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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:2298-2304.)
© 1995 American Heart Association, Inc.

Articles

Activation of Protein Kinase C Increases Adenosine Production in the Hypoxic Canine Coronary Artery Through the Extracellular Pathway

Tetsuo Minamino; Masafumi Kitakaze; Kazuo Komamura; Koichi Node; Hiroshi Takeda; Michitoshi Inoue; Masatsugu Hori; Takenobu Kamada

From the First Department of Medicine (T.M., M.K., K.K., K.N., M.H., T.K.), Osaka University School of Medicine, Osaka, and the Department of Information Science (H.T., M.I.), Osaka University Hospital, Osaka, Japan.

Correspondence to Masafumi Kitakaze, MD, The First Department of Medicine, Osaka University School of Medicine, 2-2, Yamadaoka, Suita 565, Japan.

	Abstract

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Abstract Both ischemia and hypoxia increase adenosine production in the heart. This study tested whether hypoxia increases adenosine production in the coronary artery via ecto-5'-nucleotidase and the role of protein kinase C in this condition. Canine left circumflex coronary artery was rapidly removed and incubated in 10 mL Krebs-Henseleit solution for 30 minutes. The Krebs-Henseleit solution contained 5'-iodotubercidin and 2'-deoxycoformycin, which inhibit adenosine kinase and adenosine deaminase, respectively. Adenosine production was measured in intact coronary arteries under normoxic conditions (16.2±1.2 pmol/mg protein). Adenosine production was reduced by 27% after removal of endothelium. Ecto-5'-nucleotidase activity of coronary arteries with and without endothelium was 51±6 and 41±4 nmol/mg protein per minute under normoxic conditions. Hypoxia increased adenosine production to 27.0±2.3 and 20.0±0.8 pmol/mg protein with and without endothelium. Hypoxia also increased ecto-5'-nucleotidase activity of coronary arteries with and without endothelium (74±8 and 53±5 nmol/mg protein per minute; P<.05). Increases in adenosine production under hypoxic conditions were blunted by both an inhibitor of ecto-5'-nucleotidase and inhibitors of protein kinase C. Activation of ecto-5'-nucleotidase was blunted by an inhibitor of protein kinase C. These results indicate that hypoxia increased extracellular adenosine production and activated ecto-5'-nucleotidase via activation of protein kinase C in coronary arterial smooth muscle and endothelial cells. Increased adenosine production in coronary arteries during hypoxia may contribute to coronary vasodilation and cardioprotection against ischemic injury.

Key Words: coronary artery • hypoxia • adenosine • ecto-5'-nucleotidase • protein kinase C

	Introduction

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Adenosine is known to modify several key cellular processes in the heart.^{1 2 3} Adenosine relaxes vascular smooth muscle,^{4 5} inhibits platelet aggregation,^{6 7} inhibits generation of superoxide anion from polymorphonuclear leukocytes,^{8 9} prevents adherence of polymorphonuclear leukocytes to endothelium,¹⁰ and attenuates increases in myocardial contractility due to norepinephrine.^{11 12 13} These effects of adenosine may act synergistically to attenuate the dysfunction of both myocardium and coronary arteries after ischemia and reperfusion.^{14 15 16} We recently reported¹⁷ that endogenous adenosine inhibits platelet aggregation during myocardial ischemia in dogs, suggesting that adenosine prevents thromboembolism in coronary circulation in the ischemic period. Interestingly, adenosine is released not only from cardiomyocytes¹⁸ but also from the endothelial cells¹⁹ and SMCs²⁰ of the coronary arteries. Furthermore, both hypoxia and ischemia stimulate adenosine production in the heart.^{21 22 23} If adenosine production in coronary endothelial cells and SMCs is increased under hypoxic conditions, the increased local concentration of adenosine may effectively relax the coronary vessels and attenuate thromboembolism formed by the activated platelets and polymorphonuclear cells. However, it is not known whether or how adenosine production is increased in the coronary arteries by hypoxia.

This study first determined whether hypoxia increases adenosine production in endothelial cells and/or SMCs of coronary artery. Second, we examined whether hypoxia-induced increases in adenosine production occur via ecto-5'-nucleotidase. Finally, we investigated the role of PKC activation in the increases in ecto-5'-nucleotidase activity and adenosine production in hypoxic coronary arteries.

	Methods

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Preparation of Canine Coronary Arteries
Fresh hearts were removed from healthy mongrel dogs weighing 15 to 20 kg within 10 minutes of anesthesia (sodium pentobarbital 30 mg/kg IV). The left circumflex coronary artery was dissected (about 5 cm in length) and immersed in cold, modified KHS, pH 7.4, composed of (mmol/L) NaCl 118, KCl 5.9, NaH₂PO₄ 1.2, MgSO₄ 1.2, CaCl₂ 2.0, NaHCO₃ 25, and glucose 10. Nonvascular tissues adherent to the adventitial surface of the arteries were removed carefully, and all side branches were excised. To evaluate adenosine production in coronary arteries without endothelium, some coronary arteries were cut longitudinally, and the luminal surface was rubbed gently with a cotton stick swab. We histologically confirmed that this procedure removed almost all the endothelium. To obtain a steady state, coronary arteries with and without endothelium were equilibrated for 30 minutes in a chamber filled with the oxygenated KHS (20 mL) at 37°C.

Protocol 1: Adenosine Production in Canine Coronary Arteries With and Without Endothelium Under Normoxic and Hypoxic Conditions
After a 30-minute stabilization, coronary arteries were immersed in 10 mL of KHS with and without an adenosine deaminase inhibitor, deoxycoformycin (3x10^-5 mol/L), and an adenosine kinase inhibitor, 5'-iodotubercidin (5x10^-6 mol/L). Under normoxic conditions, the incubation medium was gassed with 95% O₂-5% CO₂ for 180 minutes, and then coronary arteries with and without endothelium were placed in this incubation medium gassing with 95% O₂-5% CO₂ during experiments. Under hypoxic conditions, the incubation medium was gassed with 95% N₂-5% CO₂ for 180 minutes, and then coronary arteries with and without endothelium were placed in this incubation medium gassing with 95% N₂-5% CO₂ during experiments. The oxygen pressure in the incubation medium at the end of 30 minutes of incubation was 24±8 mm Hg for incubation medium gassed with 95% N₂-5% CO₂ and 440±26 mm Hg for incubation medium gassed with 95% O₂-5% CO₂. Oxygen pressure was measured at 37°C (ABL310, Radiometer). We confirmed that lactate dehydrogenase is not detectable even in the hypoxic incubation medium, suggesting that irreversible injury of coronary arteries does not occur when they were placed in the hypoxic incubation medium.

We observed the time course of adenosine production by obtaining a 1-mL aliquot of incubation medium 0, 5, 10, 15, and 30 minutes after immersion of the coronary arteries. We have confirmed that the concentration of adenosine in the incubation medium reaches almost a steady state in a 30-minute incubation period. We adjusted the total volume of incubation medium by adding 1-mL aliquots of incubation medium to replace the samples removed. We calculated the adenosine concentration in each sample. Coronary arteries were removed from the incubation medium 30 minutes after the onset of incubation and frozen rapidly with liquid nitrogen. We measured 5'-nucleotidase activity of coronary arteries stored in liquid nitrogen.

Protocol 2: Effect of an Ecto-5'-Nucleotidase Inhibitor on Adenosine Production in Canine Coronary Arteries
Adenosine can be produced intracellularly by cytosolic 5'-nucleotidase and by S-adenosylhomocysteine, and extracellularly by ecto-5'-nucleotidase acting on released adenine nucleotides.^{3 24} We have recently reported²⁵ that ischemia activates ecto-5'-nucleotidase in vivo. To clarify the pathway by which adenosine production is increased during hypoxia, we tested the effects of an inhibitor of ecto-5'-nucleotidase, AOPCP, on adenosine production in canine coronary arteries with and without endothelium. AOPCP (5x10^-5 mol/L) was added to the incubation medium containing deoxycoformycin and 5'-iodotubercidin at the beginning of the incubation under both normoxic and hypoxic conditions. Adenosine concentration was measured immediately before and 5, 10, 15, and 30 minutes after the onset of pharmacological interventions.

Protocol 3: Effects of PKC Inhibitors on Adenosine Production and 5'-Nucleotidase Activity in Canine Coronary Artery
We have reported²⁶ that increases in ecto-5'-nucleotidase activity are mediated by activation of PKC in the hypoxic rat cardiomyocytes. To test whether increased ecto-5'-nucleotidase activity is mediated by activation of PKC in canine coronary arteries with and without endothelium, we incubated canine coronary arteries with and without the PKC inhibitors, GF109203X {3-[1-(3-dimethylaminopropyl)-indol-3-yl]-3-(indol-3-yl)-maleimide} (1x10^-7 mol/L) and staurosporine (1x10^-7 mol/L) using the procedure described in protocol 1. GF109203X is a cell-permeable protein kinase inhibitor that is structurally similar to staurosporine and inhibits PKC by acting as a competitive inhibitor of the ATP-binding site of PKC. It is reported that GF109203X is a highly selective and potent inhibitor of PKC compared with staurosporine, since its inhibition constants for PKC (K_i=1.4x10^-8 mol/L) and protein kinase A (K_i=2x10^-6 mol/L) are quite different.^{27 28} Adenosine production and 5'-nucleotidase activity were measured after 30 minutes of incubation. We confirmed that a 30-minute incubation time under the normoxic condition does not affect ecto-5'-nucleotidase activity.

Chemical Analysis
Measurement of Adenosine
The method for measuring the adenosine concentration has been reported previously.^{23 26} Briefly, after centrifugation of 1-mL KHS, which was extracted from the incubation chamber, the supernatant was obtained and the adenosine content was determined by radioimmunoassay. Adenosine was succinylated by 100 µL of dioxane containing succinic acid anhydride and triethylamine. After a 20-minute incubation, the mixture was diluted with 100 µL of adenosine 2',3'-o-disuccinyl-3-[¹²⁵I]-iodotyrosine methyl ester (0.5 pmol), and 100 µL of diluted anti-adenosine serum. The mixture was kept in a cold water (4°C) bath for 18 hours, and a second antibody solution (goat anti-rabbit IgG antiserum, 500 µL) was added. After incubation at 4°C for 1 hour, unreacted materials were removed by centrifugation at 2500g at 4°C for 20 minutes. The radioactivity remaining in the tube was counted by a gamma counter. Adenosine production is normalized by milligrams protein of coronary artery. The protein concentration was measured by the method of Lowry et al using bovine serum albumin as the standard. In the present study, 1 g wet wt of canine coronary artery with and without endothelium corresponded to 78.4±3.2 and 73.2±4.0 mg protein, respectively.

Measurement of 5'-Nucleotidase Activity
The method for measuring 5'-nucleotidase activity has been reported previously.²⁵ Briefly, the sampled canine coronary artery was frozen and stored under liquid nitrogen, and ecto-5'- and cytosolic 5'-nucleotidase activity was measured separately. The coronary artery was separated into its membrane and cytosolic fractions as follows: The artery was homogenized with a Potter-Elvehjem homogenizer (30 strokes) for 5 minutes in 10 vol of ice-cold 10 mmol/L HEPES-KOH buffer (pH 7.4) containing 0.25 mol/L sucrose, 1 mmol/L MgCl₂, and 1 mmol/L mercaptoethanol at 0°C. To remove large unbroken tissue, the crude homogenate was strained through a double-layer nylon sieve and homogenized again for 1 minute. To remove small unbroken tissue and nuclei fraction, the homogenate was centrifuged at 1000g for 10 minutes. To prepare the ectosolic and cytosolic fractions, the 1000g supernatant was centrifuged at 200 000g for 1 hour. We defined the resulting pellet and supernatant as membrane and cytosolic fractions, respectively. The membrane and cytosolic fractions were dialyzed at 4°C for 4 hours against 10 mmol/L HEPES-KOH (pH 7.4) containing 1 mmol/L MgCl₂, 1 mmol/L mercaptoethanol, and 0.01% activated charcoal and divided into aliquots that were frozen immediately and stored at -80°C. We defined the activity of membrane and cytosolic fractions as ecto-5'- and cytosolic 5'-nucleotidase activity, respectively. In a preliminary study, we examined the recovery of 5'-nucleotidase activity in the membrane fraction and observed a 95±2% recovery in ecto-5'-nucleotidase (n=5); this recovery was highly reproducible. Ecto-5'- and cytosolic 5'-nucleotidase activities were assessed by the enzymatic assay technique and are reported as nanomoles per milligram protein per minute in the membrane and cytosolic fractions each.

Statistical Analysis
All values are expressed as mean±SEM. The data of 5'-nucleotidase activity and adenosine concentration at the points measured were compared by using Bonferroni's multiple comparison analysis. Time courses in the changes of adenosine concentration in different treatment groups were compared by performing an ANOVA for repeated measures. A value of P<.05 was considered significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Adenosine Production in Canine Coronary Arteries With and Without Endothelium Under Normoxic and Hypoxic Conditions
Fig 1 shows the time courses of adenosine production in the normoxic coronary arteries with and without endothelium. Incubation with deoxycoformycin and 5'-iodotubercidine increased adenosine production (P<.05) in coronary arteries with and without endothelium (Fig 1). Adenosine production in canine coronary arteries with endothelium was higher (P<.05) than that without endothelium from 10 minutes onward.

View larger version (15K): [in this window] [in a new window]   Figure 1. Plot shows adenosine production in the canine coronary arteries with and without endothelium under normoxic conditions. Values are expressed as mean±SEM. Adenosine production is normalized by the total protein content of the coronary arteries. De indicates deoxycoformycin; Io, 5'-iodotubercidin; and E, endothelium.

Fig 2 shows the time courses of adenosine production in the hypoxic coronary arteries with and without endothelium. Adenosine production in coronary arteries with and without endothelium was increased (P<.05) under hypoxic conditions compared with normoxic conditions (Figs 1 and 2). In hypoxic coronary arteries with and without endothelium, 5'-iodotubercidin and deoxycoformycin increased adenosine production (P<.05, Fig 2). Adenosine production in canine coronary arteries with endothelium was higher than that without endothelium (P<.05) from 5 minutes onward.

View larger version (15K): [in this window] [in a new window]   Figure 2. Plot shows adenosine production in the canine coronary arteries with and without endothelium under hypoxic conditions. Adenosine production is normalized by the total protein content of the coronary arteries. De indicates deoxycoformycin; Io, 5'-iodotubercidin; and E, endothelium.

Effects of AOPCP on Adenosine Production in Canine Coronary Arteries With and Without Endothelium in Normoxic and Hypoxic Conditions
Under normoxic conditions, inhibition of ecto-5'-nucleotidase by AOPCP did not affect adenosine production in coronary arteries either with or without endothelium (Fig 3). In contrast, under hypoxic conditions, AOPCP blunted the increases in adenosine production in coronary arteries both with and without endothelium (P<.05, Fig 4).

View larger version (18K): [in this window] [in a new window]   Figure 3. Plots show effects of AOPCP on adenosine production in canine coronary arteries with (A) and without (B) endothelium under normoxic conditions. Adenosine production is normalized by the total protein content of the coronary arteries. E indicates endothelium.

View larger version (18K): [in this window] [in a new window]   Figure 4. Plots show effects of AOPCP on adenosine production in canine coronary arteries with (A) and without (B) endothelium under hypoxic conditions. Adenosine production is normalized by the total protein content of the coronary arteries. E indicates endothelium.

5'-Nucleotidase Activity in the Canine Coronary Arteries With and Without Endothelium Under Normoxic and Hypoxic Conditions
We observed ecto-5'- and cytosolic 5'-nucleotidase activity in coronary arteries, which were decreased by removing the endothelium (Fig 5). Hypoxia increased ecto-5'-nucleotidase activity but did not increase cytosolic 5'-nucleotidase activity in canine coronary arteries with or without endothelium (Fig 5). Inhibitors of PKC, staurosporine and GF109203X, did not affect ecto-5'-nucleotidase activity under normoxic conditions but blunted the activation of ecto-5'-nucleotidase under hypoxic conditions (Table 1). Furthermore, neither GF109203X nor staurosporine affected adenosine production of coronary arteries with endothelium under normoxic conditions. However, both chemicals blunted the hypoxia-induced increases in adenosine production (Table 2).

View larger version (27K): [in this window] [in a new window]   Figure 5. Bar graphs show ecto-5'- (top) and cytosolic-5'- (bottom) nucleotidase activity in canine coronary arteries with and without endothelium under normoxic and hypoxic conditions.

View this table: [in this window] [in a new window]   Table 1. Effect of PKC Inhibitors on Ecto-5'-Nucleotidase in the Canine Coronary Arteries With and Without Endothelium

View this table: [in this window] [in a new window]   Table 2. Effect of PKC Inhibitors on Adenosine Production in the Canine Coronary Arteries With and Without Endothelium

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The present study shows that hypoxia increases adenosine production and activates ecto-5'-nucleotidase via activation of PKC in coronary arterial SMCs and endothelial cells.

Adenosine Production in the Normoxic Canine Coronary Artery
Adenosine is known to be produced in cardiomyocytes¹⁸ and coronary endothelial cells and SMCs.^{19 20} In the present study, we focused on extracellularly produced adenosine via ecto-5'-nucleotidase in coronary endothelial cells and SMCs under normoxic and hypoxic conditions. To evaluate the capacity of adenosine production, we measured the adenosine concentration in the medium free from the degradation and rephosphorylation of adenosine, ie, the medium with deoxycoformycin and 5'-iodotubercidin.

Gerlach et al²⁹ have reported that adenosine production in the lower caval veins of the rabbit is decreased by approximately 60% after removal of the endothelial cells. Sedaa et al³⁰ have also reported that purine release is diminished by 90% after mechanical removal of the endothelium of rabbit aorta. The present study showed that the isolated canine coronary artery produces adenosine and that the amount of adenosine production is decreased by 30% by removal of the endothelial cells. Although the contribution of endothelial cells to adenosine production is a function of the animal species and vessels studied, these lines of evidence support the belief that both endothelial cells and SMCs play an important role in adenosine production in the vascular system.

Interestingly, adenosine almost reached a steady state level after 30 minutes' incubation in the presence and the absence of inhibitors of adenosine kinase and deaminase. This observation is consistent with Harn et al³¹ who observed that adenosine production in hog carotid artery plateaus without inhibitors of adenosine production under normoxic and hypoxic conditions. One possible explanation of this phenomenon was a negative feedback inhibition, ie, adenosine deactivates ecto-5'-nucleotidase. Indeed, we found²⁶ that adenosine deactivates ecto-5'-nucleotidase in rat cardiomyocytes, which may serve as a negative feedback loop. Another explanation is depletion of AMP as a substrate for ecto-5'-nucleotidase. The activity of ecto-5'-nucleotidase increases in almost linear fashion with increases in AMP concentration, suggesting that depletion of AMP may deactivate ecto-5'-nucleotidase.³²

Mechanisms Underlying Increases in Adenosine Production During Hypoxia: Role of Activation of Ecto-5'-Nucleotidase
Adenosine production in the heart increases during hypoxia.^{21 22 23} The present study shows that hypoxia increases adenosine production in canine coronary arteries with and without endothelium, indicating that hypoxia increases adenosine production in both coronary endothelial cells and SMCs. However, Shryock et al³³ have reported that the release of adenosine from pig aortic endothelial cells during hypoxia does not increase. This difference may be due to the use of cultured endothelial cells in the study of Shryock et al and to the use of inhibitors of enzyme related to adenosine production and degradation in the present study. On the other hand, Harn et al³¹ have reported that adenosine production in isolated hog carotid artery is lower during hypoxia than during normoxia and suggested that adenosine is more rapidly eliminated during normoxia in the medium without inhibitors of adenosine kinase and adenosine deaminase. This study suggests that hypoxia may increase the activities of these enzymes as well as ecto-5'-nucleotidase. In the present study, we focused on the capacity of adenosine production via ecto-5'-nucleotidase and the mechanism by which adenosine production increases under hypoxic conditions. Since we observed increases of adenosine production in the presence of deoxycoformycin and 5'-iodotubercidin, we suggest that activity of ecto-5'-nucleotidase is one of the factors involved in adenosine production in the hypoxic coronary arteries.

It has been shown that AMP hydrolysis is the major pathway for myocardial adenosine production under normoxic conditions³⁴ and under ischemic and hypoxic conditions in both isolated^{35 36 37} and in vivo hearts.^{38 39} In the present study, the inhibition of ecto-5'-nucleotidase did not significantly affect adenosine production under normoxic conditions. This finding suggests that extracellular AMP hydrolysis is not the major pathway under normoxic conditions, but intracellular AMP hydrolysis due to cytosolic 5'-nucleotidase may be important. Headrick et al⁴⁰ demonstrated that AOPCP reduces adenosine levels in coronary venous effluent but not in interstitial transduate fluid under normoxic conditions, indicating that adenosine is produced via ecto-5'-nucleotidase under normoxic conditions in the isolated guinea pig model. The difference in findings between our study and that of Headrik et al may be attributable to the differences in either experimental model or choice of animals: We examined the pathways of adenosine production under normoxic and hypoxic conditions using the canine coronary arteries per se, while Headrick et al used the in vivo model of isolated guinea pig hearts in which nucleotides may be supplied from tissue cells surrounding coronary arteries.

We need to consider the possibility that a new equilibrium may be attained that may compensate for decreased production of adenosine by extracellular nucleotides under steady state conditions. Indeed, there is a possibility that adenosine production under normoxic conditions in the presence of AOPCP is nonspecific, possibly due to alkaline phosphatase activity or ATP release from cells injured in the preparation. On the other hand, under hypoxic conditions, inhibition of ecto-5'-nucleotidase by AOPCP suppressed the increase in adenosine production, suggesting that the extracellular adenosine production by ecto-5'-nucleotidase plays a crucial role in an increase in adenosine production under hypoxic conditions in coronary endothelial cells and SMCs. One of the mechanisms that increases adenosine production under hypoxic conditions is the increased ecto-5'-nucleotidase activity. Ecto-5'-nucleotidase is allosterically activated by free Mg²⁺ and is inhibited by ATP, adenosine diphosphate, and inorganic phosphate.^{41 42} We and others have shown that both ischemia and hypoxia increase ecto-5'-nucleotidase activity in the canine myocardium²⁵ and in the isolated rat heart.⁴³ The present study also shows that hypoxia increases ecto-5'-nucleotidase activity in both coronary endothelial cells and SMCs. Since hypoxia increases 5'-AMP release from vessels,²⁸ another possible mechanism for the increase in adenosine production during hypoxia is an increase in the concentration of 5'-AMP, the substrate for adenosine.

Kitakaze et al²⁶ have recently reported that increased 5'-nucleotidase activity by PKC enhances adenosine production in the hypoxic rat cardiomyocytes. Strasser et al⁴⁴ have reported that prolonged myocardial ischemia translocates PKC from the cytosolic fraction to the membrane fraction. These data suggest that hypoxia and ischemia may stimulate 5'-nucleotidase activity through activation of PKC. In the present study, we used staurosporine and GF109203X as PKC inhibitors. Both chemicals blunted activation of ecto-5'-nucleotidase in canine coronary arteries with and without endothelium during hypoxia, indicating that the increase in ecto-5'-nucleotidase activity during hypoxia is mediated via activation of PKC.

Clinical Implications
The amount of adenosine production in the coronary arteries may be small relative to cardiomyocytes.^{44 45} However, coronary adenosine production may act directly on the coronary arteries in autocrine and paracrine fashions. Furthermore, activated leukocytes and platelets in coronary vessels, which may cause thromboembolism and ischemia and reperfusion injury, initially attack the endothelium and form thromboembolisms. Thus, adenosine produced in the coronary vessels may play a key role in attenuating ischemic and reperfusion injury.^{3 47} Indeed, we demonstrated⁴⁸ that intracoronary infusion of ATP, which is degradated to adenosine, reduces the extent of no-reflow and infarct size in dogs. Clarification of the underlying mechanisms that increase adenosine production in the coronary arteries during ischemia may provide new strategies for the treatment of ischemic heart diseases.

	Selected Abbreviations and Acronyms

 

AMP = adenosine monophosphate AOPCP = ,ß-methyleneadenosine 5'-diphosphate KHS = Krebs-Henseleit bicarbonate solution PKC = protein kinase C SMC(s) = smooth muscle cell(s)

	Acknowledgments

 
We acknowledge the assistance of Noriko Tamai in measuring 5'-nucleotidase activity and of Shinya Suzuki in preparing the canine coronary arteries.

Received July 11, 1995; accepted September 21, 1995.

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