performed along with studies of the degradation of ADP in coronary vasculature using separate chromatographic columns. Thus, one can be certain that the experimental conditions (37°C, 90 minutes) do not cause significant breakdown of ADP. III. 14C-ATP Uniformly labeled 14C-ATP was obtained from New England Nuclear (Lot No. 893-209; 0.31 mg of ATP in 12.5 ml 50% ethanol; specific activity - 482 mCi/mM). The solution was stored in a freezer at -10°C upon arrival from the company. To ensure that each lot of 14C-ATP con- tained pure 14C-ATP, 50 microliters (1 uCi, or 1.24 mg ATP) of each lot of lac—ATP were chromatographically separated by rapid separation method (APPENDIX C-II) using carrier AMP, ADP, ATP (approximately 1-4 mg each) as indicators for checking the separation method and the chemical nature of the 14C radioactivities. IV. Ammonium Formate The 1.75 M ammonium formate stock solution was made by adding 18 liters of distilled water to 1985 gm of reagent grade ammonium formate CMatheson, Coleman and Bell Co.), and the pH of the solution was adjusted to 5.0 with concentrated formic acid (88%, specific gravity 1.20, Mallinckrodt Co.). This solution had a density of 1.23 gm/ml at room 69 temperature. From this stock solution, 1.0 M and 0.4 M ammonium formate solutions were made by dilution with distilled water. The 0.4 amonium formate had a pH of 4.6 at room temperature. V . Formic Acid The 4.0 N formic acid stock solution was made by diluting 262 ml of reagent grade formic acid (Mallinckrodt Co.) to 1.5 liters with distilled water. It had a pH of 1.2 and a density of 1.04 gin/ml at room temperature. From this stock solution, 0.5 N and 2.4 N formic acid solutions were made. VI. Myokinase-TEA—Mg: Buffer Solution Myokinase was obtained from Calbiochemical (Lot No. 320038, rabbit muscle, 1980 I.U./ml, protein 1.9 mg/ml, San Diego, Calif.), and was stored at 4°C. One liter of 0.1 M TEA—MgH buffer solution was made by adding 18.6 gm of TEA (triethanolamine) and 0.5 gm of MgC12-6 H20 to. water and adjusted to pH 8.5 by 1.0 N NaOH solution. The TEA—Mg“- buffer is stored at 4°C. Myokinase—TEA—MgH buffer was prepared shortly before the quantitative AMP assay by mixing 0.2 ml of myokinase to 5.0 ml of TEA-Mg“- buffer with agitation to make a solution contain- ing 73 I.U. myokinase per ml buffer. 70 VII. Anion-exchange Resin New anion-exchange resin (AG 1 x 8, 200—400 mesh, formate form) was obtained from Calbiochemical (Elk Grove, 111.). The new dry resin was washed at least 20 times with an equa1.volume of distilled water (one part of resin with one part of water for each washing), and then was stored at 4°C in hydrated form (add one part of distilled water to one part of wet resin by volume). The used resin was washed 3 times with 10% HCl, 3 times with distilled water, and 4 times with 1.75 M ammonium formate at room temperature at 24-hour intervals between each washing. The resin was then finally washed with distilled water till the supernatant had a steady pH between 4 and S. The resin was also kept in hydrated form at 4°C. APPENDIX B CORONARY REACTIVITIES T0 ARTERIAL OCCLUSIONS AND HYPOXIC PERFUSIONS OF PREPARATION III HEARTS 71 72 Figpre B—l. Coronary vascular reactivities to different durations of arterial occlusions in Prep. III hearts. Coronary reactive dilation was elicited by the occlusion of inflow for l, 2, 5, 10, 20, 30, 40, or 60 seconds. The magnitudes (peak flow) and durations of the reactive dilation were measured. The duration is the time taken from the onset of dilation to the point where coronary flow returns to the preocclusion level. 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ON. 0m cm on O 233.8 2.8%: 0. ON on (“lull II“) MO'IJNI 76 Table B-1. ATP Concentrations in the Effluents from Pulmonary Artery (PA) and from the Heart Chamber Reservoir (HC) of Prep. III Hearts Experiment PA HC No. (ng/ml) (mg/ml) 58 < 0.1 4 59 < 0.1 2 62 < 0.1 l 63 < 0.1 5 Mean i S.E. < 0.1 3 j; l All samples were not centrifuged. APPENDIX C CHEMICAL ANALYSES 77 APPENDIX C CHEMICAL ANALYSES I. Fireflnyioluminescence Assay of ATP Because of its simplicity and high sensitivity, the bioluminescent reaction has been widely used for determinations of ATP concentration (46, 59, 60, 86-88, 91, 92). The reaction can be summarized as the following equation: ATP + 02 + Luciferin Lufiferase MS > Light + AMP + 2Pi + Oxyluciferin. When 02 and luciferin are present in excess, the light emission is directly proportional to the concentration of ATP in the reaction mix- ture (59, 60, 95). The reaction is specific for ATP when purified luciferase is em? played (60). However, when the commercial firefly lantern extract is used, caution should be taken when interpreting the results. Holmsen e£_el, (46) and Silinsky (86) observed a nonspecific light emission in- duced by ADP and trinucleotides other than ATP by using the commercial firefly lantern extract (FLE-50, Sigma). They reported (46, 86) that the nonspecific phenomenon is due to the presence of an ATP—generating system and enzymes in the crude extract. Fortunately, the light pattern 78 79 induced by ATP could be differentiated from that induced by ADP and other trinucleotides (86). The sensitivity of this assay for ATP was 1.8 x 10.13 moles ATP/ml (i.e., 0.09 ng/ml) as reported by Silinsky (86). Similar sensitivity was also observed in our setups as described below. For each assay, 0.5 :_0.05 ml of buffered firefly extract (from 50 mg dried lanterns, FLE-50, Lot No. 80205, Sigma Chemical Co.. St. Louis, Mo.; reconstituted with 5 ml of freshly prepared perfusate) was pipetted by an automatic syringe with an 18 gauge needle into a cuvette (vol. 2 ml) which was placed directly above the photomultiplier (PM) tube. The PM tube was activated by a 1500 d.c. high voltage source (model 415B, Fluke 59410, John Fluke Mfg. Co., Inc. Seattle, Washington) and was housed in a light-tight black Perspex reaction chamber enclosed in a box. In a darkened room, a sample (0.25 ml) of either perfusate containing 4-10 ng/ml ATP (standard ATP) or venous effluent was injected rapidly (within one second) into the cuvette. The light produced would activate the PM tube and the voltage generated was recorded by a Grass polygraph recorder (model 5D, Grass Instrument Co. Quincy, Mass.), or a Honeywell recorder (model No. Y 153 x 18 (VA)- X-118, Minneapolis-Honeywell Reg. Co., Brown Inst. Division. Philadel- phia, Pa.). The measurement of venous ATP was determined from pen deflection in centimeters (cm) as compared to that produced by the standard ATP solution. The standard ATP solution was tested before and after assays of ATP in venous effluents. These tests were performed to check the operation of the ATP assay and to standardize the equipment as well as the pen deflection. 80 To determine the concentration of ATP in any sample, the ratio of standard ATP in ng/ml to pen deflection (cm) was calculated by averaging the values (cm) of standard ATP measurements made during that particular experiment, and this ratio was multiplied by the pen deflection (cm) produced by venous effluent samples. II. Anion Exchange Chromateggephic Separations of Nucleosides and Nucleotides Gradient elution ion exchange chromatographic procedures have been described by several investigators (l4, 17, 21-23, 25, 32, 47). Three terms used in the following descriptions of anion chromatographic separation that require clarification are: 1212222} which refers to the solution eluting the resin column, lelpepef which refers to the solu- tion after leaving the bottom of the resin column (43), and 'chromatpgram' which is a plot of A258 or cpm/m1 of eluates in the col— lection tubes (9.8 mlltube) vs. collection tube number. The gradient elution anion exchange chromatographic apparatus (A, Figure C—l) used for separation and identification of nucleosides and nucleotides in this study was identical to the one used by Collingsworth (25); however, the gradient elution procedures were con- siderably different. Furthermore, the recovery of nucleosides and nucleotides from the resin column in our experiments was consistently between 80-100%. The detail of our procedures used in this study is described below. 81 Figure C-l. The apparatus for anion exchange chromatographic A. separations. Gradient Elution Pressure of elution: 230 cm H20 The solution in the eluent mixing chamber is never emptied throughout the separation. A11 tubings connecting the reservoir, mixing chamber and the resin column were air-tight to prevent changes in elution pressure. The eluate was collected by volumetric fraction collector (approximately 9.8 ml/tube). Direct Elution This diagram shows the apparatus used for chromatographic separation of incubated samples (8 samples) in the quantita- tive assay of AMP. D: 0.9 meter when eluted with 0.4 M ammonium formate or 0.6 meter when eluted with 2.4 N formic acid. a: AG 1 x 8, 200-400 mesh anion exchange resin, the column - 1.4 cm x 1.0 cm . b: three-way stopcock with 21 gauge needle. Eluate from each column was collected by a graduated cylinder. 82 A _ THE RESERVOIR 500 ~ ml 3" RUBBER J OI TUBE . 3 MIXING r: CHAMBER n ELUENT g; LE’"L—IIIABNETIc: L STIRRER {if—As lx8 RESIN ELUATE B - [2 “—ELUENT I F L, RESERVOIR D E- l >b O ELUATE TO VOL. CYLINDERS Figure C-l 83 l) Repid Separation of ATP, ADP and AMP The separation of ATP, ADP and AMP by anion exchange resin column (6.2 cm x 1.0 cm2 anion-exchanger, AG 1 x 8, 200-400 mesh, Calbiochemical, Elk Grove, Ill.) was done according to the procedure described by Collingsworth (25). 2) Specific Separation of ATPlyADP, IMP, cAMP, AMP and Nucleosides The rapid separation of ATP, ADP and AMP described above does not separate IMP, or cAMP from AMP. Thus a more specific separation was developed to separate IMP, cAMP and AMP from one another (25). The column, containing 14 cm x 3.14 cm2 anion-exchanger (AG 1 x 8, 200-400 mesh) was loaded with standard nucleotide-nucleoside samples (3-14 mg each in 20—50 ml perfusate, APPENDIX A-II) or venous outflow samples from the heart (20-50 ml). The sample (pH - 7.4) was allowed to drain by gravity onto the resin until the level of the sample solu- tion was 1—2 cm above the resin bed. The column was steadily eluted with eluent from the mixing chamber (containing 500 ml of distilled water originally) fed 0.5 N formic acid (pH - 2.05) from the reservoir (A, Figure C-l). After 50 fractions (9.8 ml per fraction) were collected, the reservoir was filled with 4.0 N formic acid (pH - 1.22) and collec- tion continued for another 100 fractions. The content of the reservoir was then changed to 1.75 M ammonium formate (pH = 5.0) which steadily entered the mixing chamber throughout the remainder of the separation (50 fractions). The solution in the mixing chamber was never emptied throughout the separation. The order in which nucleotide peaks appeared on the chromatogram was AMP, cAMP, IMP, ADP, ATP. Nucleosides were eluted before AMP (A, Figure C—2). 84 Figure C—2. Chromatograms of standard samples and inflow perfusate analyzed by specific separation method. Resin column: 14 cm x 3.14 cm2 (AG 1 x 8, 200-400 mesh) F.A. - formic acid A.M. - ammonium formate A 58 of the eluate are expressed as dots. Crosses indicate tfie A258 had been diluted by a. A. Chromatogram of a standard sample containing the following chemicals in uM: ATP - 8.6; ADP - 10.3; cAMP - 11.4; AMP . 12; and adenosine - l6. ADP used was from a new stock. B. Chromatogram of the inflow perfusate in which no adenine nucleoside or nucleotides was added. 85 |«O.5N >I<4.0N F. A. >|I A F.A. A.M 2588 '«5 I2- 3 a ° 1 8 o. 2 (L 11. (Du o 2 ee 4 2 o I- . '2', < o - g < u 08“ g .0 .0 .... I .3 e . :‘. : .0. e . 4‘ ' ,° °’ ,° '4' , . I I :5 : k“: ..: . 0 ”‘ “E . . . I I k0.5N 4‘4 ON F A 'I‘IJSM" A - - . 25° F. A. AM. Oi ~000~eo¢99”...”. .” ”.."”Oeseeseoees. . I n A 2' 0 4O 80 IZO I60 200 COLLECTION TUBE NO. (9.8ml/tubel Figure C-2 '_7 86 Occasionally an extra peak appeared between ADP and ATP. It was thought that this extra peak was an impurity in ADP, since the absorbancy of this peak increased if ADP concentration was raised; and it disap- peared when ADP was not added to the sample, or when ADP from a freshly Opened stock was used. A chromatogram of perfusate without adenyl compounds is; shown in B, Figure C-2. 3) ModifiedlSpecific Separation of Inosine from Nucleosides When we initially eluted the column with only distilled water in the reservoir, an extra peak was present between nucleoside and AMP peaks on the chromatogram of hydrolytic 14C-ATP studies (Figure 4, right column). The extra peak was later identified to be inosine because it had a maximum absorbance at 246 nm, and the 14C radioactivi- ties of this peak emerged with indicator inosine added. The modified specific separation of inosine was as follows: The resin column (14 cm x 3.14 cmz) was loaded with standard solution (30-50 ml) containing adenyl compounds and inosine, and the column was flushed with a syringe until the meniscus of the standard solution was 1 cm above the resin bed. Then the resin was eluted with distilled water placed in the reservoir until 15 fractions (9.8 ml each fraction) were collected, and then the content of the reservoir was changed to 0.5 N formic acid. The content was changed again at the 80th fraction to 4.0 N formic acid; and changed at the 190th fraction to 1.75 M ammonium formate which fed into the mixing chamber until the 240th fraction was collected. 87 III. anntitative Assay of AMP by Myokinase and 14c: (U) -ATP The chemical reaction utilized in this method is the phosphoryla— tion of AMP to ADP by ATP and myokinase (rabbit muscle adenylate kinase, Enzyme Commission designation ATP:AMP phosphotransferase, E. C. NO. 2.7.4.3): 14C(U)-ATP + AMP < C-ADP + ADP (Myokinase and Mg++) When l4C(U)-ATP is present in constant amount, the amount of 14C-ADP formed at equilibrium is proportional to the amount of AMP added to the reaction mixture. Thus after completion of the reaction and the chroma- tographic separation, quantification of 14C-ADP allows one to estimate the quantity of AMP originally added to the reaction. This reaction is very specific for AMP as the phosphate acceptor (70, 71, 85). While ATP with IMP gives no reaction (71), ITP (inosine triphosphate) may serve as a substrate with low reactivity in this reaction in the pres- ence of AMP (13). In this study, it was assumed that no trinucleotide was present in venous effluent samples. Frequent analysis for carrier ATP (the FBA, APPENDIX C-I) in the venous effluent samples proved that endogenously released ATP was never great enough to significantly inter- fere with the AMP analysis. We also have found that adenosine did not substitute for AMP as the acceptor of phosphate. Collingsworth e£_el, (26) have used this assay method to measure venous AMP in dogs. The assay method for standard and unknown AMP H samples is summarized as follows: k ml of myokinase-TEAéMg buffer 88 (containing myokinase 73 I.U./m1, see APPENDIX A-VI, page 69) was added to each 3 m1 of sample solution. Then one uCi of 14C-ATP (0.05 ml con- taining 2.53 x 10.9 moles uniformly labeled ATP, New England Nuclear, Lot NO. 893-209) was added to each sample. The solution (pH = 8.0) was incubated for 40 (Preps. I and II) or 30 (Prep. III) minutes at 37°C with agitation. After incubation, the nucleotides in the 3.5 ml solu- tion were separated by anion exchange chromatography with direct elution (B, Figure C-l). Eight separate resin columns each containing 1.4 cm x 1.0 cm2 resin (AG 1 x 8, 200-400 mesh) were used for eight different samples. Each resin column, after loading with one incubated 3.5 m1 sample, was eluted with either 150 ml of 0.4 N ammonium formate solution (pH - 4.6, Preps.I and II), or with 10 ml distilled water by gravity and then 50 ml Of 2.4 N formic acid (Prep. III). These two eluents at these particular volumes remove nucleosides, AMP anBIADP from the column, while unreacted 14C-ATP is left bound on the resin. One hundred and fifty m1 (Preps. I and II), or 60 ml (Prep. III) of the eluate from each column was collected in a graduated cylinder. One ml of the eluate from each collection was plated on a planchet for radioactivity determin- ations. The chromatographic procedure in which 2.4 N formic acid was used as eluent is better than the other procedure because the eluted radioactivity is more concentrated, thereby giving greater cpm, and producing better counting statistics. Furthermore, less ATP is eluted with the small volume of formic acid. After the plated eluate was dried, its total radioactivities were measured and enough counting time was allowed so that the total counts 89 above background were at least 5,000 for each planchet. The AMP concen- tration of the sample was measured from the standard curve which was experimentally determined for every new stock of l[‘C-ATP or myokinase received. Frequently AMP standards were used to check the analysis at the time during which experimental samples were assayed. This assay allows measurements Of 10 or more ng/ml AMP in the samples. B IBLIOGRAPHY (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) BIBLIOGRAPHY Afonso, S., T. J. Ansfield, T. B. Berndt, and G. G. Rowe: Coronary vasodilator response to hypoxia before and after aminophylline. J. Physiol. (London), 221:589-599, 1972. Afonso, S., and G. S. O'Brien: Mechanism of enhancement of adeno- sine action by dipyridamole and lidoflazine, in dogs. Arch. int. Pharmacodyn., 194:181-196, 1971. Bacaner, M. B., F. Lioy, and M. B. 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