lllllllllI/IHHIIll/IIIUHIINIH/IIllll/IIIIIIIIHII/IIIUH 3 1293 10527 3068 RETURNING MATERIALS: )V1SSI_J PIace in book drop to LJBRARJES remove this checkout from y rrrrrrrr d. FINES wi11 .-;_. _________ be charged if book is returned after the date stamped below. l 'A _ ‘- r mix 3‘ "" ‘3'. i . " ' - 'r" .. s ‘5'“: V' J . ‘l" ADENOSINE AND EXERCISE HYPEREMIA BY Barry David Fuchs A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Physiology 1982 ABSTRACT ADENOSINE AND EXERCISE HYPEREMIA By Barry David Fuchs I measured adenosine release into venous plasma as an index of interstitial adenosine concentration during free flowr exercise hyperemia. Isolated, blood-perfused dog calf muscles were stimulated at 6 Hz for ten minutes with free flow. Plasma samples were collected before, during, and after the exercise period for analysis of pdasma adenosine concentration ([ADOJ) by HPLC. Adenosine release (R ADO) was calculated as plasma flow times venous-arterial [ADO] difference. RADO (nmole/min/100g) went from -O.1+_O.1 at rest to 6.1+_‘4.2 during 6 Hz exercise. Isoproterenol infusion, which caused an increase in blood flow equvalent to 6 Hz exercise, did not result in increased RADO' Infusion cM‘ the 5'—nucleotidase inhibitor, (x, g, methylene adenosine 5'-diphoshate (AOPCP) did not prevent the increase in RADO during exercise. These results support the hypothesis that interstitial adenosine concentration increases during sustained fTee-flow exercise and that this results in increased release of adenosine into venous plasma. Dedication To my father and to the loving memory of my mother ACKNOWLEDGMENTS I wish to express my sincere appreciation to Dr. Harvey Sparks, my thesis advisor, for his invaluable guidance during the numerous discussions concerning this work and for the insight he has given me into the study of biological processes. His limitless enthusiasm and persistent encouragement were inspiring, and made working an enjoyable experience. I would also like to thank the other members of my thesis committee Dr. Scott, Dr. Leena Mela, and Dr. Spielman, for their time and valuable suggestions toward the development of this thesis. I am indebted to Greg Romig, Joel Silver, Dave Harms, Amy Jo Bannink, and Amy Sue Abrahamsen for their expert technical assistance. I wish to express my appreciation to Mark Gorman for his assistance with the experiments, for putting up with my theoretical verbiage, and lastly for providing an endless source of snags. Thanks Gorms. I wish to thank Loren Thompson for his help with some of the experiments, for being one helluva funny guy, and for the delightful memories of these unforgettable backroom (office) discussions. I wish to thank Linda Friedsberg for her help with the typing of this thesis. Lastly, I wish to thank Liz for her help with the typing of this thesis and most importantly for her patience, selflessness and support. TABLE OF CONTENTS LIST OF TABLES . . . . . . . . . . I. LITERATURE REVIEW . . . . . . A. 9. 10. Introduction . . . . . . . The Myogenic Hypothesis . . The Metabolic Hypothesis . Direct effect of oxygen . Tissue £92 . . . . . . . PCOZL pH, and lactate . Osmolarity . . . . . . Potassium . . . . . Prostaglandins . . . . . Phosphate . . . . . . . . Local Neurons . . . . . . ATP 0 O O O O O O O O O O Adenosine . . . . . . . . II. MATERIALS AND METHODS . . . . A. B. 1. 2. D. Canine Hindlimb Preparation Protocol . . . . . . . . . QUE; free flow exercise . Isoproterenol infusion . Sample Preparation and Adenosine Assay Data Analysis and Statistics Page 10 10 11 19 19 21 21 22 23 2Q III. RESULTS 0 O O O O O O O O O O O O O O O I O O A. Adenosine Release During Free Flow at Q Twitches/Sec B. Adenosine Release During Infusions 3f Isqproterenol . . C. Effect of AOPCP on Release of Adenosine During Exercise D. BADO in the Presence of AOPCP Infusion . . Iv. DISCUSSION 0 O O O O O O O O O O O O O O O O A. Stimulus For ADO Production . . . . . . B. Interpretation of ADO Release Measurements C. Significance of ADO Release Measurements V. SUMMARY AND CONCLUSIONS . . . . . . . . . . . VI. BIBLIOGRAPHY . . . . . . . . . . . . . . 26 26 26 26 27 31 31 33 38 40 41 LIST OF TABLES Effect of 6 Hz twitch exercise and isoproterenol on . . . . . . 29 blood flow V02 and adenosine release of the canine hindlimb perfused at free flow. The effect of AOPCP on the changes of blood flow VO2 , , , , , , 30 and RADO associated with 6 Hz twitch exercise. I. LITERATURE REVIEW A. Introduction Whenever a skeletal muscle exercises, an increase in blood flow occurs which correlates with the increase in muscle metabolism. With graded exercise intensities, one observes pr0portional increases in blood flow. This phenomenon is known as exercise hyperemia. The search for the cause of exercise hyperemia dates back to 1877. During the last 100 years, many hypothetical mechanisms have been proposed to explain the phenomenon, yet at present the cause of steady state exercise hyperemia remains 2a mystery. One hypothesis, which has received much attention, and has not been definitively rejected is the adenosine (ADO) hypothesis. It is 'mna purpose of this thesis to critically test the adenosine hypothesis. This thesis is directed at the cause of steady state, twitch induced exercise hyperemia, and thus we are not searching for a factor to explain all of the dynamics of exercise hyperemia under all circumstances. It is IMNJ well established that the vasodilatory mechanism(s) which are brought into play by the muscle depend(s) on a) the exercise pattern, i.e. twitch vs. tetanic contractions, b) the duration of the exercise bout, c) the relationship between supply and demand of oxygen and d) the muscle fiber type. The most common pattern of exercise (in the laboratory) is a sustained train of twitch contractions. Such exercise patterns can be maintained for hours in skeletal muscle with high oxidative metabolism, and the increase in blood flow remains sustained. The mechanism of this sustained increase in flow over many minutes to hours is the subject of this introduction. In 1877, Caskell demonstrated that exercise hyperemia could occur normally without the presence of extrinsic nerves. Since then, investigators have searched for local chemical or physical factors to explain exercise hyperemia. First ]I will review the evidence for the physical factors, and then I will address the chemical factors. B. The Hyogenic Hypothesis Isolated blood vessels will relax when subjected to a quick decrease in initial length, and they will contract if quickly stretched (Johannson and Mellander, 1975; Sparks and Bohr, 1962). This is known as the Payliss response, named after its first observer (Bayliss, 19021, and is the basis for the myogenic hypothesis of blood flow regulation. There is no doubt that during certain conditions blood vessels in vivo are exposed to an analogous stimulus for initiation of the myogenic response i.e. changes in transmural pressure. Ibr' instance» during muscle exercise, extravascular pressure will increase due to muscle tension development, and intravascular pressure will decrease due to the extravascular compressive forces restricting arterial inflow and hastening venous outflow. The net result of these changes is that vascular transmural pressure will fall and in fact it will be reversed. This would then reduce tension in the vessel wall and initiate myogenic relaxation. Experimental evidence lends support for this hypothesis. In 51 resting muscle exposed to large increases in tissue pressure by external muscle compression, the increased vascular conductance changes elicited can account for one third to one half of the vasodilation occuring during tetanic exercise (Mohrman and Sparks, 1974). The myogenic response, however, does not appear to play an important role in the exercise hyperemia associated with twitch contractions. Bacchus et al. (1981) found that when they exposed a passive muscle to intramuscular pressure changes that were recorded during twitch contractions (up to 50 mm Hg), vascular conductance failed to increase. Thus the myogenic response may play a significant role in the hyperemia associated with tetanic contractions, however, evidence for its involvement in steady state twitch exercise hyperemia is lacking. C. The Metabolic Hypothesis It has been known for many years that changes in muscle metabolism are tightly coupled to changes in muscle blood flow, in the steady state. It has been assumed that a factor related to oxidative metabolism is responsible for the changes in vascular conductance associated with increased muscle work (Kramer efi: al., 1939). It is difficult to imagine that a parallel mechanism unrelated to metabolism could be so precise, sudh that the tight correlation between V02 and blood flow happened to be just a fortuitous relationship. Mohrman and Sparks (1973) provided additional evidence strengthening the relationship between the two variables by observing that the dynamic changes in V02 and vascular conductance were very similar in time course during sinusoidal twitch exercise» Thus factors linked directly to oxidative metabolism may indeed cause the steady state vasodilation associated with twitch exercise. This evidence, however, does not rule out the participation of factors not linked directly to oxidative metabolism. This distinction will be discussed later. 1. Direct effect of oxygen The venous outflow P02 of an exercising muscle falls during exercise. It has also been observed that vascular smooth muscle strips relax when exposed to a hypoxic bathing medium (Detar and Bohr, 1968). These findings led to the hypothesis that a decrease in vessel wall PO2 during exercise causes the steady state increase in vascular conductance (Guyton (at al., 1964). In evaluating this 'hypothesis the important question is: Does arteriolar vascular smooth muscle PO during free flow 2 exercise fall sufficiently to induce smooth muscle relaxation? Duling (1975) has determined that vessel wall P02 is primarily influenced by adjacent luminal blood P02. (Nuns, it is not intuitively obvious that vessel wall P02 will fall sufficiently, since flow delivery of oxygen increases during free flow exercise. Sparks (1980) adressed this question (n1 theoretical grounds 137 the use CH? a computer simulation compartmental model. The model predicts that as muscle work increases the vessel wall P02 first falls, but then as flOW' delivery CH? 02 increases, the vessel wall P02 rises. This prediction is iji general agreement with the experimental observation that cremaster muscle exercise dilation occurs without a change in periarteriolar P02 (Gorczynski and Duling, 1978). These results cast strong doubt on the role of vessel wall P02 in the mediation of normal exercise hyperemia. 2. Tissue P02 If lack of 02 itself is not the cause for exercise hyperemia, perhaps 0 is involved indirectly by altering the rate of an oxygen 2 dependent metabolic process within muscle cells, which then results in the release of some vasoactive metabolite into the interstitium. Alternatively, it is conceivable that the mechanism controlling vascular conductance becomes activated simultaneously' with the activation of oxidative metabolism. Both of these pathways for metabolite production would become activated with increased muscle metabolism and therefore it is difficult to define the exact nature of the stimulus responsible for metabolite release. For example: ATP hydrolysis occurs during muscle contraction. This will result in an increased ADP concentration which stimulates oxidative phosphorylation i.e. ADP+Pi ATP. At the same time AMP concentration will increase by mass reaction through the enzyme myokinase, ‘which_ catalyzes the reaction: 2ADP=ATP+AMP (McGilvery' and Murray, 1969). This in inuni may result in adenosine formation by the action of 5'nucleotidase. Thus, coincident with the increase in oxygen consumption is the formation of adenosine, a potent vasodilator metabolite. The other possible stimulus mentioned above for the formation of a vasoactive metabolite is through a decrease in tissue P02. As V02 increases with exercise the concentration of cellular oxygen declines. This is believed to occur since muscle venous blood P02, which is in equilibrium with muscle tissue PO falls during 2, exercise (Sparks, 1980). If cell PO2 falls to a critical level at which oxidative phosphorylation is reduced, the flux of ADP to ATP will decrease. This results in.51 greater net flux from ATP to ADP, which will then raise the ADP concentration. Once again AMP and adenosine will increase by the reactions previously described. Although it is not known whether this would occur under free flow conditions, this mechanimn will become operable whenever there is an imbalance between the oxygen supply and the oxygen use of the tissue. Several investigators have attempted to determine the relative importance of the latter mechanism in exercise hyperemia. Gorezynski and Imling (1978) have shown in the suffused cremaster preparation, that if the fall in tissue PO2 during exercise is prevented by increasing suffusate P02, exercise vasodilation is reduced but not abolished. Examination of the off response to exercise reveals that the dynamics for the restoration of vascular conductance is much slower than the return of tissue P02. This indicates that 'Hua PO2 decrease associated with muscle exercise determines only a portion of the overall vascular response, and that there exists another mechanism controlling vascular conductance that is unrelated to the level of tissue oxygenation. Mohrman and Sparks (1973) reached a similar conclusion in their experiments in the dog hindlimb. They observed that when sinusoidal twitch exercise was performed with excessive blood flow to the muscle, vascular conductance dynamics were quicker than the oxygen consumption dynamics. Under these conditions it appears that a control system unrelated to V0 became operable. 2 3. PCOD, pH, and lactate (— Increases iJI tissue PCO2, [H+), and (lactic acid) all accompany muscle exercise. Since each agent can cause smooth muscle relaxation in vivo and in vitro, all three agents have been proposed to be involved in exercise hyperemia. Based on two lines of evidence, however, only a relatively minor role can be attributed to these agents. The first is that skeletal. muscle resistance ‘vessels are not sensitive enough to changes in the concentration of these chemicals that physiologically occur during muscle exercise. Emerson (1974) demonstrated this point by raising the [H+] by increasing [lactic acid] of the blood perfusing a resting muscle, to levels of H+ measured in the venous blood of exercising muscle. Radawski (1975), and Stowe (1975) both present evidence on the relative inability of high PCO and thus increased [H+) 2 as ‘well, to significantly' alter the vascular conductance of resting muscle. The other line of evidence which addresses only H+ and lactic acid is the observation that in patients with monoiodoacetate poisoning or Mcardles syndrome, who are unable to produce lactic acid, exercise hyperemia still occurs (Tobin, 1965). In view (M? the recent finding that H+ potentiates the vasoactive potency of adenosine (Merrill, 1978), H+ and thus CO2 and lactic acid, may play a role in sustaining exercise hyperemia through potentiating other vasodilator systems. 4. Osmolarity In 1967, Mellander and co-workers first proposed a role for tissue osmolarity in exercise hyperemia. Evidence suggests, however, that the relative contribution (xf this proposed mediator: is species specific. For instance the potential for the release of osmotically active particles is greater in cats than in dogs (Scott et al., 1970). Humans are probably' somewhere lJl between (Lundvall eet al., 1969). Absolute increases of osmolarity in dog skeletal muscle are relatively small in comparison to the changes that occur in cat muscle at the same work intensity (Scott et al., 1970). In addition, when these venous osmolarity changes are reproduced in a resting muscle with infusions of hypertonic solutions, the vascular conductance changes are significant in cat muscle particularly at the start of the infusion, but trivial in dog muscle (Scott and Radawski, 1971). Another argument against tissue osmolarity playing a significant role in steady state exercise hyperemia in dog and human skeletal muscle is the fact that the initial increases in venous osmolarity at the start of exercise wane after a few minutes while vascular conductance remains elevated (Morganroth 6H3 al., 1975; Stowe et al., 1975). Thus tissue osmolarity may be important in cat muscle, but does not appear to play a significant role in dog or human skeletal muscle. In addition, if tissue osmolarity plays a role in exercise hyperemia, it probably occurs during the first few minutes of exercise but not during the steady state. 5. Potassium Potassium ion in low concentrations is known to be a good vasodilator (Dawes, 1941). Since it is released into the venous blood during muscle exercise, the potassium ion has been proposed to play a role in exercise hyperemia (Sparks, 1980). In light of the transitory nature of potassium induced vasodilation (Kjellmer, 1965; Chen et al., 1972; Duling, 1975; Gellai, 1974), and the observation that with extended periods of exercise, potassium release wanes with time (Morganroth et al., 1975; Stowe et al., 1975; Brace et al., 1974), potassium release can not be very important during steady state exercise hyperemia. Since potassium is released into the ISF ennui prior ‘to muscle contraction, a role for potassium in the initiation of exercise hyperemia i1; more convincing» Ouabain, in concentrations which block the vasodilation of exogenously administered potassium, slows the onset of exercise hyperemia but has no effect on the steady state flows (Chen et al., 1972). Hazeyama snui Sparks (1979) ‘have provided additional evidence in favor of potassium in the initiation of exercise hyperemia by demonstrationg that :hi potassium depleted dogs cum: of the initial phases of exercise vasodilation is abolished. These authors used a simple diffusion model to calculate the time course of ISF (K+] after the initiatbmn of exercise. The model predicts that IK+I in the ISF increases fast enough to be responsible for one of the initial phases of exercise vasodilation. ‘0 6. Prostaglandins Some prostaglandins are potent vasodilators (Bevegard and 0ro, 1969), and can be synthesized by the arterial smooth muscle (Terragno et al., 1975). It has recently been proposed that these substances play a role in the local control of blood flow. In support of this, Kilbom and Wennmalm (1976) observed that indomethacin, a Incstaglandin synthesis blocker, attenuated the hyperemia of exercising human muscle by as much as 257. Another group also demonstrated that indomethacin administration severely reduced exercise hyperemia in the canine hindlimb (Janczewsk et al., 1974). They also found measurable release during exercise. 0n the other hand, Beaty and Donald (1979), found that indomethacin had no effect on steady state exercise hyperemia of the canine hindlimb. Young and Sparks (1979) confirmed this result by demonstrating that indomethacin did not alter the relationship between V02 and blood flow, and in addition observed a dissociation between the release of PCB and the change in vascular conductance. Thus it appears that prostaglandins are not involved in normal exercise hyperemia. They may play a role, however, in the control of vascular conductance under resting conditions, and during restricted flow exercise. 7. Phosphate In 1970, Hilton proposed that inorganic phosphate caused exercise hypermia based on the observation that it was released from white muscle but not red muscle, consistent with the presence and absence of exercise hyperemia in the two muscles respectively. He also demonstrated that exogenous infusion of inorganic phosphate causes vasodilation which mimics exercise hyperemia. There are problems with this hypothesis. 10 Many other investigators have not been able to reproduce the latter result. Arterial infusions of highly concentrated inorganic phosphate do not raise vascular conductance of resting muscle significantly (0verbefl< et al., 1961; Dobsqn et al., 1971; Barcroft et al., 1971). Also exercise hyperemia is routinely demonstrated in high oxidative muscle, and as mentioned above there is 1K) evidence that inorganic phosphate is released from this muscle type. 8. Local Neurons Honig and Frierson (1976) recently proposed a local neurogenic mechanimn to account for a substantial portion of exercise hyperemia. This proposal was primarily based on the observation that arterial infusions of local anesthetics blocked the vasodilation associated with short exercise periods at 2 Hz. They concluded that the local anesthetics block ganglion cells which are intrinsic to the arteriolar vessel wall. This viewpoint, however, has not been widely accepted. It is debatable whether the effect of the anesthetics can be attributed to a specific blocking action on the intrinsic neurons. Until this specificity can be demonstrated further evaluation of this hypothesis will be difficult. This interesting hypothesis, however, deserves further investigation. 9.5T}: Abood (1962) ‘first (demonstrated that (depolarized skeletal muscle cells were capable of releasing ATP. It was subsequently demonstrated, both in man (Forrester and Lind, 1969) and dog (Chen et al., 1972), that exercising skeletal muscle released ATP into the venous effluent draining the muscle. Since the vasoactive potency of the adenine nucleotides had been known for some time (Drury and Szent-Gyorgyi, 11 1929), a role for ATP in exercise vasodilation was proposed. Although many reports have since appeared favoring this proposal, the ATP hypothesis has also received much criticimn. It appears that a major concern of many investigators is the unproven fact that a highly charged molecule such as ATP can get out of normal, intact cells. Regardless of whether or not this can be proven, one observation makes the evidence for the ATP hypothesis equivocal. Collingsworth and Selleck (1974) have demonstrated that when large amounts of ATP are infused into a dog gracilis muscle, perfused with Ringers Locke solution, only negligible quantities of ATP can be recovered in the venous effluent. Thus extracellular enzymes are capable of rapidly destroying extracellular ATP. Thus, it seems very unlikely that the ATP release seen by Forrester and Chen originated from skeletal muscle. It seems more likely that venous ATP came from some cell type (formed element) of the blood. Until the origin of the increased venous ATP seen during exercise can be determined, a role for ATP can not be substantiated. 10. Adenosine The ADO hypothesis may be stated as follows: The increases in vasculaz' conductance ‘which occur 10% of the ISF. In order to determine the potency of ADO, Phair and Sparks assumed that arterial plasma ADO equilibrated with ISF ,ADO. Since we are now aware that the endothelium has an extraordinary 17 capacity to accumulate ADO (Pearson et al., 1978), this assumption may not be valid. The ramifications of this are the following: if the vascular endothelium represents a metabolic sink for ADO, then a determination of the vasoactive potency of ADO based on exogenous ADO infusions may be a gross underestimation. This would make the detection of vasoactive ADO concentrations impossible given the current sensitivity of tissue ADO measurements. To summarize: Given the result that tissue ADO content did not increase during 10 min of free flow exercise, there are three alternative explanations which preclude rejection of the ADO hypothesis. First, the concentration of ADO in the interstitium necessary to cause exercise vasodilation might be too low to cause a detectable increase in tissue content, given the rmssibility of 2a relativeLv high backround intracellular content. Second, vasoactive ADO could be confined to a small perivascular region surrounding blood vessels, leaving most of the interstium free of ADO. Again, it might be impossible to detect this small compartment of ADO in a whole tissue measurement. Third, ADO may be released during exercise from preformed intracellular stores. One might expect to find a decrease in tissue ADO, Chm; to the subsequent metabolism of the released ADO. Although. these ‘hypotheses represent three edifferent, explanations for which a role for ADO can be reconciled, given the negative results obtained from tissue measurements, they all have one feature in common i.e. they sud. specify that 51 perivascular, vasoactive [ADO] is established during exercise. I reasoned that if in fact this occurs, some of the perivascular ADO should diffuse through the capillary wall 18 and enter the capillary pflasma. Further, this released ADO ought to appear in the venous blood, providing the blood doesn't completely degrade the ADO. It was recently shown in our laboratory, that when a good "stop" solution is used, blood degradation of ADO is slow enough to allow its quantitation in plasma by HPLC (Manfredi and Sparks, in press). With this in mind I felt a more rigorous test of the ADO hypothesis could be performed. It was the purpose of this thesis to test the following hypothesis. The increases in vascular conductance during 6 Hz free flow exercise are caused by increased ISF (ADO). I have tested the hypothesis by measuring arterial and venous plasma [ADO] and calculating ADO release. This test is based on the assumption that increased ISF (ADO) results in increased release of ADO into venous plasma which can be detected by increases in adenosine release. (ADO release measurements can be interpreted independently of the nature of the cellular ADO compartment of muscle). 19 II. MATERIALS AND METHODS A. Canine Hindlimb Preparation Male mongrel dogs weighing between 15 and 40 kg were used. Anesthesia was produced by intravenous sodium pentobarbital (30 mg/kg) supplemented throughout the experiment. To prevent clotting, an initial dose of heparin (750 units/kg) was administered just prior to cannulation of tnue muscle preparation, with hourly supplements of 100 units/kg. The dogs were ventilated so as to maintain arterial PCO 2 within the normal range. If necessary, the inspired room air was supplemented With 1007’ 02 to bring the arterial P02 into the normal range. When metabolic acidosis (pH < 7.35) occurred, it was corrected by an) intravenous drip cfi‘ 1.5% NaHCO3. Blood gases were monitored throughout the experiment and were maintained within the range of normal values supplied by Feigl and D'Alecy (1972). Esophageal temperature was maintained between 37-390C with thermostatically controlled heating pads. I used an isolated calf muscle preparation which we have previously described in detail (Mohrman and Sparks, 1973). The hindlimb was skinned by electrocautery, and the paw was vascularly isolated by ligating the anterior tibial artery and by securely tightening a hose clamp around the tibia and overlying tendons just proximal to the paw. The thigh muscles surrounding the femur were transected just proximal to the knee, as were all other structures in that region except for the major artery and vein. All small branch vessels not entering or exiting the muscles were ligated and cut. Two holes were drilled in the femur; the first was used for plugging the marrow with petroleum jelly-soaked 20 cotton balls, and the second for mechanically anchoring the limb. The sciatic nerve was ligated and cut, and the peripheral end was placed on bipolar silver electrodes. The portion of the calcaneus to which the gastrocnemius tendon inserts was transected and firmly attached to a specially adapted Grass force transducer for the measurement of gastrocnemius tension. Muscle length and stimulus voltage were adjusted to give maximum tension. The stimulation parameters were 2-5V, 0.2 msec and 6 Hz. These stimulus parameters excite skeletal muscle motor fibers but not sympathetic fibers, as confirmed by the absence of a vascular response after somatic neuromuscular blockade. The popliteal vein was cannulated just proximal to its bifurcation. The open end of the venous cannula, which was no more than 5 cm above the popliteal vein, emptied into a reservoir funnel from which a roller pump returned the blood to the contralateral femoral vein. The contralateral femoral artery supplied blood for perfusion of the calf through the cannulated popliteal artery. A constant pressure pump (Mohrman, 1980) and a Zepeda electromagnetic flow probe were interposed in the arterial perfusion line. The flowmeter was calibrated by linear regression of multiple timed collections cu‘ venous outflow cw) the corresponding oscillograph pen deflections. Perfusion pressure was monitored at the tip of the perfusion cannula via a Statham pressure transducer. Side taps in both the arterial and venous lines were used to sample blood for the analysis of oxygen content and adenosine concentration. The calf, as well as other skinned tissue exposed to the air, was wrapped in saline-soaked gauze and covered with Saran wrap to prevent evaporation. The ipsilateral brachial artery was cannulated for the measurement of arterial blood pressure and heart rate. After completion of the 21 surgery, the preparation was allowed approximately 15 minutes for equilibration. Mean aux! pulsatile blood pressure, perfusion pressure, gastrocnemius tension and blood flow were continuously recorded on a Grass Model 7 Polygraph. B. Protocol Before beginning the experiment, arterial blood gases, pH and hematocrit were measured and adjusted to the normal values if necessary. Two 3 ml blood samples were simultaneously drawn from the arterial and venous lines for the measurement of plasma adenosine concentration. In addition, a sample was drawn to determine the recovery of adenosine added to the sample, and another sample was treated with adenosine deaminase to check the specificity of the adenosine assay. Handling and processing of these samples is described below. Arterial and venous blood samples were drawn anaerobically for the determination of P02, PC02, pH, hematocrit, and hemoglobin content. Venous outflow was then measured by timed collection, and the occlusive zero of the electromagnetic flowmeter was checked by turning off the pump. In five dogs, two experimental maneuvers were performed in randomized order: 1. §.EE free flow exercise The muscle was stimulated at a rate of 6 Hz for a ten minute period during which the perfusion pressure was maintained constant at 100 mmHg. After ten minutes, blood samples were drawn as described above. A timed collection of the venous outflow from the muscle was performed. For a period of at least five minutes prior to sample collection, as well as during the sampling period, muscle blood flow was steady. The exercise 22 was then terminated and I waited until flow returned to control and took blood samples for blood gas and alveolar determinations. 2. Isoproterenol infusion Isoproterenol was infused intraarterially proximal to the pump at a rate which elicited flow increases similar to 6 Hz exercise hyperemia. The doses infused ranged from 3 to 28 ug/min. As soon as a steady state was reached with respect to blood flow, blood samples were collected as previously described. At the conclusion of each experimental intervention, flow was allowed to return to resting levels. I then waited at least ten additional minutes, and collected post-control blood samples. In six additional experiments, the effect of c.8-methylene adenosine 5'-diphosphate (AOPCP) was studied. In all dogs AOPCP (50 uM) was added to the collecting tubes for control blood samples. In four dogs AOPCP was also added to the collecting tube for the blood samples obtained during exercise. In two of these dogs and in two others, AOPCP was infused so as to establish a plasma concentration of AOPCP ranging from 20 MM to 87 pH 5 minutes after starting the exercise period. After ten minutes of exercise, and thus five minutes of AOPCP infusion blood samples were collected. After completion of the post-control period, the animals were euthanized with an overdose of pentobarbital. The calf muscles were removed and weighed so that blood flow could be normalized to the mass of tissue perfused. The tissue weights did not differ from the weights of the contralateral muscles. 23 C. Sample Preparation and Adenosine Assay Blood samples for adenosine analysis were gently drawn into 3 ml syringes and were dispensed into tared collecting tubes on ice. Each of these tubes contained 250 111 of a collection solution containing dipyridamole (26 uM), erythrononylhydroxyadenosine (EHNA) 3 HM, and methanol (5%) in isotonic saline. In seven experiments of series I, AOPCP (50 uM) was also included in some tubes. For the recovery sample in each experimental period, the collection solution also» contained approximately 0.3 nmole adenosine, which was sufficient to raise plasma adenosine concentrathmd in collected samples to about 0.30 LML Less than thirty seconds elapsed from the beginning of sample collection until the sample was mixed in the collection solution. Blood samples were centrifuged and one ml of supernatant was added to 250 pl 35% “CLO“, vortexed, and placed on ice. This procedure was followed for all samples except for those treated with adenosine deaminase (ADA). The collection solution for these samples contained 0.05 mg ADA (Sigma, Type I) in place of EHNA. These samples were incubated ten minutes at room temperature and then treated like the other samples. After treatment With ”CLO“, samples were centrifuged at 32,000 x g for fifteen minutes. The supernatant was decanted and neutralized with approximately 110 ul 9f 7'M K2C03. Samples were again centrifuged to remove the resulting precipitate and 700 1J1 of supernatant was stored at -20°C until analyzed. Adenosine was assayed by high pressure liquid chromatography (HPLC). One hundred 1 samples were injected onto either a uBondapak C18 (Waters Associates) or a partisil-S ODS (Whatman, Inc.) column. A linear gradient of 70/30 methanol/water (v/v) against AmM KHZPOA began 29 with 0% and ended twenty minutes later with “0% 70/30 methanol/water. Column flow was usually 1.5 ml/min. .For the tanalysis of certain experiments, slightly different flows (1.2 to 1.8 ml/min) gave better resolution (H? the adenosine peak. This adenosine assay has adequate sensitivity and precision (Manfredi and Sparks, in press). The limit of the sensitivity of the assay is approximately 4 pmoles of ADO in a water solution. The adenosine absorbance peak (at 254 nm) was identified by (1) correspondence of its retention time with that of standards, (2) absence of a corresponding peak in ADA-treated samples taken during the same period, and (3) an appropriately larger corresponding peak in recovery samples collected at the same time. Any period in which the recovery sample concentration was less than 80% of the predicted value was rejected. All periods in which ADA samples gave a peak greater than 30% of the corresponding adenosine peak in paired, untreated samples were rejected. When an ADA sample exhibited a measurable peak less than 30% of its paired sample, the residual peak was subtracted from the paired peak. Frequently an unknown substance which absorbs at 254 nm co-eluted with adenosine, precluding adequate resolution an? the adenosine peak. Approximately one-third of the experiments analyzed could not be accepted for this reason. Occasionally, such interference was present in one period but not others in the same animal. D. Data Analysis and Statistic Adenosine release (RADO) was calculated as the venous minus arterial difference in plasma adenosine concentration multiplied by Plasma fIOW- BIOOd 02 content was calculated from P02, pH and 25 hemoglobin concentration using a nomogram for dog blood. Oxygen consumption (V02) was calculated from arterial and venous 02 content and blood flow. In each animal, the data from an experimental intervention were accepted only when acceptable peaks were obtained from the intervention samples and either in“: pre— or post-intervention control samples. In cases where both tflwe pre— and post-intervention control samples bracketing an intervention were successfully analyzed, the results were averaged to give one control value. Each preparation was used for either one or two interventions (exercise, isoproternol, exercise plus AOPCP). Because not all experimental periods yielded acceptable adenosine measurements, data were grouped according to intervention rather than paired for each preparation. Grouped data were analyzed for statistical significance using Student's t-test. Arterial and venous plasma adenosine concentrations were compared using the paired t-test. Values stated in the text are means 1' standard deviation. 26 III. RESULTS A. Adenosine release during free flow 23.6 twitches/sec. Adenosine release during rest and steady state exercise is shown in Table I. Exercise caused vascular conductance to increase 1U-fold and V02 to increase 55-fold. RADO increased from -O.1:0.1 to 6.6_+_ll.6 nmoles/min/100g. B. Adenosine Release during infusion 32 Isoproterenol. We wished to test whether the increase in RADO seen with exercise would occur if blood flow were increased in the absence of exercise. We evaluated this possibility by infusing enough isoproterenol to raise blood flow as much as did 6 Hz exercise. The results of this experiment performed on four animals are presented in Table I. Isoproterenol caused larger changes in blood flow than were observed with exercise, but neither increased V02 nor release of adenosine was observed. C. Effect of AOPCP on release of adenosine during exercise. Another possibility for the observed exercise-induced RADO is that the increased venous plasma [ADO] resulted from the release of adenine nucleotides from formed elements in blood (e.g., platelets) and their subsequent degradation to adenosine via ecto-5'-nucleotidase. This is a possibility because aux: sample collecting solution prevents adenosine removal from plasma but not its formation. We reasoned that if this possibility is correct, addition of an inhibitor of ecto-5'-nucleotidase, AOPCP, to the collecting solution should prevent the increases in venous adenosine. We tested this hypothesis in four 27 experiments by repeating the exercise protocol with AOPCP in all the collecting tubes in a concentration (50 pH) which greatly inhibits formation of adenosine from AMP 1over the time period used in this experiment (Burger and Lowenstein, 1970; Shutz 6%: al., 1981). The values obtained for RADO in these experiments would then represent only that adenosine which has been formed prior to its mixture with the collection solution. Results from these experiments are presented in Table II. The increases in blood flow and VO2 were not significantly different from the remainder of the preparations (Table I). When AOPCP was present in the collection tubes, RADO increased from 0,210.2 to 3.812.11 nmole/min/100g during exercise (p (.05). This RADO was not significantly different from the value without AOPCP (6.6 :_ 4.6 nmol/min/lOOg). D: BADO 13 the presence 2f AOPCP infusion. Given the results of the previous experiment, we reasoned that the remaining ADO released during exercise may be the result of the extracellular degradation of released adenosine nucleotides by ecto-5'-nucleotidase occurring during intravascular transit through the muscle. We tested this possiblility by establishing 20-87 uM AOPCP in arterial plasma during exercise. Arterial samples were collected distal to the infusion site, so that AOPCP was present in both arterial and venous samples. Exercise produced statistically significant increases in flow and V02 in the presence of an AOPCP infusion (Table II). These increases were not significantly different from the values obtained in 28 the absence of the AOPCP infusion. RADO' u,3:2.7 nmole/min/lOOg. was significantly higher than rest, but also was not different from RADO in the absence of AOPCP. 29 .Amoocvmv OWH Uflm NM C003U®D OOCOLthHU QCNOHHHCme mwumcwwmmfl + .Amo.ovmv HoLunoo seem owcmno unmofiuwcmwm mopmcwwmoo c +m.mu>:.PI mo.Hmmo. :o.umoo. +s>Ho.ow + mm.Hmm. ao.:Hmo.o abo.Hom—. mo.Hm:o. mauom aom.Hm.z— FP.+:oo.I No.Homo. monwwo. mum: mo.umom. sm>.mn Poo. oeo. om we. mo.mp mpm. Nwo. om w.:P wom.l ozo. PFF. on or. =m=.mn oso. moF. om ma. Pw.F oao. omo. om >.mP w:o.| omo. mzo. s: oz. oom.m moo. mno. om :.=F wm—.I :so. oo_. 2: now. our. wmo. mmo. ow m.F =o.oP owP. omo. mm o.m_ Ppo.l ozo. mmo. om mm. Pm._ moo. mzo. om w—. omm.m mme. coo. om m.m_ moo.| omo. moo. om mop. aws.woopxmoHOE: > < m w: as aws.mmor\fie oo o> ommoaoc ocflmocoom new .om H came an oommoeaxo mosam> HH< osmuo.weH omH u>¢ .mmuo.omp xm o>< o.mnm.o azo u>< m.mep omH m.emp xm 3.5 azo em\m e.omm omH m.mmp xm 5.3. 420 mm\m .. omH m.mme xm m.w azo om\m o.ome omH F.m__ xm e.e nzo mm\= o.mme omH o.oo_ xm m.m nzo o\s ene.woop\as 30am coated some .3oHo coco um oomzocoo nswfiocfi: ocficmo on» mo m o> 30am ocean no HocoLouoLQOmH new omfiocoxo souflzu a: o oo uoommm .H canoe 30 .Amo.ovmv momo< 930:9“: ncm Sufi: xm cowsumn wocmumhmfie unmeamwcwfim mmamcwwmme + .Amo.ovmv Hegucoo Eocu omcmno ucmowoficwwm mopmcwfimoo e .om H some mm oommogaxo mo3Hm> HH¢ .momo< z om a pocfimacoo mono» ceauooaaoo oo< HH< oe.mus_m.s+.eo.uom_. mo.uso. +.summ +.o.uem.e. +oepum.P~F azH\xm u>< :=.mum>~.m =o.um>o. Po.Hmmo. amuom am.musm.mp apmum.oma xm o>< Pm.nmee. mo.umo. mo.ummo. euem eo.uomm. oeuw.m_ azu o>< mm:.e ems. mmo. In In o.mo. mzH\xm I: u- I- nu u- 1. xm oo.o oo.o oo.o a- u- m.m azo Pp\w oes.m wme. mso. om mm.o_ m.wm_ azH\xm u- -u u- -u n- u: xm AFM. woo. omo. mm New. o.o_ azo mm\e 1- I: u- u: u: nu nzH\xm ssm.m one. see. om mm.m_ w.mse xm mmm. omo. emo. ee mmm. w.em azo ep\s soe.m _mp. _mo. mm em.ee m.eoe azH\xm oem.e eme. emo. op mm.o. =.mme xm emo.u moo. mso. em ewe. n.0, azu mp\e mmo.m emp. oo_. mm e_.w_ m.PmF azH\xm eeo.m Nee. emo. em mm.sF e.ee_ xm ere. _eo. mmo. om emm. e.sm azo OM\e In I- I. u- 1- 1: dzH\xm F_m.m Pee. moo. mm me.m_ _.sme xm omo.- ems. omo. on mom. o._P azo e_\o ees.woo_\noHoEe > a m m: as see.moo_mHe ene.woo.\as conned some case A: o moose sanded cs nsoeo> o> scan m .omwoeoxo coves» n: e sea: soomnoonns case one o> goes noose eo oomemeo one so doao< do oooeoo one .HH oaooe 31 IV. DISCUSSION My experimental results demonstrate the following: (1) There is a Significant increase 1” RADO during sustained normoxic exercise hyperemia. (2) Increased blood flow caused by isoproterenol is not associated With increased RADO- (3) Exercise results in increased RADO even in the presence of an ecto-5'-nucleotidase inhibitor, AOPCP. A. Stimulus For ADO Production Previous studies have demonstrated that adenosine release increases during exercise when flow is restricted. Using a bioassay technique, Scott et al. (1965) provided evidence that adenosine (or AMP) is released from dog skeletal muscle tissue during ischemic exercise. Release of adenosine from severely ischemic muscle was then confirmed using chemical methods (Rubio et al., 1973). Release of adenosine was again demonstrated by Tominaga et al. (1980) under constant flow (but less severe) conditions. Tissue content of adenosine also increases during exercise when flow is held constant (Bockman et al., 1976). Although it is well established that hypoxia represents a sufficient stimulus for cellular increases in ADO production, these data can not be used to support a role for ADO during free flow, exercise hyperemia. It is quite clear that under the conditions of severe. cellular hypoxia, oxidative phosphorylation can not keep pace with the enhanced ATP hydrolysis associated with muscle contraction. This leads to the depletion of creatine phosphate stores, and quickly the [ADP] increases. By mass action through the myokinase reaction, an increase in [AMP] occurs (McGilvery and Murray, 1974). This supplies substrate for 32 S'Nucleotidase, for the subsequent formation of ADO. Further, S'Nucleotidase activity is simultaneously enhanced by the decrease in [CrP] and [ATP] (which normally inhibit the enzyme), as well as the increase in free [Mg++] (Rubio et al., 1979). - The important question remains: Can we invoke a role for ADO in the vasodilation seen with physiological exercise. Or more specifically: If contracting muscle cells are allowed adequate amounts of O2 to support ATP resynthesis, as occurs during free flow 90%) of the adenosine measured in tissue must not be in the interstitial space. This fits well with the current concepts concerning compartmentation of adenosine in the heart (Olsson et al., 1979; Schrader and Gerlach, 1976; Shutz et al., 1981). My data do not allow me to rule out the possibility that adenosine is limited to a relatively small perivascular space. This could be the result of localized release of adenosine from skeletal muscle cells (Rubio et al., 1973). Another possibility already discussed, is that adenosine is released from a cellular element of the vascular wall, e.g., endothelium (Nees et al., 1979). Either of these mechanisms could result in the release of adenosine observed in my experiments, but might not lead to a detectable rise in total tissue content. C. Significance 9f ADO Release Measurements Assuming that I am correct in concluding that the ISF [ADO] increased during exercise, eventually I will want to know whether it is great enough to be responsible for exercise vasodilation. Dose response curves from intraarterial infusion would suggest that an arterial plasma concentration of greater than ‘H) uM adenosine is necessary to produce the vasodilation observed during exercise (Phair and Sparks, 1979). This is much higher than the ISF concentration of ADO which I have calculated above (0.41 uM). A few years ago this analysis would have probably led to a premature rejection of the ADO hypothesis. Currently, however, the capability of endothelial cell uptake of ADO is now greatly 39 appreciated (Pearson et al., 1978). Because of this, both my estimate of ISF [ADO] as well as the vasoactive potency of ADO are suspect, since both of these estimates were determined with the assumption that the endothelium represents only a passive barrier for ADO diffusion. Providing that ADO is not transported completely through the endothelial cell, the greater the capacity for endothelial cell uptake the closer those two estimates will be. If this was the case ADO might be quite important in exercise vasodilation. On the other hand, if transendothelial ADO transport exists, the two estimates may differ by an even greater amount and thus the importance of ADO might be negligible. Till more is learned about the role of the endothelium in the transport of ADO, it will be impossible to predict the relative importance of ADO in the vasodilation associated with free flow exercise. 40 V. SUMMARY AND CONCLUSIONS In 0030193105. increased RADO «occurs during vigorous sustained exercise. This probably is the result of an increase in interstitial adenosine concentration. This increase in interstitial adenosine concentration could be confined to a small perivascular region, or distributed in the entire interstitium. Until more is known about the nature of capillary transport of adenosine, it will be difficult to state the quantitative importance of increased adenosine release during exercise vasodilation. BIBLIOGRAPHY in VI. BIBLIOGRAPHY Abood, L.G., K. Koketsu, and S. Miyamoto. Outflux of various phosphates during membrane depolarization of excitable tissues. Am. J. Physiol. 202:469-474, 1962. Bacchus, A., G. Gamble, D. Anderson, and J. Scott. Role of the myogenicresponse in exercise hyperemia. Microvasc. Res. 21:92-102, 19810 Barcroft, H., T.M. Foley and R.R. McSwiney. Experiments on the liberation of phosphate for the muscles of the human forearm during vigorous exercise and the action of sodium phosphate on forearm muscle blood vessels. J. 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