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I.' ‘1"... ~ 5'... .0 nr-"Od ' . ..‘."..’-"-<>OO--D“ - .. .. . . _ _ . , ... . .. . r» «.00.!04" '...‘,owo'lt. lo;- - . .. . . ~.- . - w- - - . or—I'--"' - ' - K _ . ‘ v __ .',' -.--.o-'. v' , . _ a ..0' - o--- " ' ' ‘ . . . , ,......’p.oo’p-.°.'-_'.","’.'.'. 1., o. .. ‘ .. — . . - '. ~.I,‘a" '_I'.""';"’.I.~.., flCP'f ‘ ' ' .' --. .rnrvl-' . I' ‘ . _.",J".f00.".r '.,",-Of'P. T .- ._ -_..i 'r MiChigafl S 3‘th 39.....- “a“ ' .- 5" . ”.11, W alumna av “3 HUM} & SUNS' BUDK BINDERY INC. [ gmnmv amnzns . ‘-' ””12: : IIEflCAfl n ABSTRACT METABOLISM OF 14C-ATP ADDED TO DOG WHOLE BLOOD, PLASMA AND DURING PASSAGE THROUGH THE LUNGS BY Ann Haviland Collingsworth Forrester and Lind (J. Physiol. 204: 347, 1969) and Chen et al. (Fed. Proc. 31: 379A, 1972) found ATP released into the venous effluent from exercising skeletal muscle and pro- posed that ATP was a chemical mediator for active hyperemia. Berne (M. J. Physiol. 204: 317, 1963) reported that adenosine may be the mediator of coronary vasodilation induced by hypoxia. The metabolism of ATP in blood, plasma and upon passage through the vasculature of the lungs was therefore investigated to determine whether or not nanogram levels of ATP or its‘degradation products could recirculate. l4C-ATP (575 ng/ml) was incubated in dog plasma or whole blood in vitro for accurately measured durations of time. 14C-labeled nucleotide degradation products were separated and quantified by gradient elution ion exchange chromatography. The approxi- mate halftimes for l4C--ATP breakdown were 3 minutes in plasma and 1 1/2 minutes in whole blood. Breakdown products of JAG-ATP in both whole blood and plasma were identified as ADP, AMP and nucleosides. After approximately 7 minutes incubation c. Ann Haviland Collingsworth in whole blood, extracellular l4C—ATP degradation products were taken up by the formed elements of the blood and re- synthesized to intracellular l4C-ATP. Heparin, barium and citrate had virtually no effect on l4C-ATP breakdown in whole blood. Cooling the blood to 3°C significantly reduced l4C-ATP breakdown. Approximately 17% of the added l4C-ATP was broken down in 1 1/2 minutes at this low temperature as compared to 55% for controls incubated at 37°C. 14C-ATP breakdown was inhibited in samples gassed with CO2 as com— pared to higher pH control samples from the same dog. A mean decrease of 0.27 pH units inhibited l4C-ATP breakdown 11% i 2% (mean : SE, N=16). Whole blood l4'C-ATP was most stable in the pH range of 7.20 to 7.42. Nucleosides and ADP were not affected in a consistent manner by whole blood pH alteration. l4C—AMP was formed at decreasing rates as whole blood pH was lowered from 7.6 to 7.2. In vitro studies of 14C-adenosine metabolism in whole blood revealed that l4C-adenosine was rapidly taken up by the cells and synthesized into 14C-AMP, 14C-ADP and l4C-ATP. Large quantities of 14C-AMP were found in the plasma as well. Plasma l4C-ATP levels were very low, if present at all. No l4C-IMP was detected in plasma or cells. In vivo studies of l4C-ATP degradation during a single passage through the vasculature of the lungs revealed that l4C-ATP injected into the right atrium was almost completely broken down or taken up by the lungs. Of the 14C activity injected l H. —-——-..___— Ann Haviland Collingsworth 83% i 3% (mean : SE, N=4) was taken up by the lungs. Of the 4 . . . C activ1ty recovered in the blood after the passage of the l4C-ATP injection through the lungs only 17% i 11% (mean i SE, N=4) was still l4C-ATP. Other labeled 14C-ATP degradation products in the blood leaving the lungs were nucleosides, 51% i 14%; AMP (and/or IMP), 24% i 6%; and ADP, 7% i_3% (mean i SE, N=4). The results indicate that ATP released from muscle tissue into the plasma could recirculate if its break— down and uptake by the lungs could be inhibited. [1 METABOLISM OF l4C-ATP ADDED TO DOG WHOLE BLOOD, PLASMA AND DURING PASSAGE THROUGH THE LUNGS BY Ann Haviland Collingsworth A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Physiology 1973 E9 ACKNOWLEDGMENTS The author wishes to express her sincere gratitude and appreciation to Dr. Burnell Selleck for his most valuable advice and guidance throughout the course of this study. The author would also like to thank Dr. C. C. Chou and Dr. J. B. Scott for their suggestions and encouragement. Finally, to her husband, Carl, the author expresses special appreciation and thanks for his assistance in the preparation of this thesis. ii TABLE OF CONTENTS LIST OF TABLES . O O O O O O O O O O O O C O 0 LIST OF FIGURES. O O O 0 O O O O O O O O O O 0 LIST OF ABBREVIATIONS. . . . . . . . . . . . . INTRODUCTION 0 O O O O I O O O O O O O O O O 0 LITERATURE REVIEW. 0 O O O O O O I O O O O O O l. Pharmacological Effects of Adenine Nucleotides and Adenosine. . . . . . . . . . . . . . 2. Adenine Nucleotide Metabolism, General . 3. Adenine Nucleotide Metabolism in Whole Blood and Plasma . . . . . . . . . . . . 4. Adenine Nucleotide Metabolism in Skeletal MuSCle O O O O I O O ‘ O O O O I O O I O 5. Adenine Nucleotide Metabolism in the Heart . 6. Metabolism of Extracellular Adenine Nucleo- tides by the Lungs . . . . . . . . . . . 7. The Passage of Adenosine and Adenine Nucleo- tides Through the Cell Membrane. . . . . . . METHODS O I O O O C O O O O O O O O O O O O O C l. Breakdown of izc-ATP in Plasma . . . . . 2. Breakdown of C-ATP in Whole Blood. . . 3. Separation of Adenine Nucleotides by Ion Exchange Chromatography. . . . . . . . . C-ATP Purity Check . . . . . . . . . . 14C-Adenosine Purity Check . . . . O‘U'Inb coo BlOOd O O O I O O 7. IMP Separation . . . . . . . . . . . . . 8. EDTA Studies . . . . . . . . . . . . . . 9. In Vivo Studies. . . . . . . . . . . . (a) Plasma l4C-ATP uptake an d br rea akdow the lungs . . . . . . . . . . . . iii Effect of pH on l4CEATP Breakdown in Whole Page vi vii 37 56 61 75 77 84 84 85 85 92 92 95 96 97 98 99 TABLE OF CONTENTS--Continued 10. (b) The effect of ATP injection and con- tinuous infusion on systemic arterial blood pressure. . . . . . . . . . . . Reagents . . . . . . . . . . . . . . . . . RESULTS 0 O Q C O C D O O O O O I O O O O O O O O O C 1. 2. 3. Breakdown of i4C-ATP in Plasma . . . . . . . . Breakdown of l C-ATP in Whole Blood. . . . . Breakdown of C-ATP in Blood with pH Con- trolled. 1 . . . . . . . . Studies of 4C-ATP Breakdown in Paired Whole Blood Samples. . . . . . . . . . . . . . . . . Effect of Barium, Citrate and Temperature on 14C-ATP Breakdown in Whole Blood . . . . . . . Effect of Hemolysis on l4C-ATP Breakdown . . . Effect of pH on l4C-ATP Breakdown. . . . . . Effect of EDTA on the Formed Elements of Blood In Vivo Studies on l4C-ATP Breakdown in the Pulmonary Circuit. . . . . . . . . . . . . . . In Vivo Studies on the Effect of ATP on Systemic Arterial Pressure . . . . . . . . . . DISCUSSION 0 O O O O O O O O O O O O O O O O O O C 0 SUMMARY AND CONCLUSIONS. . . .i. . . . . . . . . . . BIBLIOGRAPHY O O O O O O O O O O O O O O O O O O O 0 iv Page 100 101 105 105 110 121 123 123 128 128 137 140 143 147 162 164 LIST OF TABLES TABLE The Pharmacological Effects of the Adenine compounds 0 O O O O O O O O O O O O O O O O O O 14C-ATP Breakdown at Selected Incubation Times in Whole Blood at 37°C . . . . . . . . . . . . Breakdown of l4C-ATP in Whole Blood at 37°C in l l/2 Minutes with Uncontrolled pH . . . . . . 14C-ATP Breakdown in Whole Blood at 37°C in 1 1/2 Minutes at pH Approximating 7.40 . . . . 14C-ATP Breakdown in Paired Whole Blood Samples from Individual Dogs . . . . . . . . . Effect of Barium, Citrate and Temperature on 14c-ATP Breakdown in Whole Blood in 1 1/2 Minutes . O O O O O O O O C O O O O C O O O O 0 Effect of Hemolysis on 14C-ATP Breakdown in Gassed Whole Blood in 1 1/2 Minutes. . . . . . 14 Effect of pH on C-ATP Breakdown in Whole Blood in 1 1/2 Minutes . . . . . . . . . . . . The Effect of EDTA on Plasma ATP Levels. . . . Page 114 116 122 124 126 129 131 138 LIST OF FIGURES FIGURE 10. ll. 12. 13. Metabolism of the adenine compounds. . . . . . . Gradient elution ion exchange column chroma- tography apparatus . . . . . . . . . . . . . . . Gradient elution ion exchange chromatography pattern for ATP, ADP, AMP, and adenosine . . . . 14C-ATP and l4C-adenosine purity checks. . . . . l4C-ATP breakdown in isolated plasma incubated in Vitro . I O C C O O O O I O O O O O O O O O O Breakdown of 14C-ATP in isolated plasma incu- bated in Vitro O O O 0 O O O C O O O O O O O O O 14C-ATP breakdown in whole blood incubated in Vitro. O O O O O O O O O O O O O I O O O O O O 0 Distribution of 14C activity in whole blood after 20 minutes of in vitro incubation with l 4C-ATP O O O O O O O O O O O O O O O O O O O O O Breakdown of l4C-ATP in whole blood in 1 1/2 minutes at 37°C in vitro . . . . . . . . . . . . Effect Of pH on l4C-ATP breakdown in whole blood incubated in vitro at 37°C for 1 1/2 minutes . . Effect of pH on l4C-ADP, l4C-AMP, and 14C-n‘ucleo- side formation in whole blood incubated in'vitro with 14c-ATP at 37°C for 1 1/2 minutes . . . . . 14C-ATP breakdown during one passage through the vasculature of the lungs . . . . . . . . . . . . Effect of intravenous and intra-arterial ATP infusion on systemic arterial blood pressure . . vi Page 39 89 91 94 107 109 112 118 120 134 136 142 145 LI S T OF ABBREVIATIONS ATP = adenosine triphOSphate ADP = adenosine diphosphate AMP = adenosine monophosphate ITP = inosine triphosphate IDP = inosine diphosphate IMP = inosine monophosphate microcurie uCi c/n1= counts per minute C.L.A.R. = Center for Laboratory Animal Resources ng nanogram mg = milligram kg = kilogram ug = microgram x G = times gravity A = .001 milliliter SE = standard error (The standard errors in this thesis were calculated on per cent values. However, on comparison with standard errors obtained by using log values there was only a small difference observed between the two.) 2,3-DPGA = 2,3-diphosphog1yceric acid GFR = glomerular filtration rate 9 = gram AV = auricular-ventricular y = microgram vii INTRODUCTION The studies presented in this thesis were done to in— vestigate the metabolism of ATP in plasma, whole blood and during passage through the vasculature of the lungs. A more definitive understanding of adenine nucleotide metabolism in these tissues would greatly aid in determining the importance of ATP in the regulation of blood flow by allowing us to more accurately predict whether or not ATP, ADP, AMP and adenosine exist in arterial plasma. Also included in this thesis is a discussion of the possibility that extracellular ATP and other adenine compounds participate as mediators of physiological control. The evidence for the existence of adenine compounds in the extracellular fluid will be discussed, and the known pharmacological actions of these compounds will be reviewed. The most significant and most publicized pharmacological actions of the adenine compounds are related to the cardio- vascular system and the local control of organ blood flow. Conheim (1872) observed an increased blood flow in the frog's tongue following a short arrest of the circulation. The increased flow occurred in the absence of central influ- ence. Gaskell (1877) noted changes in blood flow during and after skeletal muscle contraction. It was therefore suggested that a locally produced metabolite was responsible for blood flow regulation in skeletal muscle (Gaskell, 1880, and Roy and Brown, 1879). A century later the cause of the active hyperemia in heart and skeletal muscle is still unknown. However, hypoxia, hypercapnia, hydrogen ion, potassium ion, Krebs cycle intermediates, osmolarity, prostaglandins, adenine and uridine compounds, and combinations of substances have all been suggested as participants in the increased blood flow. Adenine compounds (ATP, ADP, AMP and adenosine) have long been noted for having potent vasodilator prOperties as well as numerous other biological activities. Recent studies have provided evidence that certain adenine compounds may partici- pate in the physiological regulation of blood flow in both heart and skeletal muscle. Jacob and Berne (1961) and Rubio, Berne and Katori (1969) found evidence that adenosine is the mediator of active hyperemia in the heart. Forrester and Lind (1969) found ATP released from the exercising human fore- arm. Chen et al. (1972) found ATP released during active hyperemia in skeletal muscle in the dog. Dobson et a1. (1971), however, found adenosine and no ATP released from exercising skeletal muscle. Chen et al. (1972) and Forrester and Lind (1969) also found evidence suggesting that ATP is a normal constituent of human and dog arterial plasma. Forrester (1972a), however, attributed his finding of arterial plasma ATP to platelet damage and/or EDTA effects on the red cell. It is therefore still undetermined whether significant quantities of ATP are normally present in arterial plasma. Thus, the possibility of ATP (or other adenine compounds) in the arterial plasma entering the organs of the body and participating in the control of organ circulation and other physiological mechan- isms must be considered. Adenine compounds, if present in plasma, could produce significant vasodilation providing the organs of the body do not rapidly inactivate them. For example, the ATP found in the venous effluent of the exercising forearm could conceiv- ably recirculate, resulting in vasodilation in many areas of the body. However, evidence indicating that ATP may be metabolized far more rapidly in blood passing through an organ than in blood alone has been published. Folkow (1949) and Gordon (1961) found indirect evidence suggesting that ATP could not survive passage through the lungs. Pfleger (1969) noted that 57 per cent of perfused adenosine was taken up by the lungs in 30 seconds. Liu and Feinberg (1971) found that 20 per cent of low levels of adenosine (0.3 uM) supplied to the heart in a perfusate was taken up by the myocardial cells. In the body, therefore, organ uptake of adenine com- pounds in the plasma could be considered quantitatively more important in their removal from the circulation than their breakdown in the blood itself. LITERATURE REVIEW 1. Pharmacological Effects of Adenine Nucleotides and Adenosine The role of ATP as the driving force of many biochemical processes has been recognized and investigated since its proposal by Lipmann (1941). Also of historical interest was the recognition prior to 1930 of the importance of the adenine compounds in the glycolytic process and the relationship be- tween glycolysis and muscle contraction (Meyerhoff, 1920); see Bodansky (1927) for a brief summary of Meyerhoff's studies. Concomitant with this development of our understanding of the biochemical importance of the adenine compounds, it is noteworthy that as early as 1929 the adenine compounds were recognized to have numerous pharmacological effects on the mammalian body by Drury and Szent-Gyorgyi (1929) working at Cambridge, England. Drury and Szent-Gyorgyi isolated a sub- stance from acid extracts of heart muscle and other tissues, which produced heart block and bradycardia upon intravenous injection. This biologically active substance was identified by these investigators to be the nucleotide adenylic acid (AMP). Other pharmacological effects of intravenous AMP in- jection which were noted by Drury and Szent-Gyorgyi included: a decrease in arterial blood pressure due both to arteriolar dilation and bradycardia; arrest of intestinal peristalsis; coronary vasodilation; less forceful auricular contraction; arrest of experimentally produced auricular fibrillation; shortened auricular absolute refractory period; and decreased renal blood flow and urine formation. Adenosine, ADP, and ATP were found by these investigators to have pharmacological properties similar to those of adenylic acid; however, the removal of the ribose of adenosine to form adenine, the re- moval of the amine to form inosine, and the transfer of the amine to form guanosine all removed the biological activities of adenosine described above. Many of the initial findings reported by Drury and Szent- Gyorgyi were repeated and extended by Bennet and Drury (1931), who made the following additional observations with AMP and adenosine obtained from muscle and yeast extracts: "heart block" in the guinea-pig; dilation of .03-.05 mm diameter blood vessels in the intact and isolated perfused rabbit ear; vasoconstriction in the isolated, perfused rabbit lung; both vasoconstriction and dilation in the isolated, perfused rabbit kidney*; bronchiolar dilation; relaxation of the guinea-pig gall bladder; contraction of the isolated virgin guinea-pig uterus; decreased guinea-pig rectal temperature and localized accumulation of leucocytes in the subcutaneous A *This variability may be explained by ATP and ADP contami- nates in their relatively crude preparations of adenosine and AMP. area where nucleoside or nucleotide were applied. The rela- tion of this last observation to the inflammatory process was discussed by these investigators. After the above publications described the "heart block" and other biological effects caused by adenosine and adenine nucleotides, the obvious importance of these phenomena led to the further investigation of the pharmacology of the adenine compounds by several laboratories during the early 1930's. These studies were extensively reviewed by Drury in 1936 and Table 1 summarizes the observations reviewed by this author as well as some of the other recent findings related to the pharmacology of the adenine compounds. Some of the highlights of these studies listed in Table l which relate to this thesis will now be discussed. J. H. Gillespie published an interesting article in 1933 dealing with the "biological significance of the link- ages in adenosine triphosphoric acid." In addition to pre- senting a worthwhile review of the pharmacology of the adenine compounds known at that time (including references to several papers unavailable to us), a number of unique experimental observations were recorded. In the cat Gillespie noted a biphasic response of arterial pressure to ATP intravenous injection, i.e., first an increase followed immediately by a decrease. When a second bolus of ATP was given intravenously, only a rise in blood pressure was observed. ATP, unlike Table l. The Pharmacological Effects of the Adenine Compounds increased blood flow (forearm) decreased small vessel resistance (forelimb) generalized muscular contraction vasodilation (hindlimb) increased capillary filtration coefficient dilated capacitance vessels vasoconstriction vasodilation (transient) vasoconstriction (lasting) vasoconstriction decreased pulmonary artery pressure increased pulmonary artery pressure incmeased splanchnic nerve activity increased chemoreceptor activity initiates scratch & other reflexes increased chemoreceptor activity increased baroceptor activity Skeletal Muscle ATP AMP , ADP ATP ATP AMP, ATP ATP ATP AMP, AMP, ADP, Lung adenosine ATP (low dose) AMP, ADP, ATP (High dose) ATP ADP ADP ATP ATP ATP ATP ATP Nerves Duff et a1. Frohlich Emmelin & Feldberg Kontos et al. Kjellmer & Odelram Kjellmer & Odelram Bennet & Drury Gaddum & Holtz Gaddum & Holtz Emmelin & Feldberg Brashear & Ross Brashear et al. Dontas Dontas Emmelin & Feldberg Jarisch et al. Jarisch et a1. (1954) (1963) (1948) (1968) (1965) (1965) (1931) (1933) (1933) (1948) (1969) (1970) (1955) (1955) (1948) (1952) (1952) continued Table l--Continued heart block bradycardia coronary vasodilation arrest of auricular fibrillation shortened auricular absolute refractory period coronary vasodilation bradycardia‘ (not via vagus) impaired AV conduction bradycardia increased 0 consumption decreased amplitude of contraction increased beat strength (frog) occasional increased beat strength (rabbit) bradycardia (frog, perfused) coronary dilation decreased renal blood flow decreased urine flow AMP adenosine AMP adenosine AMP adenosine AMP adenosine AMP adenosine ATP ATP ATP adenosine ADP adenosine adenosine AMP ATP AMP ATP AMP ATP, AMP adenosine adenosine, AMP adenine Drury & Szent-Gyorgyi Bennet & Drury Bennet & Drury Bennet & Drury Bennet & Drury Bennet & Drury Wolf & Berne Emmelin & Feldberg Bielschowsky et al. Urthaler & James Brashear & Ross Wedd & Fenn Wedd & Fenn Parnas & Ostern Drury Ostern & Parnas Wedd Kidney & Bladder AMP adenosine AMP adenosine Drury & Szent—Gyorgyi Drury & Szent—Gyorgyi (1929) (1931) (1931) (1931) (1931) (1931) (1956) (1948) (1944) (1972) (1969) (1933) (1933) (1932) (1932) (1932) (1931) (1929) (1929) continued Table l--Continued vasoconstriction vasodilation vasoconstriction micturation initial decreased RBF decreased GRF sodium excretion decreased renal venous renin decreased increased RBF decreased GRF decreased Na excretion decreased renal venous renin vasoconstriction vasodilation vasoconstriction and vasodilation vasoconstriction bladder contraction contraction contraction AMP adenosine AMP adenosine adenosine ATP AMP adenosine AMP adenosine AMP adenosine AMP adenosine ATP ATP ATP ATP adenosine ATP AMP ADP adenosine ATP REL-“25’. AMP adenosine ATP Bennet & Bennet & Buyniski & Rapela Emmelin & Feldberg Tagawa & Tagawa & Tagawa & Tagawa & Tagawa & Tagawa & Tagawa & & Tagawa Scott et Scott et Scott et Marcou Burnstock et al. Bennet & Gillespie Drury Drury Vander Vander Vander Vander Vander Vander Vander Vander al. al. al. Drury (1931) (1931) (1969) (1948) (1970) (1970) (1970) (1970) (1970) (1970) (1970) (1970) (1965) (1965) (1965) (1932) (1972) (1931) (1933) continued Table l--Continued 10 relaxation decreased (rectal) increase (forearm skin) increase decreased rythmic motility vomiting decreased peristalsis increased bone defecation decreased activity decreased vascular resistance decreased compliance increased ileal motility (high dose) decreased clumping increased clumping Gall Bladder AMP Bennet & Drury adenosine Body Temperature AMP Bennet & Drury adenosine ATP Stoner & Green adenosine Richards Stomach adenine, Rehm et al. adenosine AMP, ADP, ATP ATP Emmelin & Feldberg Intestine AMP Drury & Szent-Gyorgyi adenosine Gillespie ATP Gillespie ATP Emmelin & Feldberg adenosine werle & Schievelbein adenosine Chou ATP adenosine Chou ATP Chou adenosine Platelets ATP, AMP Born & Cross adenosine ADP Born & Cross (1931) (1931) (1945) (1934) (1970) (1948) (1929) (1933) (1933) (1948) (1964) (1966) (1966) (1966) (1963) (1963) continued Table l--Continued 11 transient cessation followed by increased ventilation no effect on ventilation increased ventilation decreased tidal volume decreased arterial pressure vasodilation biphasic increase & decrease in arterial pressure decreased arterial pressure (cat) decreased arterial ‘pIessure decreased arterial piessure decreased arterial pressure (rat) decreased arterial pressure (cat) very slight vasodilation increased electrical activity dilation (rabbit ear) accumulation of leucocytes Respiratory Center ATP ATP (low dose) ADP ADP Emmelin & Feldberg Folkow Brashear & Ross Brashear & Ross Systemic Vasculature AMP adenosine AMP adenosine ATP ATP ATP AMP AMP, adenosine ADP, ATP AMP, ADP, ATP Brain adenosine ATP Skin AMP adenosine AMP adenosine Drury & Szent-Gyorgyi Bennet & Drury Drury & Szent-Gyorgyi Gillespie Emmelin & Feldberg Folkow Kalckar & Lowry Gordon & Hesse Bielschowsky et al. Buyniski & Rapela Benzi et a1. Bennet & Drury Bennet & Drury (1948) (1949) (1969) (1969) (1929) (1931) (1929) (1933) (1948) (1949) (1947) (1961) (1944) (1969) (1969) (1931) (1931) continued Table l--Continued 12 relaxation (facial artery) relaxation (renal artery) relaxation (femoral artery) decreased volume (isolated perfused) Isolated-Arterial~Strips AMP, ADP ATP, 3'5-AMP adenosine, AMP, ATP adenosine, ATP adenosine Spleen Gebert et al. Collingsworth & Selleck Collingsworth & Selleck Marcou (1969) (Unpub. Observ.) (Unpub. Observ.) (1932) l3 adenosine and AMP, was observed to frequently increase the 'tone' of the isolated small intestine. One intestinal preparation described by Gillespie "had no sign of life until the ATP was added". In studies performed on the isolated virgin guinea-pig uterus, ATP, AMP and adenosine all caused increased contraction, a phenomenon reported earlier by Bennet and Drury (1931). Isosine, IMP and ITP were also studied by Gillespie and his experiments with these latter compounds demonstrated qualitatively similar responses to those with the adenine compounds; however, a ten-fold greater dose was required in the case of the inosine compounds to give observable effects. Purity checks and anesthetics were not reported in this paper. Gaddum and Holtz (1932-1933) found that small doses of adenine compounds (5 ugram) in cats and dogs produced trans- ient vasodilation in the isolated, blood perfused lung; while larger doses (250 ugram) produced lasting pulmonary vasoconstriction. The effect was apparently on the pulmonary arterioles, but some qualitatively similar changes in pul- monary venous tone were also observed. Adenylpyrophosphate (ADP and ATP) effects were much more pronounced than those of adenosine or AMP. Emmelin and Feldberg (1948) studied systemic effects of ATP in decerbrated and chloralosed cats. They noted a steep fall in arterial blood pressure upon intravenous injection of 14 .2 to .4 mg of ATP. This was attributed to pulmonary con- striction, bradycardia, and systemic vasodilation. It was also observed that much smaller doses of ATP could cause decreased systemic arterial blood pressure when injected into the left auricle rather than intravenously. A direct effect of ATP on the respiratory center was also suggested, based upon respiratory changes after ATP injection into the carotid and vertebral arteries. These changes consisted of initial cessation of respiration or of shallow frequent respiration followed by hyperventilation. There was also evidence for indirect effects on the respiratory center mediated via the vagi. The possibility that ATP may stimu- late the chemoreceptors was discussed but not experimentally studied. Folkow (1949) observed that ATP was a powerful vasodi- lator in cats and dogs. The threshold dose for the effect was 0.05 to 0.1 Y when injected intraarterially, a potency about one-fifth to one-fifteenth that of acetylcholine. The small doses used by Folkow produced no significant effects on the heart or on respiration. Atropine, neoantergan and vessel denervation did not block the dilator effect of ATP, implying but not proving a direct effect on the vessels. Intravenous injection of low doses of ATP produced no de- crease in systemic arterial pressure, indicating inactivation of ATP by the lungs. Folkow suggested a possible role of ATP 15 in peripheral vascular regulation, a theory discussed earlier by Zipf (1931) and Rigler (1932). Although circumstantial supportive evidence for adenyl compounds being the mediators of reactive and active hyperemia was presented by these early investigators, Drury (1936) states that there is no proof. These early workers proposed the interesting theory that during muscle contraction ATP is degraded to AMP, and AMP then passes out of the cells and causes hyperemia. The finding of Dale (1933) that AMP is less active than ATP did not support the theory, but Gillespie's observation that AMP was more active than ATP did. More recently support for the proposal that ATP is the mediator comes from Duff et al. (1954), who found a three-fold increase in muscle blood flow after infusion of 16 pg ATP/min. into the human forearm. The vasodilator properties of ATP, acetylcholine, and hist- amine were compared. Mg-ATP caused as large an increase in forearm blood flow as did acetylcholine or histamine, but ‘without the uncomfortable side effects associated with the latter two drugs. Mg-ATP was a more powerful vasodilator than Na—ATP. This was attributed to the potentiated vaso— dilator action of ATP when combined chemically with magnesium. Mg-ATP did not appear to be inactivated by the circulation as rapidly as acetylcholine but was inactivated more rapidly than histamine. Interestingly, Mg++ is a dilator even when injected unbound to ATP. Wolf and Berne (1956) achieved 16 maximal vasodilation with an ATP infusion of .2 to .3 uM/min into the coronary arteries. It is thus obviously possible that small quantities of ATP released from endogenous sources could participate in regulation of blood flow. Gordon (1961) and Frohlich (1963) localized the vasodi— lator effect of the adenine nucleotides to small vessels, particularly the arterioles. Kjellmer and Odelram (1965) found changes in both arterial and venous resistance associ- ated with ATP-induced dilation. A slow movement of intra- vascular fluid into the tissue spaces was also found. Scott et al. (1965) provided evidence that ATP may be involved in regulating blood flow through kidney, hindlimb, and heart. Using a bioassay organ technique, they found that bioassay organ resistance changed in the same direction when the assay organ was the forelimb and in the opposite direc- tion when the assay organ was the kidney. This finding suggested that active hyperemia, reactive hyperemia, and autoregulation of blood flow may result from a change in the chemical environment of the vessels--an extension of the theory prOposed by Gaskell (1880). Large doses of ATP affect the heart. Green and Stoner (1950) found that ATP initially reduced cardiac contractility, followed by a period of augmented contraction. Urthaler and James (1972) found a negative dromotrOpic action of ATP on the heart. This effect was directly related to the number of 17 attached phosphate groups. A purine nucleus and 6-amino group aided this effect. Urthaler and James (1972) found further effects of adenine compounds on the heart. All adenine nucleotides impaired AV conduction. ATP and adenosine produced negative chronotropic action. Transient ectOpic beats of AV junctional origin were observed at the onset of AV block. Phosphate bonds were not necessary for the chronotropic effect of ATP. It was suggested that ATP and adenosine exerted their effect by modification of the myocardial cell membranes. The negative chronotropic effect of ATP occurred at much lower concentrations than the nega- tive dromotropic effect. ATP injected in a concentration of l to 10 mg/ml produced an immediate heart block of 5 to 30 ,seconds duration. It was suggested that since ATP is con- centrated within myocardial cells, their destruction during an infarct could release sufficient ATP to cause further heart block. Although the pharmacological effects of the adenine compounds have not been studied extensively in the kidney, several papers of interest have appeared. Decreased renal blood flow and urine flow with intravenous AMP were observed by Drury and Szent-Gyorgyi (1929). Similar findings (adeno- sine and AMP increase and ATP decreases renal resistance) have been reported by Scott et a1. (1965), Thurau (1964), Hashimoto and Kumakura (1965), Nechay (1966) and Harvey (1964). 18 Gordon (1962) found AMP in venous plasma from the rabbit kidney after brief arterial occlusion, implicating AMP in the autoregulation of renal blood flow. However, in these studies of Gordon rate of renal blood flow influenced the amount of AMP, with AMP present at high rates of flow only. This could indicate rapid breakdown of extracellular AMP in the kidney. Recently a more complete study of the effects of the adenine compounds on renal blood flow has been pub- lished by Tagawa and Vander (1970). These investigators also found initially a decrease in renal blood flow with adenosine and AMP; however, steady infusion of these com- pounds into the renal artery did not change or caused a slight increase in renal blood flow. ATP infusion increased renal blood flow with no initial decrease. Adenosine and .AMP infusion lowered GFR and sodium excretion and renal venous renin activity. ATP infusion also decreased GFR, sodium excretion and renal venous renin. It seems, therefore, that ATP dilates the efferent arterioles. On the basis of these findings, Tagawa and Vander suggest that adenosine and/or AMP may be the normal mediators of both autoregulation and renin secretion--thus overlapping but also conflicting their pro- posal with that of Gordon's (1962). This hypothesis suggests that AMP or adenosine released during increased renal perfu- sion pressure could cause afferent arteriolar vasoconstric- tion in an effort to maintain GFR and reduce renal blood flow l9 simultaneously. Also suggested was the possibility that when renal perfusion pressure is increased; more sodium is fil- tered; more sodium is reabsorbed; and thus more ATP breaks down to AMP. AMP and adenosine thus formed then pass out of the tubular cells, cause autoregulatory vasoconstriction and also cause decreased renin. It is difficult to understand if renal venous plasma AMP increases with brief renal arterial occlusion, as reported by Gordon, how renin release increases in this situation because Tagawa and Vander report that AMP inhibits renin production by the kidney. Further evidence implicating adenosine and AMP in renal blood flow autoregula- tion is discussed by Ono et a1. (1966). These investigators observed that dipyridamole decreases renal blood flow. Several investigators have studied the action of adenine compounds in the lung. Brashear and Ross (1969) found that injection of 12 mg/kg of ADP into the pulmonary artery caused a sustained fall in pulmonary artery pressure lasting for 30 minutes and systemic hypotension lasting 5 minutes. ADP disappeared much more rapidly in vivo (one minute) than in vitro (20 minutes), although it did survive passage through the lungs, since arterial ADP levels were significantly higher than controls immediately after ADP injection into the pul- monary artery. Heart rate decreased for five minutes after injection. However, Brashear et al. (1970) found increased pulmonary artery pressure, cardiac output, central blood 20 volume, stroke volume and heart rate upon injecting .4 mg/min/kg into the femoral vein. Decreases in aortic pres- sure, systemic resistance, and platelets also occurred. The increased pulmonary artery pressure resulted from in- creased cardiac output rather than increased resistance, as no change in pulmonary vascular resistance was recorded and pulmonary artery pressure promptly returned to control levels although circulating platelet levels remained depressed. Therefore, the decrease in platelets did not seem to be re- lated to the rise in pulmonary artery pressure. Gordon (1961) found that ADP was a more potent vasodilator than ATP, AMP, or adenosine upon intravenous injection. With intra-arterial injection, ATP and ADP were equally potent, indicating that ADP may pass through the lungs whereas ATP may not. During the 1930's the finding that adenine compounds lowered arterial blood pressure led to the proposal that these compounds may also be responsible for traumatic shock. This theory was supported by the large quantities of adenyl com— pounds in tissue--injury of the tissue would supposedly re- lease these compounds into the blood and thus cause decreased blood pressure. Bennet and Drury (1931) observed release of adenosine-like substances from burned, perfused rabbit heart using a guinea pig heart as a bioassay. Similar findings were made by Zipf (1932), who in 1931 published the above suggestion, as did Konig (1930). H. N. Green (1943), 21 Bielschowsky and Green (1943) and Bielschowsky and Green (1944) discuss and provide experimental support for the theory that ATP is one of the chemically labile shock-producing factors from striated muscle--these studies perhaps were stimulated by war injuries. With the development of sophis- ticated assays for the adenine nucleotides, Kalckar and Lowry (1947) attempted to chemically assay these materials in plasma during 'traumatic' shock. Anesthetized dogs and rabbits were submitted to leg injuries (500-800 blows with a mallet). Four to five minutes after such traumatization blood pressure decreased, and the animals became weak and drowsy. In several cases "slight but distinct" increases in the concentration of adenine compounds were observed in venous plasma from the traumatized extremity. No increase in systemic arterial plasma adenine compounds was observed even though blood pressure was greatly decreased. Furthermore, injection of adenosine deaminase had no effect on blood pres- sure in these animals. Although Kalckar and Lowry state that it is unlikely that adenylic acid compounds play a primary role in traumatic shock, their findings did not rule out these substances as "secondary factors". Staples et al. (1969) have noted decreased ATP in skeletal muscle, liver and kidney with both hemorrhagic and endotoxin shock. This review of the pharmacological actions of the adenine compounds has not included cyclic 3,5-AMP and adenosine 22 tetraphosphate. A vast amount of information concerning the biological actions of cyclic 3,5-AMP has recently developed, research in this area being stimulated perhaps by the finding of Sutherland and Rall (1960) that cyclic 3,5-AMP is an intracellular mediator for the glycogenolytic action of epinephrine. A short review dealing with the biological effects of cyclic AMP has been published by Butcher (1968). No information concerning the pharmacology of adenosine tetra- phosphate was found, although it is present in tissue and is available commercially. Due to the vasodilator property of the adenine compounds, it has been suggested that they may be involved in peripheral vascular regulation. Stainsby (1973) presents an excellent review of the control of peripheral blood flow. Although oxygen, carbon dioxide, potassium ion, Krebs cycle inter- mediates, hydrogen ion and osmolarity have also been suggested as possible regulators of blood flow, Scott et a1. (1965) found evidence that physiological changes in oxygen, hydrogen ion, sodium and potassium were not adequate to produce the increased blood flow which accompanies exercise. Frolich (1965) found a significant vasodilator effect of Krebs cycle intermediates on small vessels. However, the degree of change of'these intermediates during physiological alteration of blood flow is not known. 23 Jacob and Berne (1961) proposed that adenosine may be the mediator of coronary vasodilation in the hypoxic heart. Also, Berne (1963) in his early studies suggested that AMP may be involved in the regulation of skeletal muscle blood flow since no adenosine was found in either normal or anoxic muscle. However, adenosine has recently been reported to be released during ischemic contraction in skeletal muscle (Dobson et al., 1971). IMP was found in normal muscle and increased during anoxia, with an associated increase in inosine and hypoxanthine levels. A sharp drop in ADP also occurred in exercising skeletal muscle, possibly due to IDP formation (Imai et al., 1964) as well as degradation to AMP. Kontos et a1. (1968), however, presented evidence but not proof that AMP and ATP were not responsible for the vasodi- lation which occurred in skeletal muscle during short periods of ischemia. They found that dipyridamole, which potentiated the vasodilator effect of AMP and ATP, did not augment the vasodilator response to short periods of ischemia. Adenosine, like AMP, has numerous effects on the mammal- ian cardiovascular system, as summarized in Table 1. In addition to causing vasodilation throughout most of the body, adenosine was found to vasoconstrict the kidney (Scott et a1. , 11965), the lung (Bennet and Drury, 1931) and possibly the spleen (Marcou, 1932). Bradycardia and a negative inotrOpic effect on the heart have also been noted with adenosine. 24 Wblf and Berne (1956) found that adenosine and AMP were equally potent vasodilators, Inn: were only one-fourth as effective as ADP and ATP in increasing coronary blood flow. One wonders if the more potent dilators are not the ones most active normally in the body. Several drugs have been found to potentiate the vaso- dilatory effect of adenosine. Persantin (dipyridamole) and lidoflazine are two such drugs. Bunag et al. (1964) found that the drug Persantin prevented adenosine deamination by erythrocytes, presumably due to reduced permeability of the erythrocyte membrane to adenosine. Afonso and O'Brien (1971) found that dipyridamole and lidoflazine delayed the disappearance of adenosine in blood. The main effect of these drugs, however, was a decrease in tissue permeability to adenosine, especially in the lungs. Since these drugs cause vasodilation, it is tempting to speculate that adeno- sine compounds are normally present in the extracellular fluid. Forrester (1966) provided the first direct evidence that ATP is released from active frog skeletal muscle in vitro. Further studies by Boyd and Forrester (1968) supported the concept that the ATP did not come from muscle cell damage, as potassium ion level in the plasma did not increase. They suggested that ATP release may proceed via the transverse tubular system of the sarcoplasmic reticulum in skeletal 25 muscle. Forrester and Lind (1969), using firefly extract analysis for ATP, identified ATP in human plasma, both in resting and exercising subjects. Venous ATP levels of ex- ercising subjects rose consistently above resting values, indicating addition of ATP to blood passing through the muscle bed. Forrester (1972a) found that with a more re- fined technique no ATP could be identified in the venous effluent plasma from an occluded forearm without exercise. In the venous effluent from exercising skeletal muscle, 0.033 to 1.0 nmole ATP/ml of plasma was observed. After taking into account the amount of ATP lost by degradation, it was concluded that ATP could be the mediator of the active hyperemia seen in the human forearm. Chen et al. (1972) also found ATP in the venous effluent during active hyperemia in skeletal muscle. Control ATP .levels were 206 ng/ml plasma in the femoral artery and 165 Lug/ml plasma in the femoral vein. During active hyperemia, :femoral venous ATP levels rose to approximately 450 ng/ml Exlasma. Femoral venous AMP levels also increased from 52 to approximately 188 ng/ml plasma. Increased flow correlated vmith increased ATP levels. ADP released from platelets and/or tissue cells at a siste of injury or hemorrhage initiate a series of events MfliiCh participate in hemostasis (Ganong, 1969). In the presence of ADP, platelets clump together at the hemorrhage 26 site forming a temporary plug. Also occurring simultaneously with the release of ADP from the platelets is the release of serotonin, which theoretically causes a potent vasoconstric- tion in the area of the hemorrhage. In addition to ADP, other adenine nucleotides have been observed to affect the clumping of platelets-~some of these observations relating the adenine compounds to platelet function are described below. Zuker and Borrelli (1960) demonstrated that ADP added to plasma resulted in platelet swelling. Born and Cross (1963) found that ADP added to plasma caused rapid platelet aggrega- tion. The platelets dispersed with time, and it was proposed that ADP breakdown was the cause. It was suggested that ADP associated with "aggregating sites" on the platelet surface. AMP and adenosine inhibited aggregation, adenosine ten times as effectively as AMP. ATP was slightly inhibitory. The inhibition could have resulted from competition for the aggregation sites. The ADP, AMP, adenosine and ATP exerted their effects in very low concentrations. The reactions were highly specific. EDTA inhibited the aggregation effect. Calcium addition sometimes caused aggregation. Salzman et al. (1966) further investigated the effect of ADP on platelets. They proposed that ADP caused platelet aggregation by product inhibition of ATP breakdown by ecto- ATPases in the platelet membrane. Blocking this energy- supplying reaction would result in the platelets losing their unique shape and becoming spherical. This could expose 27 adhesive sites on the platelet membrane. ADP breakdown would stop product inhibition of the ATP breakdown and allow the platelet to assume its normal shape. Evidence for this theory was found in the facts that neither ADP breakdown nor ADP binding to the platelets was required for aggregation. AMP was inhibitory only after dephosphorylation to adenosine. The adenosine inhibition may be due to an adenosine-carrier complex crossing the platelet membrane. ATP also inhibited platelet aggregation. Spaet and Lejneiks (1966) proposed that the breakdown of ADP to AMP was the cause of ADP-induced aggregation. EDTA, AMP, and removal of plasma all prevented platelet clumping, thus supporting this theory. Hellem and Owren (1964) proposed a complex bridging be- tween platelets occurred during aggregation. Calcium ions, ADP and von Willebrand's factor presumably composed the bridge. Experimental evidence did not support this theory, however (Spaet, 1965). Haslam (1964) showed that the aggregating effect of thrombin on platelets was mediated by ADP release from the platelets. Ireland (1967) further studied thrombin effects on platelet adenine nucleotides. The results showed that two nucleotide pools probably exist in platelets. Thrombin treat— ment caused release of largely non-radioactive nucleotide pools. The two pools contained approximately the same amounts 28 of ATP and ADP. The ATPzADP ratio in the thrombin-released pool was 0.7-0.8. Karpatkin and Langer (1968) found that both thrombin and epinephrine caused release of sufficient ADP to account for platelet agglutination. They also found a high rate of ATP utilization in platelets. Holmsen (1967) found further evidence for the existence of two nucleotide pools in platelets. One pool participated in metabolism and was not lost during clumping. P32 taken up by the platelets was found in this nucleotide pool, and a large proportion of these nucleotides was protein bound. The second pool did not participate in metabolism but was released upon external stimulation. Approximately 2/3 of platelet nucleotides belonged to the second pool. ADP re- .1eased by collagen did not come from platelet ATP but presum- ably from the large platelet ADP stores. Born and Cross (1963) found that repeated addition of small amounts of ADP to plasma diminished the aggregating effect of ADP. Rosenberg and Holmsen (1968) also found a loss of platelet aggregability upon repeated addition of ADP. This effect was termed the refractory state of the platelets. The refractory condition remained long after ADP breakdown and was due to ADP rather than its breakdown products. When viewed together, the pharmacological effects of the adenine compounds seem to indicate that in many prepara- tions these compounds depress muscle contraction. In the 29 gall bladder relaxation was observed by Bennet and Drury (1931); in the stomach decreased rhythmic motility was noted by Rehm et al. (1970); in many tissues vasodilation was reported; in the intestine decreased activity was noted by Werle and Schievelbein (1964); and in isolated arterial strips Gebert et al. (1969) observed relaxation. These findings suggest that the adenine compounds may have a basic action on smooth muscle in general and perhaps are similar in biological function to acetylcholine. The release of ATP from nerves has been reported by Holton (1959), from the adrenal medulla with adrenal nerve stimulation by Douglas (1966), from frog nerve muscle preparations with electrical stimulation by Abood et al. (1962) and-from non-adrenergic inhibitory nerves in the gut by Burnstock et al. (1970). Therefore, nerves may serve as a direct source of ATP or at least an indirect source by mediating its release from other cells. For many years there have been hints that nerves other than the classical adrenergic and cholinergic types are present in the autonomic nervous system. However, their effects were frequently attributed to the presence of sympa- thetic nerves running in vagal trunks or to Special receptor sites rather than to a third type of nerve. It has recently been demonstrated that a third type of nerve does exist in the autonomic nervous system (Robinson et al., 1971). These 30 nerves were distinguished by a different type of vesicle than that found in adrenergic, cholinergic or sensory nerves. The vesicles were large, granular and opaque. Application of 6-hydroxydopamine, which resulted in degeneration of adrenergic nerve fibers, did not affect the non-adrenergic nerve profile; the vesicles were still present and the inhikdtory response to stimulation remained. Reserpine, which depletes catecholamine content, also had no effect on these nerves. It was therefore concluded that this type of nerve was neither cholinergic nor adrenergic but represented a third and distinct type of nerve. Burnstock (1972), in an eXcellent review of the subject, terms this class of nerves "Sharinergic", since there is evidence that ATP is their neurotransmitter (Burnstock et a1. , 1970) . Extensive evidence for the existence of purinergic nerves comes from studies on the gastro-intestinal tract. Atropine did not affect inhibitory potentials in the guinea-pig taenia coli (Burnstock et al., 1963). Campbell (1966) found that low frequency vagal stimulation of the atropinized guinea-pig stomach was much faster and more effective in producing relaxation than perivascular (sympathetic) nerve stimulation. Bretylium abolished the response to perivascular stimulation but did not affect the response to vagal stimulation. Beani et a1. (1971) also studied purinergic nerve effects in the guinea-pig stomach. Atropinization reversed vagal stimula- tory effects from excitatory to inhibitory. Sympathetic 31 blocking agents (guanethedine, bretylium, reserpine) did not abolish vagal inhibition. The results of these studies suggest, therefore, that vagal purinergic nerves may be present in the stomach; their function here is probably related to control of gastric motility. Studies on the intestine have indicated that purinergic nerves are located in this tissue also. Ambache (1951) noted that addition of botulinum toxin, which paralyzes cholinergic nerves, produced evidence which revealed a group of inhibi- tory nerves in the enteric plexuses of mice and rabbits. The response to nicotine in a normal intestine was excitatory, whereas in a botulinum-poisoned preparation nicotine produced inhibition of intestinal motility. However, these inhibitory nerves were considered to be sympathetic in origin by Ambache. Burnstock et a1. (1964) found electrophysiological evidence for two types of inhibitory nerves in the guinea-pig taenia coli. Intramural inhibitory nerves responded to lower trans- mural stimulatory frequencies than sympathetic nerves and were not affected by adrenergic blocking agents, as were the sympathetic nerves. Everett (1968) reported evidence for the presence of purinergic nerves in the ileum and rectal caecum of the chick. Burnstock et a1. (1966) identified two types of inhibitory nerves in the smooth muscle of the guinea— pig taenia coli: perivascular and intramural inhibitory nerves. The cell bodies of the intramural inhibitory nerves 32 were localized in Auerbach's plexus. Both circular and longi- tudinal muscle were innervated by the intramural nerves (Furness, 1969). Bulbring and Tomita (1967) noted that the intrinsic (intramural) nerve fibers were quite short (a few mm), based on electrOphysiological studies. Addition of tetrodotoxin, which blocks nerve but not smooth muscle func- tion, abolished inhibitory potentials in the smooth muscle, thereby ruling out the possibility that muscle stimulation by itself produced the inhibitory potential. The inhibitory potentials were not blocked by guanethedine (Furness, 1969) and therefore were not of sympathetic origin. Electrophysiological studies have revealed some char- acteristics of the inhibitory potentials produced by 'purinergic' nerves. Bennett et a1. (1966) found that stimu- lation of short duration (200 usec) excited intramural inhibi- tory nerves of the guinea-pig taenia coli but not the muscle. Hyperpolarization of the smooth muscle occurred in response to single stimuli applied to the intramural nerves but not to single stimuli applied to the perivascular nerves. The latency of reSponse was longer in intramural nerves. Beani et a1. (1971) found a rebound excitation following an inhibitory potential; this consisted of depolarization beyond the normal level and therefore a state of increased contraction. Simultaneous stimulation of excitatory and inhibitory neurones in the stomach results in contraction (Beani et al., 1971) 'whereas it causes relaxation in the colon (Furness, 1969). 33 Purinergic nerves have also been implicated in other organs. Robinson et al. (1971) found evidence to suggest the existence of such nerves in the lung. Burnstock et al. (1972) found the urinary bladder contained atropine-resistant nerves which were excitatory. Nerves with purinergic char- acteristics have also been located in the avian gizzard (Bennett, 1969). The presence of purinergic nerves in the cardiovascular system is as yet only speculative. The func— tion of these nerves is uncertain; however, their participa- tion in the regulation of organ motility seems highly probable. Evidence has accumulated which indicates that ATP is the neurotransmitter released by purinergic nerves. Indirect evidence comes from studies of the effect of ATP on specific organs. Where the stimulation of the proposed purinergic nerves results in relaxation, infusion of ATP also results in relaxation. This was demonstrated in the gut by Burnstock et a1. (1970). In organs where stimulation of the proposed purinergic nerves results in contraction, ATP infusion also results in contraction, as shown by Burnstock et al. (1972) in the urinary bladder. The bladder reSponse to ATP was also similar to the nerve-mediated response; both were characterized by rapid contraction and rapid return to the original condition. Quinidine blocked both reSponse to stimu- lation of the proposed purinergic nerves and to ATP. ATP was the mmst potent adenine compound. Satchell et al. (1969) 34 identified adenosine in the perfusate of stomachs in which inhibitory nerves had been stimulated, suggesting that the adenosine may have been coming from the nerves; however, it could have been coming from the other areas of the stomach as well. More direct evidence comes from studies of Burnstock et al. (1970). Stimulation of inhibitory nerves was accom- panied by the presence of adenosine and inosine in stomach perfusates. Satchell and Burnstock (1971) found that ATP breakdown by the stomach could account for observed levels of adenosine and inosine. ATP, ADP and AMP were found in the medium upon stimulation of Auerbach's plexus in the gizzard; since the Auerbach's plexus is mostly nerve tissue, this evidence strongly supports the proposal that the adenine compounds came from nerves. Tritiated adenosine uptake studies provide further evi- dence that ATP is the neurotransmitter (Su et al., 1971). Tritiated adenosine added to a medium in which strips of taenia coli were immersed was taken up by nerves and muscle to a considerable extent. Approximately 60 per cent of intracellular 3H activity was 3H-ATP. More of the 3H-ATP activity was in nerve than in muscle. Little 3H—ADP was formed; small amounts of 3H—AMP (25%) and 3H-inosine (10%) were present. Release studies were done on sections of taenia coli pre-incubated with tritiated adenosine. The lnedium contained atrOpine and guanethidine, thereby inhibiting 35 cholinergic and adrenergic nerves. Transmural stimulation resulted in an outflow of tritium activity into the medium; the activity could only have come from the intramural inhibitory nerves or the smooth muscle. Tetrodotoxin abol- ished both the muscle relaxation and tritium activity outflow normally resulting from intramural inhibitory nerve stimulation. Smooth muscle relaxation due to adrenergic nerve stimulation was not accompanied by tritium outflux. Therefore, the above evidence suggests that tritiated adenine compounds stored in intramural inhibitory nerves are released from the nerves during stimulation. From the evidence, therefore, it can be concluded that nerves are present in the body which are neither adrenergic nor cholinergic. ATP seems to be the most probable candidate for the neurotransmitter substance of these nerves. The pro— posed purinergic nerves have thus far been implicated in the control of gastric, bladder and uterine motility through their effect on smooth muscle. Their function in organs such as the lungs is as yet unclear. The ATP released by these nerves seems to act by changing membrane premeability to potassium, with resultant changes in cell responsiveness. Another group of compounds not discussed in this thesis but perhaps indirectly related to the pharmacology of the adenine nucleotides are the inosine, uridine, guanidine and cytidine nucleotides. The biological potency of some of these 36 compounds was reviewed by Drury (1936); and at that time the activity of these compounds in most preparations was con- sidered to be less compared to the adenine compounds. However, cytidylic acid dilates the coronary vessels to about the same degree as adenylic acid (Wedd and Drury, 1934); and Flossner (1934) reported that members of the guanine series had a potency similar to the adenyl compounds. Hashimoto et a1. (1964) found that uridine compounds produce vasodilation, UTP being somewhat less potent than ATP. Geiger (1956) has reported that a uridine compound produced in the liver is necessary for adequate perfusion of the brain. Wolf and Berne (1956) report that UTP has about the same effect as adenosine and AMP as a coronary vasodilator; however, these authors found that other derivatives of hypoxanthine, guanine, cytosine and uracil bases lacked vasodilator properties. Magnesium ITP was found to be only slightly less potent as a shock-inducing agent than magnesium ATP (Bielschowsky and Green, 1944). Phosphate groups are readily transferable via kinases from one nucleotide to another (Beyer, 1968; Glaze and Wadkin, 1967; Goffeau et al., 1967); thus, even though a particular nucleotide is not biologically active, transfer of its phos- phate may cause the formation of a nucleotide which can elicit a biological reSponse. One can therefore imagine the possible extension of the physiological mechanisms proposed to be 37 mediated by ATP to include other nucleotides as well. Also, the possibility that different organs utilize different nucleotides as mediators should be considered. 2. Adenine Nucleotide Metabolism, General The known pathways for the metabolism of the adenine compounds are diagramed in Figure l. A review of the litera- ture dealing with the metabolism of these compounds has revealed that the series of metabolic steps shown in Figure l is present in virtually all organs of the mammalian body. Some exceptions to the ubiquitious nature of these pathways may occur, however; and there is definite evidence that the quantitative importance of the alternative pathways shown in the figure may vary from one organ or tissue to another. The following sections devoted to individual organs and tissues describe our present understanding of these variations and also briefly describe the theoretical relationship of these pathways of adenosine metabolism to the local control of blood flow. 3. Adenine Nucleotide Metabolism in WEoIe Blood and Plasma Drury et a1. (1937) found that adenosine was inactivated by plasma of the cat and other species; AMP and ADP were also inactivated in plasma but less readily than adenosine. Following the studies of Drury, our understanding of the 38 Figure 1. Metabolism of the adenine compounds. 5'-nucleotidase = adenylate phosphatase = AMP phosphatase; apyrase = a—B-ATPase; adenylate kinase = myokinase = adenosine diphosphoric acid phosphomutase = adenosine tri— phosphate-adenosine 5'-monophosphate phosphotransferase; purine nucleoside phosphorylase = purine ribosyltransferase; ' hypoxanthine phOSphoribosyltransferase = nucleotide pyro- phosphorylase; adenine phosphoribosyltransferase = purine nucleotide pyrophosphorylase. ) ATP Phosphoryl Transferase ADP a apyrase ADPase Myokinase adenine phospho- ribosyltransf erase I LN ._______A 5'-nuc1eotidase- purine nucleoside phosphorylase , I) cyclaseE 39 adenyl ,5 AMP phospho- diesteraseL 8-y-ATPase ADP deaminase Adenylo succinase adenylic deaminase fiadenosine kinase IDP Adenylosuccinic acid IMP IF "1 adenylosuccinic acid synthetase _Adenine Adenosine .7— adenosine deaminase Inosine purine nucleoside phosphorylase 5'—nucleotidase hypoxanthine Hypoxanthine xanthine oxidase Xanthine Uric Acid phosphoribosyltransferase Figure l - l ' .u‘v" I .ul.\t. u‘ I fl 5' ads: - ‘ulvdl ' Q 'ppnu' a Its-I I c - u u... l (1' nu. I) 'o‘vou O . I-th .. H "luau *- .Vv‘ h. N u“ v ‘ ~| : . "‘1 ' “A 0‘ a n. .- \,_ 40 definitive pathways for metabolism of the adenine compounds by blood was initiated by the studies of Conway and Cooke (1939). These investigations indicated that the enzymes adenylic deaminase and adenosine deaminase are present in whole blood. Conway and Cooke also found these enzymes in plasma, but to a lesser degree than in whole blood. Purine nucleoside phosphorylase has been found in erythrocytes by Kim, Cha and Parks (1968). In regard to synthesis of nucleo- tides by blood, Miech and Santos (1969) have demonstrated the presence of adenosine kinase in erythrocytes, and adeny- late kinase has been identified in erythrocytes by Tatibana, Nakao and Yoshikawa (1958) and Kashket and Denstedt (1958) and in platelets by Holmsen and Rozenberg (1968). The more indirect routes for nucleotide synthesis catalyzed by purine ribosyl- and phosphoribosyltransferase have been found in blood cells by Krenitsky (1969). -The evidence for the pres- ence of d-B-ATPase, B-y-ATPase, ADPase and 5'-nuc1eotidase in plasma and platelets is discussed by Holmsen and Day (1971); and ATPase and apyrase have been reported to be in erythrocytes by Clarkson and Maizels (1952). Thus, virtually all the pathways for nucleotide metabolism have been identi- fied in blood. The following paragraphs discuss additional findings that have been made regarding the metabolism of these substances in whole blood. 41 Early studies on stored human blood indicated that as the length of storage increased, the blood became less viable; cellular ATP levels fell and the normally discoidal erythro- cytes became spherical. Jorgensen (1955) found that xanthine and hypoxanthine accumulated in stored human blood, apparently as a result of nucleotide degradation. Also upon storage, human blood became less viable after a time due to loss of acid—hydrolysable phosphate from nucleotides (Mills and Summers, 1959). Mills and Summers noted, in addition, that glucose and inosine were important in maintenance of cell viability, as cellular ATP levels increased when inosine or glucose was added to the blood. ATP is a required coenzyme in the conversion of glucose to g1ucose-6-phosphate, catalyzed by hexokinase, an important reaction for maintenance of cell viability. Guanine, cytidine, and 2,3-DPGA levels fell con- currently with a fall in ATP. Guanine and cytidine levels presumably fell as ATP may be necessary to maintain them. They suggested that the fall in 2,3-DPGA was contributing to the maintenance of the ATP level during storage of whole blood. Nakao et al. (1959) found that addition of adenine plus inosine to long-stored erythrocytes resulted in increased 3 ATP levels and a return of characteristic disc shape to the erythrocytes, which had become geometrically distorted with aging. Neither adenine nor inosine added alone was effective in restoring ATP levels in long-stored erythrocytes. Inosine 4|- .. ‘I ‘5 Vi ‘ I ‘tl . V. ‘II '3 42 was probably necessary to provide the ribose residue of the nucleotides and the glycolytic intermediates required for generating a high-energy phosphate supply, such as hexose monophosphate and hexose diphosphate. Jorgensen and Poulsen (1955) found 1-3 UQ/ml of hypo- xanthine plus xanthine in the plasma of freshly withdrawn human blood. The concentration rose to 90:100 ug/ml upon standing at 37°C for 24 hours; intracellular ATP levels fell concurrently. There was also some increase at 4°C. Free phOSphate liberation was slow, presumably due to its transfer to other substances. ATP levels also fell in stored human whole blood (Jorgensen, 1957). Hemolysis or stirring of the blood accelerated xanthine and hypoxanthine formation. ATP disappeared ten times faster in mechanically hemolysed blood as compared to non-hemolysed blood (Chen and Jorgensen, 1956). Chen and Jorgensen (1957) found that AMP conversion to hypoxanthine occurred at the same rate in hemolysed and non-hemolysed blood. A solution of erythrocytes in Tyrode- Locke solution also showed oxypurine (xanthine and hypOr xanthine) accumulation. Leucocytes had no effect on oxypurine accumulation. There was no xanthine oxidase activity, as no uric acid was formed. Siliconized glassware reduced the rate of oxypurine formation. These studies on stored whole blood therefore indicate that there is a continual degradation of ATP and simultaneous 43 formation of xanthine and hypoxanthine. In these experiments the specific intermediates between ATP and xanthine were not identified. Nonetheless, these initial findings with whole blood in conjunction with those of Conway and Cooke (1939) led to future investigation of the details of the metabolism of the adenine compounds in blood. Jorgensen (1956) studied the breakdown of ATP in human platelet-rich plasma and whole blood. Nucleotide analysis was done by the spectrOphotometric technique of Jorgensen and Chen (1956). ATP was added to whole blood or plasma in a concentration of about 0.4 mM. It was degraded to hypoxanthine in both plasma and whole blood. ADP, AMP, adenosine and inosine were identified as intermediates in this conversion. The half-time in plasma was approximately two hours. In whole blood the breakdown proceeded 7-8 times faster than in plasma, since the half-time here was approximately 1/2 hour. Since free phosphate concentration rose only half as rapidly as ATP was broken down, ATP breakdown was not one of simple ortho- phosphate liberation. Adenine added to plasma was not broken down, implying conversion of adenosine to hypoxanthine via inosine. The presence of adenosine deaminase, which converts adenosine to inosine, in plasma was thus implied; and nucleo- side phosphorylase, which converts inosine to hypoxanthine, was also localized in plasma. The inosine-hypoxanthine con- version was found to be the rate determining step. 44 After eight hours at least 50 per cent of added inosine was converted to hypoxanthine with no decrease in the velocity of the reaction during this time. 'The reaction evidently did not reach equilibrium, as the velocity of hypoxanthine formation did not decrease in an eight hour period. Since hypoxanthine largely remained in the medium after its forma- tion, ribose phosphate must have been continually removed to account for the maintenance of reaction velocity. The split- ting of the ribose group from inosine by nucleoside phos- phorylase was therefore the true determinant of the velocity of inosine breakdown to hypoxanthine in plasma. Forrester (1969) studied the breakdown rate of ATP in diluted human plasma at room temperature. He found a 34 per cent fall in ATP concentration during the ten minute period following centrifugation of the blood. He also found that no ATP could be detected in plasma left standing at room temperature for one hour. Forrester (1972) further studied ATP breakdown in citrated human whole blood at 37°C. He found a linear relation between the amount of ATP added and the amount of ATP recovered from the plasma after centrifu— gation. Addition of low concentrations (1, 5, and 10 Ug/ml) of ATP to whole blood at 37°C produced a consistent pattern of degradation with each concentration. For example, it took 32 minutes for 5 pg ATP/ml blood to be degraded beyond the threshold of detection, whereas 1 ug/ml required only about 12 minutes. 45 Holton (1959) found that 10 nmoles of ATP injected into rabbit ear arteries was both broken down in the blood and taken up by the tissues. Approximately 46 per cent of the adenine of ATP injected was not recovered in the venous effluent and was presumably taken up by the ear. Of the 54 per cent recovered, only 8 per cent was ATP; the remaining 46 per cent was ATP breakdown products. ADP degradation in plasma has also been investigated. Holmsen and Stormorken (1964) found that an adenylate kinase reaction was the first step in ADP breakdown in human plasma, as both ATP and AMP were formed. AMP was inhibitory, while adenosine was not. However, Holmsen (1967) and Flatow et a1. (1965) could not localize adenylate kinase in plasma. Plasma adenylate kinase activity found by earlier investigators was I attributed to hemolysis. Holmsen and Stormorken (1964) found that EDTA in equimolar concentration with magnesium completely blocked ADP breakdown, while citrate had to be 18 times the magnesium concentration to be inhibitory. Pyrophos- phate also inhibited breakdown, probably due to magnesium chelation.f Heparin had no effect. Magnesium was required. Optimal pH was 8.5 to 8.8, although variation with substrate concentration probably occurred. Odegaard et al. (1964) also studied ADP inactivation (removal of platelet clumping effect) in plasma. The inacti— vating system was heat labile and destroyed at 58°C. Maximal 46 activity was at 37°C. Dialysis had no effect and inactivation occurred in serum as well as plasma. AMP was formed. ADP dephosphorylation to AMP by an ADPase in the plasma was found by Ireland and Mills (1966) to have a half—time of about 8 minutes. The KIn of the ADPase for ADP was 1-2 uM 1 (Mills, 1966). Platelets did not enhance the breakdown. Adenylate kinase may also have been acting, as ATP was formed 1~‘_‘_‘ — “7:- upon addition of 200 UM of ADP to platelet-poor plasma. Smaller quantities of added ADP, however, did not result in significant ATP accumulation. No IMP was detected. Use of citrate rather than heparin as anticoagulant resulted in approximately doubling the half-time of ADP (200 uM) break- down in plasma; AMP also accumulated to a greater extent be- fore disappearing. At lower initial ADP concentrations (1-2 PM), however, AMP accumulation differed little between heparin and citrate-treated samples. With heparin as anticoagulant more hypoxanthine accumulated in platelet-rich than in platelet-poor plasma. The path of ATP breakdown in whole blood has been dis- puted. Mills and Summers (1959) found that IMP accumulated in the erythrocyte during an 8 hour incubation period. It was suggested that the IMP resulted from hypoxanthine ana- bolism rather than deamination of AMP, since IMP accumulated rapidly upon addition of inosine to the blood. Bishop (1960) found IMP formation in blood exposed to air and in hemolyzed 47 blood. The IMP was presumably formed from hypoxanthine in the presence of nucleotide pyrophosphorylase rather than by isosine formation, as inosine was not detected in stored or incubated blood. IMP was also presumed to be formed from the deamination of AMP. The equilibrium between IMP and AMP 53 was in favor of IMP. Bishop therefore proposed that the FF pathway of ATP breakdown in whole blood proceeds via ADP, AMP, IMP and hypoxanthine. Ireland and Mills (1966), however, found no IMP formed during l4C-ADP breakdown in plasma. In addition Conway and Cooke (1939) found that erythrocyte adenylic acid deaminase was normally inhibited due to the CO2 and bicarbonate system of the blood. Also, Chen and Jorgensen (1957) found that addition of adenylic acid deaminase to blood caused a considerable change in intermediary compounds, suggesting that AMP deamination is a slow process normally. AMP dephosphorylation via 5'-nucleotidase seemed to occur much more rapidly. It was thus concluded that ATP breakdown in human blood takes place primarily via AMP, adenosine, and inosine to hypoxanthine. Additional information concerning the breakdown path was found by Chen and Jorgensen (1957). IMP added to an erythro- cyte suspension was converted to hypoxanthine two to three times as rapidly as AMP. This indicated that if AMP was deaminated to IMP, the resulting IMP could readily form inosine and hypoxanthine. Adenosine and inosine were converted to 48 hypoxanthine 50 to 75 times as rapidly as AMP. Therefore, adenosine deaminase acts more rapidly than 5'-nucleotidase in the erythrocyte. Added adenine resulted in no measurable hypoxanthine formation; therefore, it could not be an inter- mediate in ATP breakdown in whole blood unless it was formed in an isolated compartment. The paths of ATP breakdown in whole blood and plasma described by Jorgensen (1956) and Bishop (1960) conflict, therefore, as to whether AMP is deaminated or dephosphorylated, Bishop's evidence suggesting deamination, while Jorgensen found evidence indicating dephosphorylation. Perhaps the concentration of AMP in part determines the pathway for its breakdown; the above discrepancies, therefore, may be caused by the different substrate levels studied by these investiga- tors. Hemolysis and duration of the incubation are other factors which may affect the path of AMP breakdown in blood. The enzymes involved in adenine nucleotide degradation in blood have been studied by many investigators. Mills (1966) found that 20 per cent of ATP added to human plasma was dephosphorylated to ADP by a plasma B-y-ATPase, with a half-time of approximately 8 minutes, while a plasma a-B- ATPase dephosphorylated 80 per cent of the ATP directly to AMP with a.half-time of about 3 minutes. The Km of the a-BeATPase for ATP was approximately 2 x 10-7M. Increasing initial ATP concentration to 20 uM resulted in increased 49 formation of orthophosphate rather than pyrophosphate. Therefore, the a-B-ATPase appeared to be more easily saturated. Parker (1970), in studies on human erythrocytes, found that ATP was degraded via ADP and AMP to adenosine by the outer surface of the erythrocyte membrane. Clarkson and Maizels (1952) isolated an apyrase from the stroma of human erythrocytes, which converted ATP to AMP. The apyrase had a pH optimum of about 6.8 and was stimulated by magnesium. Calcium had no effect. The apyrase acted on both extracell- ular and intracellular ATP. It is interesting to speculate that these enzymes in the erythrocyte membrane probably have some physiological significance related to membrane function. Herbert (1956) also studied a human erythrocyte apyrase located on the outer surface of the membrane. This apyrase liberated orthophosphate from ATP and thus may be different from the apyrase* described by Clarkson and Maizels (1952). Herbert found that both calcium and magnesium acti— vated his apyrase. The concentration range for magnesium was lower than that for calcium, however, possibly explaining why Clarkson and Maizels found no calcium effect. Calcium antag- onized magnesium in a competitive manner. The pH optimum was 7.0 to 7.4. ADP was also dephosphorylated by the outer sur- face of erythrocytes. Caffrey et al. (1956) found a pH Optimum of 7.3 to 7.6 for human erythrocyte ATPase. ~ *It should be noted that the term apyrase refers usually to the enzyme producing AMP and perphosphate (rather than (IrthophOSphate) from ATP (Caffrey et al., 1956). 50 Cysteine increased the enzyme's activity. ADP and ITP were also substrates, but with lower Kms than ATP. A 1:1 ratio of magnesium to ATP was optimal for enzyme activity. The presence of carbon dioxide, bicarbonate, and phosphate in the erythrocyte reduced apyrase activity. Adenyl cyclase, which converts ATP irreversibly to cyc1ic AMP, to the best of the author's knowledge, has not been found in dog erythrocytes (Davoren and Sutherland, 1963). Conway and Cooke (1939) found adenylic acid deaminase, which converts AMP to IMP, in erythrocytes. The enzyme was normally inhibited, however, by the CO2 and bicarbonate of the blood. They also found adenosine deaminase, which converts adenosine to inosine, in blood. Baer et a1. (1966) found and characterized adenosine deaminase in erythrocytes. The optimal pH was between 6.5 and 7.0. Schrader et al. (1972) located adenosine deaminase in both the cytoplasm and stroma of the human erythrocyte. The enzyme was evidently not on the outer surface of the membrane, however, since dipyridamole prevented adenosine deamination and uptake but did not interfere with adenosine deaminase activity. Nucleoside phosphorylase, which reversibly splits the ribose from inosine to form hypoxanthine, was originally isolated by Kalckar (1945) from liver. Kim et al. (1968) 51 found nucleoside phosphorylase in high concentration in human erythrocytes. The enzyme had a broad range of optimal pH, from 6.5 to 8.0 and was quite stable. The paths of adenine nucleotide synthesis in blood have also been investigated. The conversion of adenosine to AMP was studied by Miech and Santos (1969) in rat erythrocytes. They found that adenosine was converted to AMP without cleavage of the glycosidic bond, indicating the presence of adenosine kinase. Lowy and Williams (1966) also found direct phosphorylation of adenosine to AMP. Lerner and Rubinstein (1970) established the presence of adenosine kinase in erythrocytes and found that it was inhibited by ATP. Schrader et al. (1972) localized it in both the erythrocyte membrane and cytoplasm. Schrader et al. (1972) suggest that direct phosphorylation of adenosine occurs because the Km of adenosine kinase is significantly lower than that of adenosine deaminase. Since both adenosine kinase and adeno- sine deaminase are present in erythrocytes, the fate of adenosine here is determined by the relative activities and Kms of the two enzymes and the adenosine concentration. Lowy and Williams (1966) found that the human erythrocyte is unable to convert IMP to AMP. Bishop (1960) found adenine uptake by human erythrocytes. The adenine was then converted to AMP, presumably by reaction with ribosylpyrophosphate S-phosphate catalyzed by nucleotide 52 pyrophosphorylase. Reaction of adenine with ribose-l-phos- phate to form adenosine and thence AMP was considered unlikely due to high adenosine deaminase activity in blood; however, this may not be valid reasoning because low levels of adeno- sine rapidly go to AMP via adenosine kinase in blood. ATP synthesis has been for the most part reported to occur intracellularly. Ronquist (1968) found extracellular ATP formed by human erythrocytes. However, the synthesis only occurred in a medium supplied with the substrates and co- factors of the glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase reactions. Tatibana et a1. (1958) found a highly active adenylate kinase (myokinase) in human erythrocytes. This enzyme converted two molecules of ADP to AMP and ATP in a reversible reaction. Adenylate kinase activity was much higher than that of AMP deaminase. The enzyme was somewhat resistant to acid and quite resistant to heat. Flatow et al. (1965) found adenylate kinase activity in erythrocytes but not in leucocytes. Kashket and Denstedt (1958) isolated adenylate kinase from both the membrane and cyt0p1asm of rat and human erythrocytes. The presence of the enzyme in the cytoplasm permitted rapid interconversion of cellular nucleotides. Membrane-bound adenylate kinase be— lieved located just below the surface could account for the conversion of extracellular ADP to intracellular ATP. ADP added to a suspension of washed erythrocytes appeared to enter w I‘I vv. fi.‘ I u”. - ‘Oh ‘h. tn. ~ "I I g“ 53 the external part of the erythrocyte membrane, whereas ATP or AMP apparently did not. The evidence suggested that the ATP or AMP formed from ADP could then pass freely into or out of the cell. AMP which entered the cells could evidently be converted to IMP, as large amounts of IMP were detected in Stroma-free hemolyzate. Incubation of cells with ATP plus AMP resulted in ADP formation. Addition of AMP inhibited ATP formation from ADP. Clarkson and Maizels (1952) found that AMP phosphorylation could occur only within the erythrocyte membrane or intracellularly and required glucose. It has been questioned as to whether de novo purine syn- thesis can occur in the erythrocyte. Evidence seems to indicate that it cannot. Lerner and Rubinstein (1970) found that adenine and glucose added to human erythrocytes resulted in increased intracellular ATP levels. The amount of the increase depended on the intracellular level of ATP prior to incubation. Addition of adenosine also resulted in increased intracellular ATP levels. Elevated ATP levels inhibited adenosine kinase. The maximum ATP level obtained with any cells or substrates was twice that found in fresh cells. Iowy and Williams (1966) also studied effects of adenosine and adenine on human erythrocyte ATP levels. They noted that adenine was only incorporated into ATP in the presence of inosine. Adenosine formed ATP at both high and low adenosine concentrations; however, due to the rapid deamination of 54 adenosine which occurred at low concentrations, much more ATP was formed at the higher concentrations.* Bishop (1960) found no evidence for de novo synthesis of purines from gly- cine in human erythrocytes. Lowy et a1. (1958) could not find evidence for de novo purine synthesis in the rabbit erythrocyte. Platelets contain large quantities of adenine nucleotides (Karpatkin and Langer, 1968). However, the role of platelet adenine nucleotides in the control of blood flow is not yet clear. Holmsen (1967) and Holmsen and Rozenberg (1968) found evidence for a metabolically inert ADP pool in platelets which was released in response to collagen, whereas a second ADP pool was metabolically active and not released by col- lagen. Karpatkin and Langer (1968) found considerable ATP generated with no increment in platelet ATP stores, implying a high rate of platelet ATP utilization. During the release reaction ATP goes irreversibly to IMP within the platelets and to hypoxanthine in the plasma (Ireland, 1967). AMP de- aminase, therefore, appears to be much more important in platelet function than 5'—nuc1eotidase. Holmsen and Rozenberg (1968) found that adenosine was taken up by platelets to form .ATP via AMP and ADP. Virtually no AMP was stored, and less *Another interpretation of this observation would be that more adenosine present at high adenosine levels caused more ATP formation by mass action. Schrader et al. (1972) found that adenosine kinase had a lower Km than adenosine deaminase. 55 ADP was formed than ATP. ADP added to a suspension of washed platelets was converted to AMP extracellularly. Platelets enhanced hypoxanthine formation from ADP added to plasma (Ireland and Mills, 1964). ATPases have been located on the surface of platelets (Chambers et al., 1967, and Mason and Saba, 1969). Chambers et al. (1967) characterized ecto—ATPase activity of human platelets. The enzyme was calcium and magnesium dependent, blocked by EDTA, and was maximally active in a pH range of 7.0 to 8.5. ADP competitively inhibited the enzyme. Ouabain had,no effect, thereby differentiating this ATPase from the one involved in active tranSport of sodium and potassium. The enzyme was more active at 37°C than at 20°C. This ecto- ATPase may be the thrombosthenin described by Nachman et al. (1967). Thrombosthenin was characterized by its contractile properties and was implicated in clot formation and retrac- tion, maintenance of platelet shape and ATPase activity. It was magnesium dependent and was located in both platelet membranes and granules. However, membrane thrombosthenin had greater ATPase activity than granule thrombosthenin. It resembled the protein which composes cellular microtubules, as it consisted of multiple polypeptide subunits of a polymeric nature. Mason and Saba (1969) found that the platelet ecto- .ATPase thrombosthenin was inhibited by sulfhydryl compounds. 56 Saba et a1. (1969) isolated a light and a heavy platelet membrane fraction consisting of membrane bound vesicles. Thrombosthenin ATPase activity was associated with the light fraction. Flatow et al. (1965) found adenylate kinase activity in platelets from an unspecified species, and Holmsen and Rozenberg (1968) found adenosine kinase in human platelets. Holmsen and Rozenberg also found that platelet lysates con- tained adenosine deaminase, AMP deaminase and purine nucleo- side phosphorylase. Platelets were impermeable to AMP. 4. Adenine Nucleotide Metabolism in Skeletal Muscle Conway and Cooke (1939) investigated adenylic acid deaminase in body tissues. Skeletal muscle had forty times more of the enzyme than other tissues, but the activity of the enzyme was 500-1000 times greater due to the absence of enzyme inhibitors in skeletal muscle. AMP was directly deaminated to IMP by this enzyme. Conway and Cooke found very low levels of adenosine deaminase compared to adenylic deaminase in skeletal muscle. Thus, in skeletal muscle the breakdown path of ATP differs somewhat from that occurring in whole blood and heart. In this regard Imai et al. (1964) noted a striking absence of adenosine in anoxic skeletal muscle; however, more recent reports from the same laboratory state that adenosine is normally present in skeletal muscle 57 and that its concentration increases with ischemic contrac— tion (Dobson et al., 1971). In Imai's et a1. studies there was a sharp drop in ADP during skeletal muscle ischemia which was not found in myocardial ischemia. IMP levels in' skeletal muscle were also relatively high and increased shortly after ischemia due to ATP degradation and high adenylic acid deaminase levels. Creatine phosphate levels were 225 per cent higher in skeletal muscle than in cardiac muscle; and the authors provide evidence that this large pool of creatine phOSphate in skeletal muscle helps maintain the ATP level in this tissue during ischemia. The IMP formed during ischemia was subsequently converted in these studies to inosine, presumably by a 5'-nucleotidase. Gerlach and Dreisbach (1962) found that the adenine nucleotide breakdown path in the rat kidney was similar to that in skeletal muscle, i.e., ATP, ADP, AMP, IMP, inosine and hypoxanthine. Dobson et al. (1971) detected no ATP in the venous plasma from ischemic or normal skeletal muscle. However, no precautions (of significance*) were taken to prevent ATP breakdown during the isolation of the plasma. Adenosine, however, was found to increase fivefold in the venous plasma *Blood samples in these studies were cooled before centrifuga- tion; however, plasma ATP degradation continues in whole 'blOOd at an appreciable rate even when iced; see Table 6, this'theSis. Also, the samples were exposed to room air, which results in faster ATP breakdown than when physiological pH is maintained (see Figure 10, this thesis). 58 during ischemic contraction. Inosine and hypoxanthine in- creased 22 and 270-fold, reSpectively. It was therefore proposed that adenosine may regulate skeletal muscle blood flow in a manner similar to its role in coronary blood flow regulation; however, it was also concluded by these authors "that the adenine nucleotides are not directly involved in skeletal muscle blood flow." Sutherland et al. (1962) localized adenyl cyclase in dog skeletal muscle and suggested that this enzyme was strongly influenced by circulating hormones. The role of adenyl cyclase here was presumably to influence cellular metabolism rather than to regulate blood flow. Webster (1953) found that direct deamination of ADP by rabbit skeletal muscle myofibrils occurred, with resultant production of IDP and small amounts of IMP. Thus, in the skeletal muscle studies of Imai et a1. cited above one must also consider the possibility of IDP formation. However, Webster also reports that as pH was increased from 5 to 7, dismutation of ADP was favored over deamination. In regard to the synthesis of adenine compounds in skeletal muscle, Davey (1961) observed that AMP could be formed from IMP. The initial reaction involved a condensa- tion of IMP and aspartic acid to form adenylosuccinic acid, catalyzed by adenylosuccinic acid synthetase. This was then converted to AMP. The pH optimum for this reaction was 59 between 6.9 and 7.2. GTP was the energy source and was found to be bound to sarcoplasmic proteins. Adenylosuccinic acid synthetase was detected in skeletal muscle and liver but not in heart, lung or kidney. The enzyme was implicated in the rapid deamination-reamination cycle of the adenine nucleotides prOposed to occur during muscular contraction. Newton and Perry (1960) also studied the reamination of IMP in skeletal muscle. They noted that muscle potential for IMP reamination is less than for AMP deamination. Kashket and Denstedt (1958) isolated adenylate kinase from skeletal muscle. The presence of the enzyme in the cytoplasm permitted rapid interconversion of cellular adenine nucleotides. Krenitsky (1969) found a high level of adenine phosphoribosyltransferase in skeletal muscle. This enzyme converts adenine to adenylate, which in turn could be con- verted to adenosine via 5'-nuc1eotidase or to ADP and ATP via adenylate kinase. Inosine kinase, responsible for converting inosine directly to IMP, was characterized by Allan and Bennett (1971). The enzyme was isolated from E. coli. It required magnesium and was stimulated by potassium. It has not been found in mammalian tissues to the best of this author's knowledge. Cell membranes have generally been considered to be im- permeable to ATP. Chaudry and Gould (1970), however, found evidence that ATP may be taken up by rat soleus muscle. H .1 _ ”.1713 I no. N? ‘9 W". tidal An. 'o'EIE as 1"Y: «Q ‘1DOIy‘ it ‘5‘ V‘ a '5" v. Iv 60 l4C-ATP added to soleus muscle was extensively degraded to ADP, AMP, and IMP. Small amounts of inosine and adenosine were also formed. AMP and IMP were found only extracellu- larly. Addition of l4C-adenosine did not result in as much intracellular l4C-ATP accumulation as did l4C-ATP.* Addition of 14C-ADP resulted in increased intracellular 14C-ADP con- centration. The evidence therefore suggested that rat soleus muscle was permeable to ATP, ADP and adenosine but not AMP or IMP. It was also determined that ATPase, adenylic acid deaminase and 5'-nuc1eotidase were located on the muscle surface, as their activities were not found in the incubation medium but the products of their reactions were. Also sup- porting nucleotide extrusion from the skeletal muscle cells, Boyd and Forrester (1968) found ATP released from exercising skeletal muscle. The authors argue that the ATP was not a result of damaged muscle cells, as potassium levels in the venous effluent did not rise; however, this conclusion is disputed by Dobson et al. (1971). Further consideration of this rather crucial prOposal that these pharmacologically active adenine compounds can pass through the cell membrane is reviewed in more detail in a later section of this litera- ture review. *This observation leads one to wonder if extracellular ATP can aid cellular adenosine uptake by supplying ATP for adenosine kinase in the cell membrane. 61 5. Adenine Nucleotide Metabolism in the Heart The metabolism of the adenine compounds has been studied in the heart, perhaps to a greater degree than any other organ of the body. Evidence for the existence of several enzymes responsible for the metabolism of these substances in the heart has been published. Burger and Lowenstein (1967), Conway and Cooke (1939) and Baer et al. (1966) found adenylic deaminase and adenosine deaminase activity in this organ. Baer et a1. (1966) also purified and studied 5'-nucleotidase in the rat heart. Colowick and Kalckar (1943) found low levels of adenylate kinase in rabbit heart muscle. Purine ribosyl- and phosphoribosyltransferases in the Rhesus monkey heart have been described by Krenitsky (1969). Cardiac muscle adenosine kinase activity was noted by Goldthwait (1957), and its purification has been reported by Alma and Feinberg (1971). Sutherland et al. (1962) found adenyl cyclase in the dog heart. Many investigations have been performed to determine the location and factors controlling the activities of cer- tain of these enzymes in the heart and the theoretical rela- tionship of their respective activities to the regulation of the coronary blood flow. The following discussion describes some of the observations and theories regarding these enzymes. AMP is dephosphorylated to adenosine by 5'-nucleotidase. In the studies of Baer et a1. (1966) optimal activity was at pH 9.5, and ATP was a powerful competitive inhibitor. 62 Since ATP levels fall during hypoxia, 5'-nuc1eotidase could be activated during this state, thereby increasing AMP break- down to adenosine with resulting vasodilation rather than conversion to IMP. However, Burger and Lowenstein (1967) found that the fall in ATP during hypoxia did not appear to be sufficient to stimulate 5'-nuc1eotidase. AMP deaminase would presumably still be activated by the ATP levels present during hypoxia. However, adenosine formation may result from the increased rate of AMP formation during hypoxia or possibly a compartmentalization of 5'-nucleotidase, AMP deaminase and ATP. In the latter case ATP levels could con- ceivably fall low enough in localized areas or compartments of the membrane to activate 5'-nucleotidase. In regard to the intracellular localization of 5'-nucleotidase, Edwards and Maguire (1970) refer to unpublished studies of Maguire and Steggel, who found 5'-nucleotidase largely bound to cell membranes, i.e., the microsomal fraction of homogenized rat heart. The observations of Maguire and Steggel confirmed the similar findings reported earlier by Baer et al. (1966). Unlike Baer et al., the pH optimum reported by Edwards and Maguire (1970) for purified 5'-nucleotidase was 7.6 rather than 9.5 with AMP as substrate. Magnesium was found to induce shifts in the pH optimum, possibly explaining the different (9.5) pH Optimum found by Baer et al. for their purified rat heart 5'-nucleotidase. Also, according to 63 Edwards and Maguire (1970) calcium inhibited this enzyme more strongly than magnesium; however, Baer et al. found 7 mM Mg activated and 7 mM Ca inhibited. ATP strongly inhibited rat heart 5'-nuc1eotidase in a competitive (Baer et al., 1966) and non-competitive (Edwards and Maguire, 1970) manner. Alkaline phosphatases also probably contributed to AMP hydrol- ysis in the studies of Baer et al., since their 5'-nuc1eo- tidase preparations hydrolyzed glucose-6-phosphate to a small extent. Burger and Lowenstein (1967) also have confirmed the finding that ATP inhibits "adenylate phOSphatase" (5'-nuc1eo- tidase) from rat hearts. Histochemical studies have also localized 5'-nucleoti— dase in the cell membranes of the rat heart (Bajusz and Jasmin, 1964, and Rostgaard and Behnke, 1965). Rostgaard and Behnke found AMP dephosphorylated in the interspace separating plasma membranes at the intercalated disks and at pinocytotic vessels but in no other myofibril structures but the T system. These observations have led Jacob and Berne (1960) to propose that 5'-nucleotidase in the cell membrane converts intracellular AMP to extracellular adenosine. Observations of others (Borgers et al., 1971) regarding the histochemical localization of 5'-nuc1eotidase in the dog heart indicate that this enzyme is more or less restricted to the perivascular and interstitial mesenchymal cell membranes of the heart. Thus, this latter author suggests that the Berne theory for regulation of coronary blood flow is not a7 4‘4? 64 valid since adenosine would be degraded to inosine by adeno- sine deaminase before it could get out of the cardiac muscle cell. Borgers et al. (1971) argue that adenosine from the perivascular cells controls the coronary blood flow. Berne currently has found 5'-nuc1eotidase in dog cardiac muscle membrane (in press) and thus substantiates his theory. Another enzyme of key importance in the relationship between adenine nucleotide metabolism and the control of coronary blood flow is adenosine deaminase. By the conver- sion of adenosine to inosine the vaso-activity of the nucleo— side is removed, and therefore the activity of adenosine deaminase could affect the coronary blood flow by influencing the cardiac adenosine levels. Its presence in virtually all tissues of the body was described by the Irish investigators Conway and Cooke (1939), and some of the properties of cardiac adenosine deaminase have been recently characterized. Baer et al. (1966) isolated and partially purified adenosine deaminase from rat hearts and noted that (1) its activity was solely in the supernatant cellular fraction; (2) its pH optimum was between 6.5 and 7.0; and (3) persantin 4 x 10-4 M, a coronary vasodilator, did not inhibit its activity. The finding that adenosine deaminase is in the heart tissue super- natant fraction does not necessarily indicate that this enzyme is present in the cardiac muscle cells; however, the low Jheart tissue concentration of adenosine may suggest that such Iv '1 P '- (I) r!) N 3*. "n. I (1 My: 65 is the case. The loss of adenosine deaminase from the heart with Tyrode's solution perfusion is not easy to explain if adenosine deaminase is only intracellularly located (Jacob and Berne, 1960). Evidence that ATP and GTP do not alter the activity of rat heart adenosine deaminase has been reported by Burger and Lowenstein (1967). It is rather difficult to propose a definite physio- logical role for adenosine deaminase in the heart. When 14C-adenosine is infused into the isolated Tyrode-perfused cat heart, 50 per cent enters the adenine nucleotide pool of the heart and the remainder leaves the heart as l4C-inosine and hypoxanthine in the perfusate (Jacob and Berne, 1960, and Jacob and Berne, 1961). Similar findings were made by Weidmeier, Rubio and Berne (1972) in the isolated perfused guinea-pig heart, and these authors state that they believe that virtually all of the adenosine entering the cardiac mmscle cell is incorporated into nucleotides. Since adenosine seems to go into nucleotides rather than inosine in the heart, the biological role for adenosine deaminase in the heart is vague. The cardiac enzymes converting adenosine to AMP, ADP and ATP appear to be of greater importance even when the .heart is anoxic (Jacob and Berne, 1961). Perhaps anatomically separate compartments of adenosine exist in the heart, and intra-arterially injected adenosine does not enter the adeno- sine compartment exposed to adenosine deaminase. 66 Rubio, Weidmeier and Berne (1972) state that the appear- ance of greater amounts of l4C-isosine and l4C-hypoxanthine in the perfusate than in the tissue of isolated l4C-adenosine perfused hearts is an indication of the presence of adenosine deaminase in the capillary endothelium. In support of this hypothesis they refer to the finding of Conway and Cooke (1939) that adenosine deaminase is present in a non-specified artery. In regard to this rationalization for the low adenosine levels found in coronary venous blood, the fact that adenosine deaminase and nucleoside phosphorylase are found in the perfusate of isolated perfused hearts (Jacob and Berne, 1960) should at least be considered. Should these enzymes have been present in the perfusate of the isolated perfused hearts of Rubio, Weidmeier and Berne (1972), it is quite obvious that considerable amounts of l4C-adenosine could have been converted to inosine and hypoxanthine directly in their perfusates. In order to eventually determine and clearly understand the mechanisms for the chemical control of an organ's blood flow, it may be best not to favor any one single theory at this time. Indeed, the selection and dis- cussion of data only in terms of its support of one's pet theory may be detrimental. The observation described in the previous paragraphs that (exogenous 14C-adenosine enters primarily adenine nucleotides .in heart tissue brings focus upon the enzymes responsible 67 (primarily adenosine kinase) for permitting such conversion. Although several enzymes to be discussed later can influence the conversion of adenosine to nucleotides, adenosine kinase is of primary importance because it directly converts adenosine to AMP. The presence of adenosine kinase has been shown in dialyzed pig heart homogenates by Goldthwait _ 1---1§~ (1957) by demonstrating the capacity of such preparations to i rapidly convert 14C-adenosine to AMP in the presence of ATP. Sister Alma and Feinberg (1971) characterized cardiac muscle adenosine kinase. Magnesium enhanced its activity but was not required. Optimal pH was 5.8. Dipyridamole had no effect on adenosine kinase activity. Low ATP concentrations (2-8 x 10-4 M) stimulated the enzyme while higher concentrations (> 8 x 10.4 M) inhibited. Thus, cardiac anoxia with low cellular ATP levels may enhance the conversion of adenosine to AMP in spite of lower levels of ATP as a substrate for adenosine conversion to AMP. Indeed, the studies of Jacob and Berne (1961) seem to confirm this possibility. Lindgerg et al. (1967) found that activity of adenosine kinase de- creased with time at 37°C and pH 7.4. Other enzymes can also participate in the conversion of (adenosine to the adenine nucleotides. .Should adenosine be (monverted to adenine in the heart, cardiac adenine phosphori- lxasyltransferase (Krenitsky, 1969) could act on the adenine tx) form AMP. Another alternative would be for adenosine to 68 be deaminated to inosine by adenosine deaminase and subse- quently converted to hypoxanthine and IMP by the cardiac enzymes nucleoside phosphorylase (Rubio, Weidmeier and Berne, 1972) and hypoxanthine phosphoribosyltransferase, respectively, as indicated in Figure 1. The quantitative insignificance of these enzymic pathways for conversion of adenosine to —4 nucleotides in comparison to the adenosine kinase step has been demonstrated by Goldthwait (1957) in the pig heart and Weidmeier et al. (1972) in the isolated, perfused guinea-pig heart. The lack of conversion of adenosine to adenine in the rabbit heart was reported by Liu and Feinberg (1971) on the basis of conversion of adenosine-8-14C to nucleotides without formation of l4C-adenine. However, Jacob and Berne (1960) report adenine formation from l4C-adenosine in the isolated, perfused cat heart. In spite of their relative insignificance in adenosine metabolism, the presence of these alternate routes for nucleotide synthesis has been shown by Goldthwait (1957) in the heart and may indicate that the nucleotide pool .in the heart is in a dynamic state with continual breakdown and resynthesis occurring. The construction of the metabolic scheme for these events may involve compartmentalization of ‘the different enzymes; and within a tissue or organ synthesis (and.degradation of the nucleotides conceivably could occur lmath in the same or different sites. Thus, exogenous adenosine Inay'be directed to adenosine kinase, whereas some endogenous 'v- ’1 “u- h. ‘F nah ‘I B. a. b 69 adenosine may possibly be converted to adenine or hypoxan- thine before conversion to AMP. The proposed dynamic state of the cardiac nucleotides alluded to in the previous paragraph may also involve the interconversion between AMP and IMP, as indicated in Figure 1. The enzyme adenylic deaminase, which converts AMP to IMP, is certainly present in heart tissue (Goldthwait, 1957; Conway and Cooke, 1939; and Baer et al., 1966). In rat heart homo- genates Baer et al. (1966) found adenylic deaminase in nuclear, mitochondrial, microsomal and supernatant fractions; its activity was mostly found in the nuclear and supernatant fractions. Burger and Lowenstein (1967), using a semi—purified preparation of rat heart adenylic deaminase, demonstrated that ATP activates and GTP inhibits this enzyme. Thus, with decreased ATP in the heart AMP conversion to adenosine rather than IMP could be favored due to decreased adenylic deaminase activity and increased 5'-nucleotidase activity in localized areas. These findings therefore support the Berne theory that increased adenosine production and release during anoxia cause cardiac vasodilation. However, these findings also bring into focus the criticism that studies concerning the quantitative .importance of adenylic deaminase as compared to adenosine «deaminase (Conway and Cooke, 1939) must be reviewed with con- sideration of the ATP level in the enzyme preparation. In studies of adenine nucleotide degradation in the iso- Jxated perfused rabbit heart by Richman and Wyborny (1964), it iii 5‘. i . ‘P'.‘ a «(UP A - vVuV "E w hi U C an ’. U' L a "Wam- d-hrVu v}. o. A I \ ‘I U I ”A [1‘ Va ‘ ,- s‘u 70 was suggested that the principle path for AMP degradation is initial dephosphorylation to adenosine. The basis for this conclusion was the appearance of inosine and hypoxanthine in the perfusate concomitant with nucleotide breakdown triggered by anoxia and uncoupled oxidative phOSphorylatiOn. Nonethe- less, since IMP increased in the cardiac tissue with uncoupling, the authors indicate that direct adenylic acid deamination must at least be operative. It is difficult to understand why Richman and Wyborny (1964) did not find cardiac tissue IMP in their controls. With high tissue ATP in the control state adenylic deaminase should be most active; perhaps low control tissue AMP did not permit IMP formation. One also wonders if the increased IMP formed during anoxia is the cause of increased inosine in the venous effluent during hypoxia reported by Berne (1963), since IMP like AMP is a substrate for 5'-nuc1eotidase. As indicated in Figure l, adenylosuccinic synthetase and adenylosuccinase can convert IMP to AMP. To the best of our knowledge these enzymes have not been identified in the heart; however, their possible existence in this tissue has been discussed by weidmeier, Rubio and Berne (1972). On the basis of the observation that the specific activity ratios of nucleoside:base in heart perfusion fluid were the same as heart tissue nucleotides, these authors conclude "that the degradation of adenosine to base prior to its incorporation H.“ “II" I I I. (I. I '- nfih “LL 1'2: “in. (2 '1 (D h’. 5“ '04 v u .1 71 into adenine nucleotides is minimal". Again, however, one must consider the possibility that exogenous adenosine may not reach the sites for its enzymic degradation. The adenosine diphosphate deaminase reported by Webster (1953) to exist in rabbit skeletal muscle fibrils was not' found in rabbit heart homogenates, actin or myosin. Adenyl cyclase, localized in the dog heart by Sutherland et al. (1962), converts ATP to cyclic AMP. The enzyme in heart tissue was very sensitive to catecholamines; an in- creased catecholamine level stimulated adenyl cyclase. The primary function of this enzyme in the heart, therefore, is probably to increase cellular metabolism and thus support an increased force of contraction. No direct role of adenyl cyclase in the regulation of coronary blood flow was found. The intracellular and intratissue location of one of the major enzymes for nucleoside degradation has been studied by Rubio, Weidmeier and Berne (1972). These investigators, using a new histochemical method for nucleoside phOSphorylase, observed this enzyme in the cytoplasm and nuclei of endo- thelial cells and pericytes but not in the myocardial or smooth muscle cells. These findings apparently explain the arppearance of relatively large quantities of hypoxanthine in 'the perfusate of the isolated heart compared to the tissue .itself. It should be considered, however, that the hypoxan- 'thine could arise directly from interstitial inosine as well he a" .- I; -4» ?:~. ‘nv 72 as indirectly from interstitial adenosine as suggested by the authors. If adenosine deaminase and nucleoside phos- phorylase are in the capillary endothelium, one wonders why adenosine entering the isolated, perfused heart does not become completely degraded to hypoxanthine before it reaches the cardiac cells. Should Schrader's proposal that the low F Km of adenosine kinase causes adenosine phosphorylation rather ( than deamination apply to the heart as it does for the erythro- cyte ghost, then adenosine leaving the cardiac cell should be rapidly reincorporated into AMP rather than being washed out of the heart as hypoxanthine. As mentioned previously, Berne has proposed that adeno- sine may be the chemical mediator of coronary vasodilation in the hypoxic heart (Berne, 1963). This proposal is based on the observation that inosine and hypoxanthine were present in the perfusate and coronary sinus blood of severely hypoxic hearts. Due to the rapid breakdown of adenosine to these products in the body, it was suggested that adenosine was the substance released by hypoxic myocardial cells, with resultant vasodilation. Berne (1963) found that 3 to 27 times more :moles of inosine and hypoxanthine were released from the hypoxic heart than were required to double coronary flow when infused as moles of adenosine into the left coronary artery. These substances were not detected in arterial blood or in 'venous blood from a well-oxygenated heart. Berne proposed, I l . v ##r :- .Eb“). Run», v .3: h (A): ';ll :. futuqsh 1‘ 391.5 a) 563 I . V! “we \- . n “~"Q‘ I ‘tflh “a; I 73 therefore, that during hypoxia the decreased oxygen tension caused breakdown of heart muscle adenine nucleotides to adenosine. The adenosine then would diffuse out of the cells and reach the coronary arterioles via the interstitial fluid, with resultant dilation. The increased coronary blood flow which would occur would raise the oxygen tension, thereby reducing the rate of degradation of adenine nucleo— tides and washing out high levels of adenosine. The feedback mechanism would thus provide a way to adjust coronary flow to meet the heart's oxygen needs. Richman and Wyborny (1964) found evidence to support Berne's theory. Adenosine was recovered in the perfusate during adenine nucleotide degradation in the heart resulting from hypoxia or uncoupled oxidative phosphorylation. The study showed that adenosine can leave cells of the heart in the presence of either adenosine deaminase saturation or inhibition with 8-azaguanine, since adenosine appeared in 'the perfusate under these conditions. Large quantities of iruosine were recovered in spite of adenosine deaminase inhibition by 8-azaguanine. Therefore, the direct deamina- tion of AMP is an Operational pathway in the intact heart, atlthough these investigators suggest that it is not the one used normally. Further evidence in support of Berne's theory cxnnes from the studies of Imai et al. (1964), who under condi- tixxns of complete ischemia found adenosine in the underperfused ”Ada; w' «v: 6 up.“ "vs. '5‘... Nv‘“. .l' (I) _‘ I 74 myocardium of the rabbit. In addition, Chen et al. (unpub— lished observation) found that the ischemic myocardium released a substance which vasoconstricted a bioassay kidney; adenosine injected into the kidney was found to have the same effect. Therefore, adenosine perhaps was in the venous effluent of the ischemic myocardium. Katori and Berne (1966) found adenosine in the perfusate of hypoxic, anoxic or epinephrine-treated cat hearts in the presence of 8-azaguanine. Graded hypoxia showed that increased coronary flow was roughly proportional to the sum of adenosine, inosine and hypoxan- thine released (Wiedmeier et al., 1970). Rubio and Berne (1969) found adenosine released by the normal myocardium continuously into the surrounding inter- stitial fluid (pericardial fluid). The normal adenosine concentration of the pericardial fluid was approximately 10.9 x 10-7 M, presumably a basal level. These results indi- cated that interstitial fluid adenosine levels could regulate coronary blood flow to maintain a proper oxygen balance. Rubio et a1. (1969) found that the amount of adenosine re- leased during short periods of reactive hyperemia in the dog heart was sufficient to account for observed dilation. The adenosine was believed to accumulate only in the extracellular space of the ischemic myocardium, because heart tissue adenosine content was quantitatively accounted for by an estimation of interstitial fluid adenosine. r11 3 (vHQAO hgibvh ”-5,. H: o'Uu {JV 3.". :18 Ugly"; Y ebui“ - 1 ‘0. Q In ' ‘Nbo‘ ' 2"; DA ‘nnl “U "1 Vflu; vfi. ‘1“ 75 6. Metabolism of Extracellular Adenine Nucleotides'by'the'Lung§ Folkow, as early as 1949, reported that intravenously injected ATP was inactivated (its hypotensive action removed) upon passage through the lungs. This conclusion was based on the observation that a much greater injection of ATP was required to lower systemic arterial pressure when injected intravenously than when injected intra-arterially. Brashear and Ross (1969) found that ADP passed through the pulmonary circuit without complete breakdown, since intravenously injected ADP was recoverable in arterial blood. Gordon (1961), repeating Folkow's experiments, found that ATP and adenosine were inactivated in the lungs; however, adenosine inactivation was to a lesser extent. According to Gordon ADP and AMP were not subject to this pulmonary inactivation. Pfleger et a1. (1969) found that 57 per cent of 14C-aenosine intravenously administered was taken up by the lungs in 30 seconds. Inosine addition reduced this uptake. The im— portance of organ uptake was shown by estimates which sug- gested that more than 90 per cent of injected adenosine was taken up by organs and that only 10 per cent was broken down by the blood. Afonso and O'Brien (1971) found that dipyridamole greatly decreased uptake of adenosine by organs, especially the lungs, suggesting in view of Schrader's red cell ghost model that adenosine enters the lung and other .IAAH “‘.‘ UfiiUH '1‘”), J :ub‘ek 76 tissues of the body via facilitated diffusion. A comparison of adenosine disappearance rates in different tissues showed the following: .76 ug/min/ml in blood; 156 ug/min/gm in heart; and 2117 ug/min/gam in lung. Clarke et al. (1952) showed that the adonosine deaminase content of the lungs was very high; and Burger and Lowenstein (1967) identified both adenylic and adenosine deaminase in lung tissue. Considering the avid metabolism of adenosine by the lungs, this organ may therefore function to provide the body with arterial plasma free of adenosine, as well as fulfilling its more publicized gas exchange and angiotensin II formation functions. Of course, the validity of this speculation regarding adenosine hinges on whether or not adenosine and/or adenine nucleotides exist physiologically in venous plasma, and whether or not the blood itself is capable of rapidly destroying these pharmacologically active compounds. Another source of adenine compounds in the lungs may be the non-adrenergic inhibitory nerves supplying the interw stitial smooth muscle identified by Robinson et al. (1971) in the toad lung. Relaxation of the lung upon vagal stimulation was unaffected by adrenergic and cholinergic blocking agents. Although the neurotransmitter in this case has not yet been identified, Burnstock et al. (1970) found evidence that ATP is the transmitter released by similar nerves in the gut. n..- W lh:.. 1 v... I‘Hv l ”"1 in ‘ 2R°FCS “Hint . I .W‘W‘n with. T'InLi um“. .5 . Here I -“H‘ 77 7. The Passage of Adenosine and Adenine NucleotideS'ThrOugh the Cell Membrane The ability of adenosine to pass through cell membrane is well documented. According to Rubio, Berne and Katori (1969), adenosine passes out of the cardiac muscle cells into the blood perfusing the heart. Regarding the movement of 1 adenosine in the opposite direction, Su et al. (1971) have found 3H-adenosine taken up by enteric nerves in considerable quantities. The 3H-adenosine was rapidly metabolized in the nerve, largely to 3H-ATP. Jacob and Berne (1960) demonstrated the uptake of adenosine by the isolated perfused heart, and Schrader et al. (1972) have provided evidence that 14C- adenosine rapidly enters the red cell ghost. In the red cell ghost adenosine uptake had two components: a facilitated diffusion system which saturated at about 10 mM and a simple diffusion system which did not saturate. At low extracellu— lar adenosine concentrations (below 3 uM), most of it was taken up by facilitated diffusion and converted to adenine nucleotides in the erythrocyte. At higher concentrations there was much degradation to inosine and hypoxanthine, since the Km of adenosine deaminase was much higher than that of adenosine kinase. Adenosine was found intracellularly only ‘when external concentrations were as high as 1 mM. Intra- «cellular adenosine metabolism was so fast that a concentration flan-7y vb‘.‘ A)! V q Stu. a :w. 5t c, ‘9. N.“ E‘s _4_ I [u I) l .1' LJ: 78 gradient from out to in would exist under physiological conditions. Adenosine uptake would therefore occur whenever extracellular adenosine was present. Berne suggests that in the anoxic heart outflux rather than uptake of adenosine occurs--a theory which may conflict with the proposal of Schrader. Parker (1970) found that the adenosine formed from ATP degradation in blood did not accumulate but was rapidly taken up by the erythrocytes and rephosphorylated directly to AMP. The adenosine uptake showed kinetics of a saturable carrier transport system, in agreement with Schrader et al. (1972). The purine portion of the adenosine taken up by the erythrocyte was homogenously distributed through the cell adenine nucleotide pool. Extrapolation of erythrocyte data to cardiac muscle indicates that at physiological concentrations, adenosine enters the cells by facilitated diffusion, since such concen- trations extracellularly would be much less than 10 mM. In support of this proposal the uptake of adenosine by the heart and its incorporation into the adenine nucleotide pool of the heart is well documented (Jacob and Berne, 1960, and weidmeier, JRubio and Berne, 1972). Thus, it is undoubtedly evident that adenosine is capable of passing through the membranes of the . n-v- View '1‘ r. 50‘ pa. ‘I it. "a; ‘5“ . nu. A.“ V» .‘I it. I I. 8. h“ ‘A 'v 79 cardiac cells. However, the actual mechanism by which adeno- sine enters the cells of the heart, like other tissues of the body, has not been determined. Nonetheless, in this latter regard the studies of Afonso and O'Brien (1971) have demon- strated that both dipyridamole and lidoflazine greatly reduce E the uptake of adenosine by the dog heart. Since these drugs F did not reduce the destruction of adenosine by myocardial L homogenates, it appears that they reduce cardiac adenosine uptake by decreasing the permeability of the cells to this nucleoside. These findings may be indicative of a carrier- mediated transport mechanism for adenosine in the heart similar to that proposed by Schrader, Berne and Rubio (1972) for the red cell ghost. A relationship between red cell adenosine uptake and red cell deaminase activity has been described by Van Belle (1969). He found that both platelets and erythrocytes were responsible for the disappearance of adenosine in the dog and guinea pig. Ireland and Mills (1966) also found that platelets were per- meable to adenosine. However, in human blood Van Belle found that the erythrocytes were more important in regard to adenosine uptake and deamination. Variations in pH between 6 and 8 and ions did not affect adenosine uptake or deamina— tion in dog or human blood. Increasing temperature from 20°C to 35°C resulted in increased adenosine uptake and uvzn nun: "J“- l i~ n u n t (I) Y‘ n ‘5‘ I "A ' A H . *5 “A Hg ‘ .l,’ I): (I) 80 deamination in both human and dog blood. Use of metabolic inhibitors showed no dependence of adenosine uptake on gen- eral metabolism, glycolysis, oxidative phosphorylation or transport ATPase. Adenosine uptake therefore appeared to depend on intracellular adenosine activity unless thelcell. ;§au membrane was changed in.a way which altered sulfhydryl groups. ‘_‘a_un.-; Membrane sulfhydryl group integrity was necessary for normal ‘1‘“: t uptake. Evidence for rapid entry of adenosine into red cells has also been reported by Whittam (1960). Whittam studied uptake of inosine, adenosine, hypoxanthine and adenine by human erythrocytes. Inosine was taken up in direct proportion to its concentration in the medium. Equilibrium between cells and medium was reached within five minutes. Adenosine was also rapidly taken up by erythrocytes and deaminated to inosine. Less formation of nucleotides occurred, probably because of the high levels of adenosine used by Whittam. Hypoxanthine, regardless of the initial concentration in the Inedium, reached equilibrium across the red cell membrane; and rub appreciable concentration was established between cells (and.medium. Temperature, glycolysis, or the presence of sindlar compounds had no effect on uptake. Adenine in low (noncentrations, in the presence of a substrate for glycolysis, Vfiis avidly taken up by erythrocytes at 37°C. Only a small annount (12%) of the adenine taken up leaked back into the [LI LU 81 medium. At high adenine concentrations (3-7 mM) glycolysis had no effect on uptake. Adenine was presumably converted into adenine nucleotides within the erythrocyte, since iodo- acetate blocked glycolysis and also significantly decreased 14C-adenine uptake. In addition, iodoacetate added after 30 minutes incubation resulted in very little leakage of intra- cellular'14C-adenine or its derivatives back into the medium. Therefore, the derivatives of the adenine may have been adenine nucleotides, as these compounds are presumably none diffusable through cell membranes. The adenine uptake was not due to active transport, based on this evidence. Bishop (1960) also found adenine taken up by human erythrocytes. Henderson and LePage (1959) found that adenine-B-Cl4 was taken up by mouse erythrocytes in vitro. This adenine was lost in some measure to body tissues upon injecting the blood into an animal. Hamilton (1953) found adenine-8-C14 taken up by human leucocytes. The passage of adenine nucleotides through cell mem- branes is less well documented and indeed, some investigators (Jacob and Berne, 1960, Lowry, Romot and London, 1958, and“ others) have stated that the cell membranes of various tissues are relatively impermeable to these phosphorylated compounds. Ireland and Mills (1966) presented evidence that platelets were impermeable to ADP and AMP. Nonetheless, studies of others indicate that nucleotides may escape in at least small In f ‘1 (D f 82 amounts from the intracellular to the extracellular fluid. Forrester (1972b) and Chen et al. (1972) report values for ATP and AMP in plasma. Abood et al. (1962) report efflux of ATP from muscle and nerve and relate this phenomenon to depolarization. The release of ATP from non-adrenergic inhibitory nerves in the gut was noted by Burnstock et al. (1970) and Su et al. (1971). They also found uptake of tritiated AMP, ADP and ATP by these nerves but to a lesser extent that of tritiated adenosine. However, the possibility that the nucleotides were first converted to adenosine before uptake was not considered. Kuperman et a1. (1964) found tritiated AMP released from frog sciatic nerve; they also stated that ATP behaves similarly. Chaudry and Gould (1970) report evidence for the direct entry of ATP and ADP into the rat soleus muscle; whereas, Hoffman and Okita (1965) report that the penetration of ATP into the myocardium is initiated and takes place through ATP breakdown followed by intracellu— lar resynthesis. Conway and Cook (1939) stated that AMP could readily pass through the red cell membrane and 'voluntary muscle'; however, we could not find the authors' experimental evidence for these statements. Levine et al. (1969) found that rat liver cells were slightly permeable to exogenous cyclic adenosine 3',5'-monophosphate-8-14C, and Davoren and Sutherland (1963) found extracellular cyclic AMP accumulation following its intracellular synthesis. Douglas et al. (1966) (J (n a“) 83 found a high ATP concentration in the chromaffin granules in adrenal medulla cells and described evidence for its release into the extracellular fluid. Apparently, ATP and other adenine nucleotides were discharged into the blood vessels along with catecholamines upon medullary stimulation; however, ATP was quickly degraded to AMP in the adrenal blood vessels. Taken as a whole the studies described above indicate the possibility that small quantities of adenine nucleotides may leak out of the cells of the body. If such is the case, the large number of pharmacological effects of extracellularly administered adenine nucleotides which have been listed in Table I may have some physiological significance. It thus becomes of prime importance and one of the objectives of this thesis to re-examine the possibility of outflux of intra- cellular adenine nucleotides. The studies described in this literature review indicate a need for further investigation of the role of adenine com- pounds in the body. It should be determined whether or not they exist in normal arterial plasma, affect respiration under physiological conditions, and/or mediate hyperemia. The studies presented in the Results of this thesis are a prelimi— nary attempt to answer and formulate approaches to these questions by investigating adenine compound metabolism in blood and during passage through the lungs. METHODS 1. Breakdown of 14C-ATP in Plasma Male and female mongrel dogs of both sexes were obtained from C.L.A.R. and anesthetized with 25 mg sodium pentobarti— tal per kg body weight intravenously. The femoral artery was cannulated with polyethylene tubing; and approximately 10 ml of arterial blood was collected into a Siliconized tube which contained 100 units of heparin. The blood was immediately centrifuged twice in a Sorvall centrifuge at 10,000 x G for 10 minutes each time. Two 2 m1 platelet-free plasma samples were then pipetted into Siliconized test tubes. A 50 A sample of 14C-ATP (1 uCi; 1150 ng ATP) was added to each. The plasma was then incubated in a constant temperature water bath at 37.5° C for a selected incubation time. One-half'ml of carrier nucleotides (60 mg ATP, 25 mg ADP, 20 mg AMP and 5 mg adenosine) were added to each sample in order to be able to determine subsequently by optical density measurements the Specific fraction collector sample tubes containing each nucleotide. The plasma, with carrier nucleotides added, was then poured directly onto a column of Dowex 1 resin, .9 cm in diameter and packed to 6.2 cm, and a gradient elution with ammonium formate immediately commenced. 84 85 2. Breakdown of l4C-ATP in Whole Blood Approximately 10 ml of arterial blood was collected into a Siliconized tube containing 100 units of heparin. After gently swirling the blood, two 2 ml samples were pipetted with Siliconized pipettes into Siliconized test tubes and allowed to equilibrate in a 37.5° C constant temperature water bath for one to two minutes. A 50 A sample of l4C-ATP (l nC; 1150 ng ATP) was then added to each and the mixture incubated for an accurately measured time. Seven ml of 6% perchloric acid was added to each sample at the end of the incubation period to denature proteins and stOp ATP breakdown. One-half ml of carrier nucleotides was added to each sample. The samples were then centrifuged at 10,000 x G for 10 minutes at -3° C for denatured protein removal. The supernatants were decanted into test tubes and placed in an ice bath. Perchloric acid was removed by titration with 5 M potassium carbonate, using methyl orange as the end point indicator. The protein-free supernatant was then poured directly onto a column of Dowex l anion exchange resin for nucleotide separa- tion by gradient elution ion exchange chromatography. Hematocrit and pH were determined on the excess blood which was carried through identical incubation procedures. 3. Separation of Adenine Nucleotides by Ion Exchange Chromatography» A gradient elution ion exchange chromatographic apparatus similar to that described previously by Busch et a1. (1952) 86 for organic acids was used to separate the adenine nucleo— tides. Similar procedures for separating nucleotides have been described by Egawa and Neuman (1964), Cohn and Carter (1950), Cohn and Bollum (1961), Cohn (1950), Hurlbert et al. (1954), Blattner and Erickson (1967) and Burger and Lowen— stein (1967). Problems related to reaction of ATP with the anion exchange resin utilized in these procedures have been described by Lund et al. (1954); however, to the best of our knowledge such chemical alteration of ATP did not occur in our procedures since both carrier and l4C-labeled ATP were completely recoverable from the resin columns. The procedures modified from those reported above which were used in the studies described in this thesis were as follows. A 500 ml reservoir bottle approximately 15 feet from the floor was filled with a l M ammonium formate solu- tion. Tubing which could be clamped connected the reservoir bottle to a mixing chamber containing 500 ml of distilled water and a magnetic stirrer. The mixing chamber was approxi- mately 5 feet from the floor and was connected by tubing to a chromatography column of .9 cm inner diameter packed with 6.2 cm of Dowex l anion exchange resin in the formate form. This tubing could also be clamped when perfusion of the resin column had to be interrupted. As the ammonium formate from the reservoir bottle was gradually added to the mixing chamber, an increasing concentration of ammonium formate was gradually 87 delivered to the resin column. As the slowly increasing ammonium formate solution perfused through the resin column, the adenine nucleotides which had been previously added to the column were sequentially eluted. The resin column was positioned over an automatic fraction collector which con- tained 90 or more tubes. The solution flowed from the column into a volumetric siphon which emptied approximately 10 m1 aliquots into each test tube of the fraction collector. Adenosine and other nucleosides, which bind very weakly (if at all) to the resin, came off first, followed in order by AMP, ADP and finally ATP. The reservoir, initially filled with 1 M ammonium formate, was refilled with 500 ml of 1.75 M ammonium formate after fraction collector sample #50 in order to elute the ATP. The apparatus used for this chroma- tographic separation is shown in Figure 2 and the pattern of nucleotide elution is shown in the upper diagram of Figure 3. In certain experiments an elution with distilled water for the first 30 fraction collector tubes before adding 1 M ammonium formate was used to more completely separate the nucleosides from nucleotides. Since it was observed that carrier IMP and AMP came off the resin in the same fraction collector samples with the above procedures, a gradient elution procedure utilizing formic acid was modified from that of Hurlbert et al. (1954) to separate AMP from IMP. The details of the elution pattern 88 Figure 2. Gradient elution ion exchange column chromatography apparatus. 89 ELUANT RESERVOIR —- (500 m1.) [=1 bi!) 2.3m -— E LUANT MIXING CHAMBE R L c: (5004);!) -———MAGNETIC MIXER -—-6.2 CM RESIN COLUMN 6 TO VOLUME CONTROLLED FRACTION COLLECTOR Figure 2 9O .cEsHoo demos was 0p poops mmz ASH com mad suoo ocesflmpsoo coeusaom Hmfluumo 8 mo HE m.o sm£3 mEH some mad SufismCHume on com: soaumummom mo moms may msocm musmflm HmBOH one .58 com um mmHmEMm was no msflommu muflmsmo Hmoeumo so an conflmuno mo3 pom cowum>nmmno moo mucmmmnmmu ucfiom comm .N mnsmflm ca QBOSm we poms msumummmm one .wsHHMm mo HE N CH sEDHoo was on cover was mossomsoo on» OCHGHMPGOO coflusaom M NO HE “amalmco .msHmocmpm cam .mzd .mQ¢ .mefi How sumppmm mommnmoumaonso mmcmsoxm now coepsHm usmflomnw .m mnsmflm 91 m unseen cumin: m4a34w om. o¢_ o~_ 00. ca om 0¢ ou _ l ._ _ t. _ _ _. .. 0 go... Hf... 0 o 3' o ao< a:. m r¢ . . N . . m ae< To az< rm; whine“. 2:.zozz< :2... + 93 0.3.9. 2.. I'm—0.: 8. Arc (L Li _ e1. _ 1 _ p .: . ao< . I¢. .. azIa<¢00h<20mxo moreowqoaz 92 for this procedure are described in Figure 3 (lower diagram) and Section 7 of Methods. 4. 14C-ATP Purity Check A purity check was done on the l4c-ATP by adding a 50 A sample of the isotope in 2 m1 of saline with 0.5 m1 of carrier nucleotides to the resin column described above and chroma- tographically separating the ATP. One ml of each fraction collector sample was plated on a 1 cm aluminum planchet and dried to infinite thinness. Each planchet was then counted in a Baird Atomic counter for one minute to determine 14C-activity. Identification of 14C-labeled adenine compounds present was inferred by comparison of 14C-labeled peaks with carrier nucleotide peaks determined by optical density read? ings. The results of a purity check on the l4C-ATP used in the present studies is shown in Figure 4. Ninety-three per cent of the total 14C activity in the sample was l4C-ATP. ADP, AMP, and nucleosides each accounted for approximately 2% of the total 14C activity added to the resin column. 5. 14C-Adenosine Purity Check one uCi of 14C-adenosine in 1 m1 of saline with 0.5 ml of carrier nucleotides was chromatographically separated as described above. Identification of adenine compounds present was inferred by comparison of l4C-labeled peaks with carrier 93 . l4 . . Figure 4. C-ATP and l4C-adenOSine purity checks. l4C-ATP (l uCi) or l4C-adenosine (l pCi) were added to Dowex 1 ion exchange resin and eluted with l M ammonium formate to sample #50; 1.75 M ammonium formate to sample #90. One ml of each sample was then plated, dried, and counted. The separation pattern of the adenosine com- pounds for this chromatography procedure is shown in the upper diagram of Figure 3. 94 3000- 2000- AT P 1000- o...“ ."°ou.'""”“”:.'o.-.oocanon“. 0"°°"'~'...'. "b"- 30004 2000- . A D E N O S l N E 10001 ' “'0'”... 0000'..0o°u‘0..0'0.'oo'0 .00..."o‘~°.°""°'°"°o"°"°~'° '0""’°° T I I l 20 4O 60 80 FRACTION COLLECTOR SAMPLE NUMBER Figure 4 95 nucleotide peaks. The results of one l4C-adenosine purity check are presented in Figure 4. Since adenosine, inosine, and hypoxanthine were all eluted with distilled water in the first fraction collector samples, it was not possible to differentiate individual nucleosides with the separation procedure used. Calculation of nucleoside 14C activity as per cent of total showed that 95% of total 14C activity was clearly nucleosides. The remaining 5% of the counts was distributed in the areas of AMP, ADP and ATP. However, the 14C activity in these nucleo- tides may actually have been (hue to a "tailing off" of the large amount of l4C-adenosine added to the column, as no definitive 14C-labeled peaks were identifiable in the areas containing carrier AMP, ADP and ATP. 6. Effect of pH on 14C-ATP Breakdown 'in WhoIe Blood The effect of pH on l4CuATP breakdown in whole blood was studied by continuously gassing blood samples with gas mixtures containing varied amounts of carbon dioxide (5, 10, 20 or 30% carbon dioxide in air). _The gas mixture was saturated with water by bubbling through distilled water to prevent drying of the blood. To compare 14C-ATP breakdown in high versus low pH whole blood, in each experiment one 2 ml whole blood sample was 96 continuously gassed with C02, while a second 2 ml sample from the same animal was exposed continuously to room air. Two other blood samples from the same animal were simultaneously treated in the same manner for pH determination. For quanti- tative determination of l4C-ATP degradation the samples were treated as previously described in the section on breakdown of l4C-ATP in whole blood. 7. IMP Separation The chromatographic separation previously described was not able to separate individual nucleosides or AMP from IMP. Since it is controversial as to whether AMP is dephosphory— 1ated or deaminated in blood, a separation similar to that of Hurlbert et al. (1954) was utilized to separate AMP and IMP and is shown in the lower portion of Figure 3. Dowex 1 anion exchange resin in the formate form was packed in a 2 cm inner diameter column to a height of 12.1 cm. The elution solution reservoir was filled with 500 m1 of 4 N formic acid. One-half m1 of a carrier nucleotide solu- tion containing ATP, ADP, AMP, IMP and adenosine was added to a 2 ml blood sample gassed with 5% C02, balance air, and incubated for 1 1/2 minutes with a 50 A sample of l4C-ATP. .After treatment with perchloric acid and titration with potassium carbonate, the sample was added to the column for chromatographic separation. At approximately tube 90 the 97 elution solution was changed to 1.0 M ammonium formate to elute ADP. The elution solution was changed to 1.75 M ammonium formate at approximately tube 120 to elute ATP. Optical density readings were measured at 260 mu. Peak read— ings were re-read at 248 mu, the wavelength of maximum molar absorbancy of IMP, to identify the IMP peak. The results of a separation of carrier nucleotides using this system are shown in the lower diagram of Figure 3. 8. EDTA Studies The effect of EDTA on the formed elements of blood was studied to determine if EDTA could cause release of intra- cellular ATP. A 5 ml sample of blood was incubated with 50 HCi of l4C-adenosizne (0.24 mg) 13,1 m1 of saline for 20 minutes at 37.5° C with gentle swirling. The tube was Siliconized and sealed with parafilm to minimize drying and hemolysis. Three-fourths m1 of a 3% EDTA solution (experi- mental) or 0.75 ml of isotonic saline (control) was then added to the blood and gently mixed. To allow comparison to the studies of Chen et a1. (1972), the sample was then imme- diately placed in ice and incubated for an additional 5 minutes. The blood was then centrifuged at 42,000 x G for 10 minutes at -3°C. Plasma was carefully removed with a Pasteur pipette and recentrifuged at 42,000 x G for 10 minutes. The isolated formed elements were immediately treated with 98 7 ml of perchloric acid. The supernatant plasma was removed with a Pasteur pipette after the second centrifugation and 7 m1 of perchloric acid was then added to this supernatant. One-half ml of carrier nucleotides were added to both the perchloric acid-treated plasma and to the perchloric acid— treated formed elements. Both samples were then centrifuged at 10,000 x G for 10 minutes at -3°C to remove denatured proteins. Supernatants were decanted, titrated with 5 M potassium carbonate and immediately added to columns for nucleotide separation by ion exchange chromatography. To determine if pH caused the release of adenine nucleo- tides from the formed elements of the blood, a 5 ml whole blood sample treated in the same manner as described above, except for omitting the addition of saline or EDTA, was gassed with 5% carbon dioxide in air during the incubation period. 9. In Vivo Studies Four mongrel dogs of both sexes weighing between 7 and 14 kg (mean = 10.5 kg) were obtained from C.L.A.R. and anesthetized initially with 25 mg sodium pentobarbital per kg body weight intravenously. A sustaining dosage of 6 mg/kg body weight was administered intravenously when a strong corneal reflex was observed. 99 (a) Plasma 14C-ATP uptake and breakdown by the lungs The external jugular vein was cannulated proximally with polyethylene tubing (PE 160) for injection into the cir- culation to the lungs. The tip of the cannula was threaded to the right atrium. The left carotid artery was cannu- lated with PE 320 proximally with the cannula tip inserted to the aortic arch for collection of blood from the lungs. An injection solution was prepared by mixing 1 ml l4C-ATP (20 uCi; 23 ug ATP) and 2 m1 of an isotonic sucrose solution. The sucrose was used as an indicator of dilution of the in- jection solution in the extracellular fluid volume (Mulrow et al., 1956). Seven-hundredths ml of the injection solution was added to 5 ml of distilled water for sucrose and 14C analysis. The remaining 2.9 ml of the injection solution was injected via the external jugular cannula into the right atrium. Three seconds after injection, blood was collected from the aortic arch directly into 25 ml of 6% perchloric acid in a 50 m1 graduated cylinder; collection was for approxi- mately 5 seconds, until the 50 m1 mark of the graduated cylinder was reached. One-half ml of carrier nucleotides were added to the sample, and then it was mixed and centrifuged at 10,000 x G for 10 minutes at -3°C. The protein-free fil- trate was decanted and titrated with 5 M potassium carbonate. The protein-free supernatant was then added to a resin column for nucleotide separation. A small (1 ml) sample of the pro- tein free filtrate was used for l4C-recovery and sucrose 100 determination. Injection solution and arterial whole blood sucrose analyses were performed simultaneously using the method of Walser et al. (1955). Per cent l4C by-passing the lungs was calculated by dividing the per cent of the injected l4C recovered in the blood collected by the per cent sucrose recovered in the blood collected, times 100. Subtraction of the per cent l4C by-passing the lungs from 100 gave the estimated per cent l4C taken up by the lungs. (b) The effect of ATP injection and continuous infu- ‘Eion on systemic arterial blood pressure Arterial blood pressure was measured from a PE 90 cannula in the femoral artery using a pressure transducer and a Grass polygraph. The injection and infusion sites for ATP were the aortic arch, femoral vein and right atrium, which were cannulated with PE tubing inserted into the dog via the carotid artery, lateral saphenous vein and external jugular vein, respectively. The solution for single injection and continuous infusion of ATP was prepared by adding 100 mg of ATP to 100 m1 of iso- tonic saline. This solution was injected in quantities rang- ing from 0.1 ml to 2 ml and was infused at rates ranging from 1 ml/min. to 8 ml/min., depending on the amount required to give an easily recordable change in arterial blood pressure. 101 10. Reagents 1. l4C-ATP.---Uniformly labeled l4C—ATP was obtained from New England Nuclear (Lot #651-162; 0.345 mg ATP/15 ml 50% ethanol; specific activity 520.9 mCi/mM; and Lot #767-052; 0.43 mg ATP/15 ml 50% ethanol; specific activity 405.4 mCi/mM). The solution was shipped in a dry ice container and stored as suggested by New England Nuclear at -15°C. Purity checks were made on each lot of l4C-ATP; however, the specific activities were not checked in our laboratory. 2. l4C-Adenosine.--Adenosine-8-14C was obtained from Cal Atomic (Lot #000-981; 50 uCi; specific activity 53.5 mCi/mM). The shipment of l4C-adenosine arrived as a dried powder and was maintained as such at -15°C until use. Just prior to use 1 ml of isotonic saline was added to a 50 uCi sample (one vial) for convenience when adding the isotope to the blood. 3. Carrier Nucleotides.--Adenosine and Ba-ADP were obtained from Sigma Chemical Company. ATP and AMP were ob- tained from Nutritional Biochemical Corporation. All were stored at -15°C. A solution of carrier nucleotides for determination of ion exchange chromatographic peak location was prepared in the following way. Five mg adenosine, 20 mg AMP, and 60 mg ATP were mixed with 4 ml distilled water. The solution was heated slightly with hot tap water to dissolve the compounds completely. Twenty-five mg of Ba-ADP were mixed with 4 ml of 0.2 M sodium sulfate. The solution was then centrifuged for 7 minutes. The supernatant was decanted 102 and added to the nucleotide solution previously prepared, thus producing a solution containing 0.625 mg/ml adenosine, 2.5 mg/ml AMP, 3.1 mg/ml ADP, and 7.5 mg/ml ATP. One—half m1 of this solution was added to each sample that was to be fractionated by ion exchange chromatography. In experiments in which IMP was studied, 35 mg of IMP were dissolved in the solution described above. The IMP was obtained from Sigma Chemical Company. 4. Heparin.--Fifty mg of sodium heparin obtained from Nutritional Biochemical Corporation were added to 1 ml of distilled water. A 20 A pipette of this solution was added to the blood collection and dried before each experiment. A 1% solution of heparin in saline was prepared. Four ml of this solution were added to 500 ml of saline for preventing coagulation in the polyethylene cannulas used in the in vivo experiments. 5. Ammonium Formate.--A 1.75 M ammonium formate elution solution was prepared by adding 18 liters of distilled water to 1980 g of reagent grade ammonium formate obtained from Matheson, Coleman and Bell Company. Reagent grade formic acid obtained from Mallinckrodt Company was added to this solution until pH 5 was reached. The pH was measured on a Beckman Expandomatic pH meter. One M ammonium formate elution solution was prepared by adding 857 m1 of distilled water to 1143 m1 of 1.75 M ammon- ium formate. 103 6. 6% Perchloric Acid.--A 6% perchloric acid solution was prepared by adding 7.7 m1 of a reagent grade 70% per- chloric acid solution obtained from Mallinckrodt Company to a 150 ml graduated cylinder and diluting to 150 ml with distilled water. 7. 2% Sodium citrate.--A solution of sodium citrate was prepared by adding 3.16 g of sodium citrate to a volumetric flask and filling to the 100 ml level with distilled water. The osmolality of the resulting solution was measured on an Ckmmette osmometer and was approximately 289 mOsm. A 0.22 ml sannple of this solution was added to 2 ml of whole blood in order to determine the effect of citrate upon 14C-ATP break- down. 8. 3% EDTA.--A 10% EDTA solution was prepared by adding 100 ml of distilled water to 10 g of diNa-EDTA. A 33.3 ml sample of the 10% solution was added to a 100 ml graduated cylinder and diluted to the 100 ml level with isotonic saline. Three-fourths ml of the 3% solution was added to 5 ml of whole blood for determining the effect of EDTA on 14C-ATP breakdown in whole blood. 9. Sucrose.--A 300 mosm/l isotonic sucrose solution was prepared by adding 1.03 g of sucrose to 10 m1 of distilled water. Approximately 2 ml of this solution along with 1 ml of l4C-ATP were injected into the blood going to the lungs. 104 10. 4 N Formic acid.--A 4 N formic acid elution solution for gradient elution ion exchange chromatography was prepared by diluting 209 g of 88% formic acid to 1 liter with dis- tilled water. RESULTS 1. Breakdown of 14C-ATP in Plasma l4C-ATP was added to heparinized fresh dog platelet- free plasma and incubated at 37°C for accurately measured intervals of time. The breakdown of l4C-ATP was determined by measuring the plasma l4C activity in nucleosides, AMP, ADP and ATP after the separation of these compounds by ion exchange chromatography (Figure 5). The per cent of the total 14C activity in each of the individual adenine compound peaks was calculated from the raw data shown in Figure 5, and these per cents of total activity plotted against time . of incubation are shown in Figure 6. It is evident from both of these figures that l4C-ATP is rapidly broken down in dog plasma, and the metabolic degradation appears to proceed via ADP, AMP and nucleosides. It is also evident that the l4C-ATP is completely destroyed after twenty minutes of incu- bation; and no evidence of reformation of 14C-ATP was observed as was the case with whole blood described in the following Section 2. The halftime for l4C-ATP breakdown in plasma was estimated to be approXimately three minutes. Since the breakdown of 14C-ATP was difficult to measure accurately in incubation periods of less than one minute, this halftime can only be an estimate and may indeed consist of reactions having 105 106 Figure 5. l4 C-ATP breakdown in isolated plasma incubated in vitro. One uCi of l4C-ATP was added to 2 m1 of plasma and incubated for accurately measured durations of time at 37°C. The plasma pH was approximately 7.8 in each experi— ment. One ml of each fraction collector sample was plated, dried and counted for one minute. 14C-activity peaks were identified by comparison with carrier nucleotide peaks. Each point represents one observation. 107 "c—ATP BREAKDOWN IN PLASMA 3 ATP I MINUTE 1% S 6 3 AM? a. AOP , .. Junk”: I“, T F T If 1 ‘ . 2 MINUTES ~ ."--......-"'”‘~...,... w- 4 _T r T 7" ' . 4 MINUTES ' 8 MINUTES UJ ‘ o +— I D 0 g '4 2 r. .. .‘ .‘ 3 a: ..".'L".. #fii‘hd} _ 2%-? E 4 ~ ,, :2 MINUTES m '— 2 i 3 .° 0 O O -1 ° ISBMNUTES J O .b .' ”00‘ 25 MINUTES .4 O 2000-4 . I IOO SAMPLE NUMBER Figure 5 108 .cEsHoo gammy was Eoum omusHo macsoo Hmuou was we moss xmom Hmflunmo powMHommm was ca .cHE\mussoo Hmuou unflofl>flo mo pmGHmuoo was com coeum>nomoo moo mucmmonmmu usflom comm .mnsmem wooe>oum mop CH mucmefiummxw was Eoum om>fiumo we whomem were .OHHH> cH UUPMQDOCH mammHm owPMHOmH CH medlUva mo GBOUMDMHm .w musmflh 109 w mnsmflm mme32=2 0N ON 0. o. 0 ,_. _ _ _ _ u/u o\\\\\\o\.\\|\\\I\I mmo_mow.._032 <2m<4a z_ a._.>OoxHp an owsHmuoo mum3 moon> .muscHE woo How poucsoo pom .UOHHU .UOPMHO mmB OHQEMM HOPOOHHOO coHuomum sumo mo HE moo .omsmmnmoumEosno can OONHGHOPOHQOO SHOOMHOOEEH mm3 OOOHQ was soHumosocH Hmumm .m.h Op m.e Eoum cwmsmu mm one .oEHu mo mHm>HoucH OossmmmE mHmumssoom MOM erm um OOPMOSOGH pom OOOHQ mHoss ou cocoa was mB¢IUvH HO HO: moo .OHuH> EH cosmosocH UOOHQ OHO£3 EH Esopxmmuo mBEIUVH .5 musmHm 112 e OHOmHm mmeazi ON m. N— e _ e QOOJm wI_OI>> 2_ *——0 .128... to zzooxfiimm 10. 1ON 1.00 10b 100 Pom foo. W/Q ‘IVlOJ. °/. 113 lack of pH control. These experiments were performed with the bloods incubated in air; thus, with different durations of preincubation different CO2 losses from the blood would occur; hence, the pH varied between 7.5 and 7.7. The hemato- crits of the bloods used in the experiments shown in Figure 7 were also measured; however, no correlation between l4C-ATP breakdown rate and hematocrit was observed. Table 2 shows the breakdown of 14C-ATP and resulting products in whole blood expressed as per cent of the total 4C activity remaining at selected incubation times. l4C-ATP underwent rapid breakdown and then resynthesis. 14C-ADP levels increase slightly and then decline. This observation supports data obtained by Mills (1966), showing that 80 per cent of ATP dephosphorylation in isolated plasma occurs by the direct splitting off of the two terminal phosphates. Also this observation in conjunction with results to be described later (Table 9) seems to indicate a relatively small intracellular ADP pool. 14C-AMP, after an initial increase, is rapidly converted to nucleosides, probably adenosine. 14C-nucleoside levels seem to gradually increase initially and then decline after 15 minutes due to uptake into cells and subsequent resynthesis into l4C—ATP. It should be noted that the values cited in Table 2 are means. A large variation was noted in the individual observa- tions at the specified times. An example of the degree of 114 Table 2.--14C-ATP Breakdown at Selected Incubation Times in Whole Blood at 37°C Per cent of Initial C/M 1 1/2 4 15 22 minutes minutes minutes minutes ATP 42 20 41 50 ADP 15 20 6 7 AMP 41 26 ll 10 Nucleosides 2 32 41 32 115 variation at One time of incubation is shown in Table 3. Thus, the data can only indicate qualitative trends in the formation and breakdown of the l4C-adenosine compounds and acceptance of these data as quantitative would be risky. An experiment was done to determine whether l4C-ATP resynthesis was occurring in the cells, in the plasma, or both. 14C-ATP was incubated in whole blood at 37°C for twenty minutes. The blood was then centrifuged at 0°C at 10,000 X G for ten minutes. Plasma was decanted and 7 ml of perchloric acid was added to the separated formed elements and plasma, and the mixtures were then centrifuged at 10,000 X G for ten minutes to remove coagulated protein. The protein-free supernatants were decanted. Both supernatants were then poured onto columns of Dowex 1 resin for nucleotide separation. Figure 8 shows that l4C-ATP was found only in the cells. Although only a single experiment was done in this manner to establish the intracellular reformation of 14C-ATP and lack of reformation of 14C-ATP in the plasma, a series of experiments with l4C-adenosine confirmed this find- ing. These latter experiments are described in Section 8 of these results. Two experiments were done using an elution procedure which would distinguish AMP from IMP. Figure 9 shows the data from one of these two similar experiments and indicates that virtually no IMP is formed during l4C-ATP breakdown in gassed whole blood after 1 1/2 minutes. 14 Table 3.--Breakdown of 116 C-ATP in Whole Blood at 37°C in 1 1/2 minutes with Uncontrolled pH Per cent of Initial C/M Exp. pH ATP ADP AMP Nucleosides E 1 7.80 30 24 44 2 it 2 7.75 38 12 49 l 3 7.60 63 16 19 2 4 7.60 32 12 55 l 5 7.40 34 11 53 2 6 7.80 33 26 39 2 7 7.60 68 ll 17 4 8 7.60 58 10 31 1 9 7.60 22 ll 65 2 Mean i>SE 42 + 5.5 14.8 i 2.0 41.3 i 5.5_ 1.9 i 0.3 117 Figure 8. Distribution of 14C activity in whole blood affln 20 minutes of in vitro incubation with l4C-ATP. One mCicfi l4C-ATP was added to 2 ml of whole blood and incubated at 37°C for 20 minutes. The pH was 7.6. The blood was then immediately centrifuged at -3°C to separate plasma and ceth Each fraction was then treated with perchloric acid, neutraL- ized and chromatographically separated. One ml of each fraction collector sample was plated, dried and counted for one minute. Each point represents one observation. The pattern of nucleotide elution is the same as the upper curve in Figure 3. SOOCh 20004 [($00- "c-ATP 2000- I000- 20 . ' .-’~..... MM ‘ '00:, o. I 118 INCUBATED IN WHOLE BLOOD 20 MINUTES PLASMA 00...... . “Mwmip T 40 SAM PLE Figure 8 119 .soHum>HomQO moo mucmm Imsmmu ucHom room .mzH Eoum Ase mumummmm Op m OHOmHm mo EMHmMHO Hoon was CH UOQHHOmOU mm mHHMOHSQMHmOumEOHSO ooumummmm mums mmUHpOOHOOC one .vm.e was me one .HHD EH Nov mm SOHB common mm3 UOOHQ may use» umooxm .5 musmHm CH OOQHHOmmO mm OOHpsum mm3 UOOHQ OHO£3 :H ssooxmouo medlovH .OHuH> CH o.sm um mauscss N\H H as eooHn mHoez as ABEIOSH mo czoexmmum .m musmsm 120 Om. P Om. _ m musmHm mmmZDz m4a2 An N «N mH mm mv.h Am N mH Nm mH vH om.h Ab HH mm OH 0N Hm.n Am H mmonOOHOsz mad EOE med mm pcoEHHOmxm E\U HMHUHGH mo usmo Hum mmuscHz N\H H CH OOOHm OHOQB EH QSOUxmonm mefllo SH so an MC DOOMMMII.m OHQMB 132 N 0 mm mm [\m \DO‘ fi'l‘ M'd‘ V‘kD NM MQ‘ Or-l NN LOB mm MN (0&0 mm ON m¢ mv mm mN ON 5H mm mN ON HH mN 5N ow mv «N mH vm mm mm ow mm mm mm om om NN mN hN NN mH HN mH NH mm mm mm mm mm Nm mm mm vm om mH vH mN mH VH mN NN MN mm mm m¢ mm Nm mv om mm Ho mm vv vm mm mm mm mm mm mm mv wv om mm mm vm Hv NM NM NH HN mn.> vv.h m5.h mv.h Nm.h mN.h mh.h m¢.h om.h mv.h Hw.b mb.m mm.b mm.w wo.w vm.w om.h 00.5 mw.h ma.h mm.b hH.b 3.5 NN.> mw.h mN.h nm.n mN.n Am mN E «N E mN Cm NN Am HN Am 0N Am mH Am mH in NH Am mH Am 3. Am VH 133 Figure 10. Effect of pH on 14C-ATP breakdown in whole blood incubated in vitro at 37°C for l l/2 minutes. One uCi of l4C-ATP was added to 2 ml of whole blood which was continu- ously gassed with 5, 10, 20 or 30% C02. A second 2 ml blood sample from the same dog was incubated with l uCi of 14C-ATP in an unstoppered tube. pH of the gassed samples ranged from 6.78 to 7.47; pH of the non-gassed samples ranged from 7.50 to 7.79. At the end of the incubation each sample was treated with perchloric acid, neutralized, and chroma- tographically separated. One ml of each sample was plated, dried and counted for 1 minute. Each point in the upper diagram represents one observation and was obtained by dividing the total counts in the l4C-ATP peak by the total counts eluted from the resin column. An x indicates an experiment which may not have been valid since unusually high l4C-ATP levels were found in the non-gassed as well as the gassed sample. Each point in the lower diagram was obtained by plotting the difference in the per cent 14C-ATP against the difference in pH between paired gassed and non- gassed samples for the same experiment shown in the upper diagram. Each point represents one observation. EFFECT or PH 0» "c-ATP BREAKDOWN 134 IN WHOLE BLOOD 18°— '2 2: -60- o x 0 xx 0 2%; . . .’ . ‘. . o (40- . . ... E - - °'.- . . a! o .’ o :5:Z()“ . o. 8' I l T I l l 6.8 7.0 7.2 7.4 7.6 7.8 P" 30" '0 0' 0 i520“ g 0 <1 ' . IO— . : o . o no . o . . I I 1 P l I l .2 .4 .6 .8 I L2 APH Figure 10 135 Figure ll. Effect of pH on 14c-ADP, l4C-AMP, and 14C- nucleoside formation in whole blood incubated in vitro with l4C-ATP at 37°C for 1 1/2 minutes. The data in these figures was obtained from the same experiments described in Figure 10. The per cent of total counts/min. for each adenine compound was calculated in the same manner as ATP in Figure 10. Each point represents one observation. 136 EFFECT OF PH ON ADP,AMP AND NUCLEOSIDE FORMATION FROM "c-ATF IN WHOLE BLOOD 40— ADP x . . C O . O C .0. ..C .5 . O «O 2K3-‘ .. O ' OfizOe: . "Ib; . W t I T T I 1 1 "J. z 6 O _J . AMP 2>\ ..O. O -. . . . . <( O O l- ‘ND—‘ O B O . . g .. .. u. . ‘D ..O.. O . (3 2K}—‘ . 0‘; xx 39 x I I I I l I NUCLEOSIDES <3" O O "x .0 0. g x ‘4-4 O . OO . O O O O OO OO O O O OO O O I I I I I I 6.3 7.0 7.2 7.4 7.6 7.8 WHOLE BLOOD P H Figure 11 137 Tables 3 and 4, 14C-AMP levels appear to vary directly with pH; however, in this series of experiments, unlike those in Tables 3 and 4, pH does not appear to affect l4C-ADP or l4C-nucleoside levels. 8. Effect Of EDTA on the Formed Elements F“ of Blood ‘ Studies were done to determine if EDTA can cause release I] of ATP from the formed elements of blood. Intracellular ATP 14C by adding 50 uCi of l4C-adenosine to was labeled with 5 ml whole blood and incubating for 20 minutes; 0.75 ml of EDTA solution or isotonic saline was then added to the labeled blood. There is no consistent difference in plasma l4C-ATP levels between bloods treated with EDTA or isotonic saline in Table 9. In one experiment the blood was gassed with 5% CO2 and neither saline nor EDTA was added; the plasma 14C-ATP level again did not significantly differ from either saline- or EDTA-treated bloods. Due to the quantitative Isimilarity of the 14C activity in the ATP area to background counting levels and the chromatographic tailing effect of the large amount (Hi l4C-adenosine added, it is probable that this method would not be able to detect the very small dif- l4 ferences in plasma C-ATP levels which we would suspect to be due to EDTA. In one of the EDTA experiments, however, a 14 . . C peak was clearly defined in the ATP area of the chromato- gram. Such a well-defined plasma 14C-ATP peak did not occur 138 .©O>meno on pHsoo xmmm OHHGHMOU os #53 .moum xmom HOHHHOO may CH ucmmmum muo3 mpcooo A«v .mucsoo OGHmoampm mo mOHHm>o 0p Odo OHQOHMHucmsw you won ucommum Mama ouHaHmwfi MW mOHOOHOGH A+V .UO>HOmQO xmmm O>HpHchmU oz moumOHch HIV oooumom oooummH nu: oom.m oom.mH ooe.NNH mHHmo ooo MHm ooo mam «Hem oom.Hm mmm oov.H mammHm oooHomN oomumw In: ooo.o ooe.mm ooo.meH mHHOO coo Nam ooo com In: oom.mH oo~.m oom.H mammHm oooUNem oooHHHN In: ooo.m ooo.om ooo.mNH mHHmo ooo mam 000 com II: + FOOH.N oo~.H mamMHm occumom ooounHH nu: oom.m ooe.eH ooo.mmH mHHmo ooo mew 000 was In: : *mmm mmm mammHm emummue meow oooueem oooueem .oom ooo.vH ooo.NN ooo.mo mHHmo ooo mmw ooo mam «Han ooe.mm OOH.H com mammHm Houuaou Ummmmo oooummm oooHemH nu: oom.OH ooe.mm ooo.eHH mHHmo ooo Hem ooo «mm In: oom.~H oom.~. OOH.H mammHm ooo.mmm ooo.mmH nun oom.NH oom.mm oom.NmH mHHmo ooo.em¢ ooo.mHv an- + .ooo.m oom.H mammHm Honucoo OGHHmm Hmuoe mmonomHosz mzH axe maH me< pamEHummxm OHSGHZ mom muasoo mHm>mq nae mammHm co 49am mo pommmm Osauu.m mHnma 139 in any of the other experiments, although counts above back— ground were present in the ATP area in all experiments. The per cent of intracellular ATP l4C activity found in the plasma was 0.76 in the gassed control, a mean of 0.92 in the saline controls, and a mean of 0.85 in the EDTA-treated samples. Only 0.1 per cent of the intracellular ATP, if . found in the plasma, would account for the plasma ATP levels found by Chen et al. (1972) and Forrester (1969). Therefore, the normal existence of ATP in arterial plasma cannot be ruled out by these studies. Likewise, this group of experi— ments was not able to determine if the 14C activity in the chromatographic ATP area of the EDTA-treated blood was due to an effect of EDTA on the membrane causing outflux of intra- cellular 14C-ATP. In these studies considerable 14C-AMP in arterial plasma was observed in six of the seven experiments performed. In the one experiment which did not show l4C—AMP in the plasma, intracellular l4C-AMP levels were also very low. The large plasma 14C-AMP levels apparently indicate AMP formation from adenosine kinase on the surface of the formed elements rather than transmembrane outflux of intracellular 14C-AMP, as intra— cellular l4C-AMP levels were considerably lower than plasma 14C-AMP levels. This finding also supports the relative stability of AMP compared to ATP in plasma noted in Results, Section 1 (Figure 5), as considerable plasma l4C-AMP remained even after 20 minutes of centrifugation° 140 In two experiments a chromatographic procedure was used which separated AMP from IMP. Table 9 shows that virtually no l4C-IMP is formed from l4C-adenosine during this incuba- tion time, either intra- or extracellularly. Although l4C activity is present in the IMP area, it is probably due to HA an adenosine tailing effect, since no definable l4C-IMP peak i was observed. Another fact evident in Table 9 is that plasma 14C- “ nucleoside levels are in every case considerably higher than those observed intracellularly, suggesting that over the time course of these experiments 14C-adenosine had not equilibrated across the cell membrane. 9. In Vivo Studies on l4C-ATP Breakdown in the Pulmonary Circuit Folkow (1949) observed that intravenously injected ATP appeared to be rapidly destroyed (inactivated) in the pul- monary circuit. Gordon (1961) obtained results which also indirectly indicated a trapping or rapid breakdown of ATP in the lungs. Figure 12 shows the results of one of four similar experiments in which l4C-ATP and sucrose were injected into the thoracic vena cava and outflow was collected from the aortic arch approximately 5 seconds later. It is apparent that very little l4C-ATP passed out of the lungs and through the left side of the heart. By comparison with sucrose recovery in the aortic arch blood, 83% i’3% (mean : SE, N = 4) 141 Figure 12. l4C-ATP breakdown during one passage through the vasculature of the lungs. Twenty uCi of l4C-ATP in 2 ml of isotonic sucrose were injected into the right atrium. Blood was collected 3 seconds later from the aortic arch into perchloric acid. After neutralization and chroma- tographic separation, 1 ml of each fraction collector sample was plated, dried and counted for one minute. The upper diagram shows a l4C-ATP purity check, performed as described in Figure 4. The lower diagram shows the 14C activity 14 profile after passage of C-ATP through the lungs. Each point represents one observation. 142 "c-ATP BREAKDOWN BY LUNGS 3 4000 9 8 AMP AOP ATP “J 'o 3000— g _ _ g PURITY CHECK EOOO-H . . I IOOO-I ' C/ 0.. 3"". M (unmanuamoiuo. cm. a... “0]" 0° "0'” T I T 800-- AFTER PASSAGE 600*, THROUGH LUNGS» 4004. 200-4 . . I I I I r 20 4O 60 BO IOO SAMPLE NUMBER Figure 12 143 of the 14C injected was estimated to be taken up by the lungs. The lungs therefore avidly take up ATP from the blood or rapidly convert it to other substances which do not imme- diately leave the lungs. Of the 14C activity recovered in 14 the blood after the passage of the C-ATP injection through the lungs only 17% i 11% (mean t SE, N = 4) was still 14C-ATP. The other labeled 14C-ATP degradation products in the blood leaving the lungs were nucleosides, 51% i.14%3 AMP (and/or IMP), 24% i.6%; and ADP, 7% i 3% (mean : SE, N = 4). The breakdown of plasma ATP during passage through the lungs is . 4 . extremely rapid, as l C-nucle031des can be recovered from , 4 . . . injected 1 C-ATP in just Six seconds. 10. In Vivo Studies on the Effect of ATP 'on Systemic Arterial Pressure Figure 13 shows the results of ATP injection (A) and infusion (B & C) on systemic arterial blood pressure. The results shown are typical of those observed in four experi- ments performed on different dogs° Injection of 500 pg of ATP into the aortic arch (Figure 13,A) produced a large, transient drop in systemic pressure, whereas 500 pg of ATP injected into the vena cava adjacent to the right atrium resulted in little if any fall in pressure. Infusion of 2 mg ATP/min into the aortic arch (Figure 13,B) produced a large and continuous drop in systemic pressure. Switching the 2 mg/min infusion of ATP into the vena cava at the right atrium, however, 144 Figure 13. Effect of intravenous and intra—arterial ATP infusion on systemic arterial blood pressure. (A) Five hundred pg ATvaere injected in 1 ml saline into the aortic arch followed by 1 ml isotonic saline injected into the same site and then 500 pg ATP injection into the vena cava adjacent to the right atrium. (B) Two mg/min ATP was infused°first into the aortic arch and then into the vena cava adjacent to the right atrium. (C) Fifteen mg/min ATP was infused first into the vena cava adjacent to the right atrium, then into the femoral vein, and then back into the right atrium again. Chart speed = .5 cm/20 sec. III: V8 mm: 145 EFFECT OF INTRAVENOUS AND INTRAARTERIAL ATP INFUSION ON SYSTEMIC ARTERIAL BLOOD PRESSURE mmHg /\ + I + ATP Saline ATP Aortic Aortic Right Arch Arch Atrium 4. ATP f Aortic Right Stop Arch Atrium Infusion I ATP + + + Right Femoral Right Stop Atrium Vein Atrium Infusion Figure 13 146 resulted in a return of systemic pressure to control levels; and no further increase in systemic pressure was recorded when the infusion was stopped. It can also be seen that infusion of 15 mg ATP/min into the thoracic vena cava at the right atrium (Figure l3,C) did produce a significant drOp in systemic pressure. Switching the infusion of this amount of ATP to the femoral vein did not change the pressure drop. An increase in pressure was recorded when this infusion was stOpped. These findings support the uptake and inactivation of ATP by the lungs observed by others (Folkow, 1949, and Gordon, 1961). The slower breakdown of ATP in blood as com- pared to the lungs is also supported by the lack of changes in systemic blood pressure when a large amount of ATP being .infused into the thoracic vena cava is switChed into the femoral vein. Also of interest are the changes in ventilation observed during ATP infusion. An increased ventilation of 31 breaths/ min was observed during intravenous infusion of 15 mg ATP/min as compared to a control ventilation rate of 20 breaths/min° Also, increased ventilation was observed when 500 pg of ATP were injected into the aortic arch. DISCUSSION The results of in vitro studies on l4C-ATP metabolism in whole blood and plasma showed that added ATP was rapidly 1 broken down. Jorgensen (1956) and Forrester (1972) also found ATP degradation in whole blood and plasma. Jorgensen, :9 however, found a slower rate of ATP breakdown than observed in our studies. In his studies approximately 50% of the added ATP was degraded after two hours in plasma and one- half hour in whole blood compared to three minutes in plasma and 1 1/2 minutes in whole blood reported in the results of this thesis. Furthermore, our studies indicate that l4C-ATP is virtually completely broken down in plasma after 20 minutes, whereas Jorgensen still had measurable ATP after five hours. The slower ATP breakdown observed by Jorgensen may have been chua to his use of human rather than dog blood, but more probably was due to: (l) the large quantities of ATP which he used, and (2) his dilution of the blood and plasma with Tyrode-Locke's solution. His ATP concentration added initially to both whole blood and plasma was 400 pmoles/l of diluted plasma or whole blood. The plasma and blood were diluted by adding 1 ml of Tyrode-Locke's solution containing the proper amount of ATP to 4 m1 of whole blood. This was 147 148 approximately 240 Hg of ATP/ml of diluted whole blood or plasma, whereas in our studies only 0.57 pg ATP was added to each m1 of undiluted whole blood or plasma. The use of high l4C-ATP made it possible to add such small specific activity quantities of carrier ATP. The 0.57 pg ATP/ml plasma used approached physiological levels observed by Chen et al. I (1972) (0.45 pg ATP/ml) in post-tetany femoral venous plasma 1 and Forrester (1972) (0.2 pg ATP/ml) in venous plasma from an exercising human forearm. The dependence of ATP breakdown rate upon initial ATP concentration was shown by Forrester (1972). Approximately one—half of the ATP added to human citrated plasma at 37°C was degraded in 15 minutes when the initial ATP concentration was 10 pg/ml, but in only six minutes at an initial concentration of 1 pg/ml. Thus, the breakdown rate of low levels of ATP in human citrated plasma observed by Forrester was very close to that found in our studies with heparanized dog blood, presumably due to his addition of similar quantities of ATP. l4C-ATP added to whole blood disappeared approximately twice as fast as that added to isolated plasma. This is similar to the findings of Jorgensen (1956), who observed ATP breakdown in whole blood to be 7 to 8 times faster than in isolated plasma. Considerable plasma ATP is evidently broken down by the formed elements in blood, especially the erythro- cytes, and an "ecto" ATPase related to thrombosthenin has been 149 reported for platelets (Chambers et al., 1967, and Mason and Saba, 1969). Even though ATP breakdown in blood is rapid, there is still sufficient time for ATP released by an organ to leave that organ in the venous blood and even to recircu- late if ATP breakdown by the lungs could be inhibited. F? The resynthesis of l4C-ATP by the formed elements of the I blood which occurred in our studies after 7 minutes incuba- A ' - | ‘31... tion is in agreement with studies by Parker (1970) and 4 Schrader et al. (1972), although Schrader's studies were done on red cell ghosts. Added l4C-ATP was apparently degraded to adenosine in the plasma via ADP and AMP, as described by Chen and Jorgensen (1957), who used enzymic assays to quantify these nucleotides. 'The adenosine was then rapidly taken up by the erythrocytes and platelets and rephosphorylated to ATP. Inosine and hypoxanthine may also have been formed, but their specific formation was not detected since the chromatographic separation used in these studies was unable to distinguish between inosine, hypoxanthine and adenosine. The rapid uptake of adenosine by the formed elements and its incorporation into intracellular adenine nucleotides was also found when 14C-adenosine was added to blood. This uptake may provide a mechanism of conserving adenine nucleotides rather than de- grading them in the plasma to hypoxanthine and then to uric acid in the liver. The resynthesis of ATP by the formed elements supports Schrader's suggestion that phosphorylation 150 rather than deamination of adenosine occurs at low external adenosine concentrations due to the lower Km of adenosine kinase. The rapid rise in 14C-AMP and the consistently smaller rise in 14c-ADP that occurs during l4c-ATP degradation in plasma and whole blood is in agreement with Mills (1966), who found that 80 per cent of added ATP was directly dephos- I phorylatedvia d-B-ATPase in plasma to AMP. Although considerable variability was observed in the l4C-ATP breakdown rate in whole blood from different dogs in the present studies, l4C-ATP breakdown rates in paired whole bloods from the same dog were very similar. Thus, the analysis of paired whole bloods provided a suitable way to study the effect of various in vitro conditions on ATP break- down. In an effort to facilitate the study of the physio- logical role of extracellular ATP, several methods of inhibiting ATP breakdown were tried; however, an effective inhibitor which would be harmless to the body and not affect the cell membrane was not found. Heparin had no effect on ATP breakdown. Barium was investigated in our studies as a pos- sible inhibitor of ATP breakdown but also had no effect. Citrate inhibited breakdown only slightly in the concentration used by Forrester (1972) (15 mMoles citrate/l). Holmsen and Stormorken (1964) found that eighteen times as much citrate as magnesium (l mMole/l) had to be added to blood to cause marked inhibition of ADP breakdown. Since Forrester used a 151 citrate concentration smaller than this as an anticoagulant, any ATP present in plasma from resting subjects would pre- sumably have had time to breakdown during the approximately 30 minute period between collection and analysis. Jorgensen and Poulsen (1955) found that temperature was an important factor in oxypurine formation in blood, much faster accumulation occurring at 37°C than at 4°C. Forrester (1969) found rapid ATP decay in diluted plasma at room temper- ature. The present studies have shown that significant l4C-ATP degradation can occur even at very low temperatures. After 1 1/2 minutes, approximately 20 per cent of added ATP was degraded in whole blood incubated in an ice bath. This finding could explain why Katori and Berne (1966) found adenosine rather than ATP in the perfusate from hypoxic heart muscle and why Dobson et al. (1971) could find no ATP but sig- nificant quantities of adenosine in the cooled (0°C) venous effluent from exercising skeletal muscle, since sufficient time elapsed for much ATP breakdown to occur between sampling and ATP assays. In the present studies the presence of hemolysis seemed to greatly accelerate ATP breakdown. Chen and Jorgensen (1956) also noted this phenomenon. In our preliminary studies non-Siliconized glassware caused slight hemolysis; and also, blood taken from dogs anesthetized with chloralose- urethane was noted to be greatly hemolyzed. In both types of 152 hemolyzed blood, l4C-ATP breakdown was much faster than in non-hemolyzed blood samples. Thus, it is possible to have lower than normal ATP levels in plasma as a result of hemolysis even though ATP is released from the red cells in this circumstance. In order to estimate the ATP breakdown that occurs in 8 plasma of the intact animal, the breakdown of 14C-ATP in whole blood in vitro was studied. During the course of this study it became evident that there was considerable variabil- ity in ATP breakdown rate in bloods from different dogs. Non-gassed blood samples had a wide variation in ATP break- down rate when incubated for 1 1/2 minutes. Gassing the blood with specific mixtures of C02, however, enabled much more consistent results to be obtained. The 14C-ATP added to gassed blood was degraded 51.8 i 2.9% (Mean : SE, N = 11) in 1 1/2 minutes. This per cent breakdown in 1 1/2 minutes can now be used for comparison with other studies to determine if ATP breakdown in whole blood is significantly altered by pathological conditions such as hypertension. Bishop (1960) found that gassed blood samples lost ATP far less rapidly than non-gassed samples. This effect was attributed to pH differences between the two samples, the non- gassed sample having a higher pH and therefore losing ATP more rapidly. Jorgensen and Grove-Rasmussen (1957) found that adjustment of pH to 7.1 or lower in stored blood that was drawn through an ion-exchange column prevented oxypurine 153 accumulation and therefore ATP degradation. Beutler and Duron (1963) found that at 4°C red cell ATP disappeared rapidly at pH 7.5 to 7.9 but was quite stable at low pH (6.8 to 7.2). At 37°C, however, they noted an opposite pH effect; that is, greater stability at high pH.. Scott et al. (1969) found that red cell ATP was lost rapidly at pH values above 7.6-7.7 at 25°C. The lost ATP was recovered by lowering the pH. At 37°C ATP was rapidly lost above pH 7.4-7.5. The increased gly- colysis which occurs at 37°C could in part explain ATP preser- vation at this temperature at elevated pH values. Rosenthal (1948) found that the pH of blood in vitro was very dependent upon temperature. The pH rose with a fall in temperature in a linear manner. The present pH studies were more concerned with effects on ATP in the plasma rather than on red cell ATP per se. Therefore, it is questionable as to whether the conclusions of some of the previous studies described above can be applied here, as they dealt with pH effects on red cell ATP. pH was found to have a definite effect on added ATP breakdown rate. l4C-ATP appeared to be more stable in bloods having pH between 7.2 and 7.4 at 37°C; however, this can only be an approxima— tion due to the small number of samples below pH 7.3. Both above and below this range, breakdown rate seemed to increase. Breakdown was always inhibited in the paired sample with the lower pH down to approximately pH 6.94. Below pH 6.94 154 breakdown was inhibited only slightly when compared to the sample that was not gassed. 14C-ATP and l4C-AMP appeared to vary inversely with each other after 1 1/2 minutes incubation at different pH values. l4C-ADP and nucleosides were not affected in a consistent manner by pH. Fleishman et al. (1957) found active small vessel dila- tion in reSponse to an increase in hydrogen ion concentration in the dog foreleg, the effect occurring predominantly on the alkaline side of pH 7.3. At pH 7.6 vessel resistance was significantly increased. A similar response to pH change occurred in the kidney (Emanuel et al., 1957). The mechanism of the vessel response was not known. However, since it has been suggested that ATP may exist in arterial plasma and since the results presented in this thesis indicate that ATP breakdown is less in more acid bloods, it is possible that ATP may be the mediator of the pH effect. Stowe et al. (1973) found that pH decreased to a minimum of 7.1 during active hyperemia in skeletal muscle with constant flow. With natural flow the decrease would probably be less. The present find- ing that ATP is most stable in bloods with decreased pH at body temperature also indicates that during active hyperemia ATP released into the blood may have a somewhat longer survival time and may thus act as a dilator longer. The relatively greater instability of ATP at high pH could be another reason why Berne and Dobson found no ATP in their studies, as their 155 samples were collected in unstoppered tubes and thus would have high pH because of CO2 loss. Forrester (1969) and Chen (1972) found approximately 200 ng/ml of ATP in normal arterial human and dog plasma respectively. Forrester (1972), however, attributed his ATP levels to platelet damage and the effect of EDTA on the H erythrocyte membrane. Although EDTA is both an anticoagulant i and an inhibitor of ATP breakdown, Forrester used EDTA in his 1 early studies as an anticoagulant. After becoming aware of evidence that EDTA increased the permeability of skeletal muscle membranes to ATP (Abood et al., 1962) and after noting that EDTA increases plasma ATP, Forrester used citrate as his anticoagulant. Citrate, however, has been shown in the present studies and by Forrester (1972) to be ineffective in inhibiting ATP breakdown in the concentration used by Forrester for anticoagulation. Thus, the lack of measurable amounts of arterial plasma ATP in Forrester's later studies (1972) on citrated plasma could be caused by either increased plasma ATP breakdown, lack of release of platelet and/or red cell ATP into the plasma, or both. Abood et a1. (1962) found that the presence of EDTA caused greater release of skeletal muscle ATP than a medium without EDTA. Kuperman et al. (1964) found an increased out— flux of 3H—AMP from nerve axons immersed in a solution to which EDTA was added as compared to an EDTA-free medium. 156 However, Scott et a1. (1969) found no loss of red cell ATP in EDTA-treated blood when the pH was kept below 7.6. On the other hand, Scott observed evidence that ATP passes out of the red cell in whole blood with pH over 7.6. High pH, therefore, could be the factor causing ATP release in blood rather than EDTA. The studies reported in this thesis on EDTA-treated blood are not able to determine whether EDTA or high pH cause the release of small quantities of ATP from the formed ele- ments of the blood, since the technique used was not sensitive enough to detect differences in such low ATP levels. However, the l4C-ATP activity in the plasma did appear to vary directly with the amount of intracellular l4C-ATP formed. This pro- vides some evidence for ATP leakage from the cells in both control and EDTA-treated blood. However, the normal existence of ATP or other adenine compounds in arterial plasma is still questionable. Those investigators who have attempted but have not found ATP in plasma used methods which did not sig- nificantly inhibit its breakdown; those who did find it used substances such as EDTA which may have altered the cell mem- brane and thereby caused a leakage of ATP. The studies on EDTA-treated and control bloods revealed a significant peak of l4C-AMP (or a closely related isomer) in the plasma in both types of bloods. Therefore, AMP can evidently exist in arterial plasma for a considerable period 157 of time. This fact may indicate that AMP could be clinic- ally useful as a vasodilator, provided its metabolism in vivo is not extremely fast. In this latter regard, Gordon (1961) has reported that AMP is not inactivated by the lungs. Based on the erythrocyte ghost data of Schrader et a1. (1972) and the results from the whole blood experiments re- ported in this thesis, it seems more likely that any adeno- sine released from the heart during active hyperemia is either phOSphorylated to AMP extracellularly or enters the cellanuiforms adenine nucleotides, rather than forming extra- cellular inosine and hypoxanthine. The presence of myokinase in myocardial and other cell membranes also makes the conver- sion of this extracellularly formed AMP to ADP and ATP a possibility, should either extra— or intracellular ATP be available for this reaction. Thus, even though adenosine may be the only adenine compound passing through the cell mem- brane, extracellularly formed AMP, ADP or ATP present in the plasma or interstitial fluid could also be possible mediators of vasodilation. The significance of the IMP formation in blood described by others is questionable, as it was not formed in signifi— cant quantities during l4C-ATP breakdown in blood or during l4C-adenosine. Bishop's theory nucleotide synthesis from regarding adenylic deaminase in blood seems to be incorrect, although his bloods were hemolyzed in many cases and also O 158 were incubated considerably longer than those studied in this thesis. On the basis of Bishop's observations, it was ex- pected that the large quantity of l4C—adenosine added would l4C-IMP formation. certainly result in some intracellular On the other hand, since virtually none was found, the special inhibition of adenylic acid deaminase in blood found by Conway and Cooke (1939) may be very true indeed, although intracellular compartmentalization of adenine nucleotide formation and AMP deamination may also explain these discrep- ancies. The in vivo lung studies when compared to the in vitro whole blood studies of l4C-ATP breakdown illustrate a great difference between ATP stability in isolated blood and in blood passing through an organ. The experimental evidence described in the results of this thesis indicate conclusively that the lungs rapidly take up and degrade ATP, in agreement with the indirect observations of Folkow (1949) and Gordon (1961). Nucleosides were formed from plasma ATP by the lungs in a few seconds, whereas in isolated blood minutes were required. Although the fate of the ATP taken up by the lungs is unknown, the following comments regarding ATP metabolism by the lung can be made. Fishel et a1. (1970) have found that mouse lungs contain very little adenyl cyclase, the enzyme which converts ATP to cyclic AMP; thus, this is an unlikely route for lung ATP degradation. On the other hand, 159 Fishel et al. observed that lung tissue actively converted ATP to AMP. Since ADP and AMP apparently can pass through the lungs unchanged (Brashear and Ross, 1969, and Gordon, 1961), and since very little l4C-ADP and 14C-AMP were found in the pulmonary outflow in our studies after l4C-ATP injec- tion into the right atrium, the a-B-ATPase and B-y-ATPase reactions must be almost totally absent on the external cellular surfaces of the lungs. This suggests that ATP per se is taken up by lung tissue rather than its degradation products. Clarke et al. (1952) found high adenosine deaminase levels in lung tissue, and Pfleger (1969) found rapid adeno- sine uptake by the lungs (90 per cent uptake compared to 10 per cent breakdown). The metabolic fate of the ATP and adenosine taken up from the plasma by the lungs remains to be elucidated. ATP infusion studies presented in the Results give us indirect and inconclusive evidence that only high levels of ATP can pass through the vasculature of the lungs. ATP in_ fused into the right atrium or femoral vein produced approxi- mately the same amount of vasodilation, suggesting but not proving that little breakdown of ATP occurred in blood travelling from the hindlimb to the lung, in agreement with the reported in vitro blood studies. The lung studies, in conjunction with the breakdown rate studies in isolated blood, indicate that ATP could recirculate 160 through the body if it could survive passage through the lungs. Thus, the vasodilator potency of ATP could be uti- lized in conditions such as hypertension if a suitable inhibitor of ATP breakdown in the lung could be found. Perhaps dipyridamole, which blocks adenosine uptake by the lungs, would also block the uptake of ATP. The fate of ATP F in passing through other organs remains to be investigated. It was suggested in the Literature Review that the IA mechanism of action of ATP may be similar although not neces— sarily identical to that of actylcholine, as both compounds depress smooth muscle contraction in many body tissues. Although the actual mechanism by which the smooth muscle relaxation is produced by extracellular ATP is unknown, several distinct possibilities exist. ATP could decrease membrane permeability to calcium, thereby decreasing intra- cellular free calcium levels and thereby increasing the effectiveness of relaxing protein (troponin, tropomyosin). A second possibility would be hyperpolarization of cells by ATP. This could occur either by an increased activity of the pump extruding sodium from the cell, by decreased membrane permeability to sodium, or by increased membrane permeability to potassium. In the case of a coupled sodium-potassium pump, an increased pump rate would also cause membrane hyperpolariw zation by increasing the rate of potassium diffusion out of the cell. More evidence is needed to elicidate the actual 161 mechanism of ATP action and to relate the extracellular nucleotide profile to physiological regulation. SUMMARY AND CONCLUS IONS A gradient elution ion exchange chromatographic procedure was utilized to investigate the breakdown of l4C-ATP in dog whole blood and plasma in vitro. Metabolism of l4C-ATP by the lungs was studied in vivo; and l4C—adenosine metabolism was studied in whole blood in vitro. 14C-ATP was degraded at a faster rate in whole blood than in plasma. However, even in whole blood the survival time of the l4C-ATP was suffici- ently long to permit recirculation in the body. l4C-ATP breakdown products in both plasma and whole blood were ADP, AMP, and nucleosides. Hemolysis apparently increased l4C-ATP breakdown rate, whereas cold decreased the rate. Citrate, barium, and heparin had little, if any effect on breakdown rate. Decreasing whole blood pH consistently inhibited l4C-ATP breakdown rate except when below pH 6.94. 14C—ATP survival time in plasma of whole blood was greatest at about pH 7.2 to 7.4, indicating that any ATP released into the acidic blood flowing through a limb during exercise hyperemia may have a longer time in which to exert its vasodilatory 1 4C effects. In vivo studies indicated that -ATP was rapidly removed from the plasma by the pulmonary circuit. Eighty- three per cent of 14C intravenously injected as l4C-ATP was 162 163 taken up by the lungs during a single pass. Of the 17 per cent 14C activity recovered after a single pass through the lungs, only 17 per cent was still ATP. 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