Measurement by fluorescence of interstitial adenosine levels in normoxic, hypoxic, and ischemic perfused rat hearts

An improved assay was used to investigate the effects of hypoxia or ischemia on interstitial fluid and coronary venous effluent levels of adenosine in isolated perfused nonworking rat hearts. The adenosine in 5- to 10-microliter samples of left ventricular epicardial surface transudates and coronary effluents was reacted with chloroacetaldehyde, and the fluorescent derivative (1,N6-ethenoadenosine) was quantitated using high pressure liquid chromatography and fluorescence detection. Hearts responding to hypoxia could be separated into two groups. In one group of hearts, the control (normoxic) transudate and effluent adenosine concentrations were 94 +/- 24 and 41 +/- 6 pmol/ml, respectively. These values increased by 118 and 96%, respectively, with 5 minutes of hypoxia (30% O2), and returned to control levels 5 minutes after resumption of normoxia. In a second group of hearts, the normoxic control levels of adenosine in the transudates (42 +/- 7 pmol/ml) and coronary effluents (62 +/- 17 pmol/ml) were increased with hypoxia by 174 and 1,178%, respectively. However, the transudate levels continued to rise for 5 minutes after resumption of normoxic perfusion while effluent levels fell. In another series of hearts, global ischemia for 30 seconds elicited an elevation of transudate adenosine levels by 362 to 641% above control (58 +/- 15 pmol/ml) as determined 30 seconds after resumption of perfusion flow.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of graded perfusion and isovolumic work on epicardial and venous adenosine and cytosolic metabolism

Epicardial adenosine levels and venous adenosine release were measured in isovolumically contracting (ISO) and empty non-isovolumic (non-ISO) guinea-pig hearts subjected to graded perfusion (approximately 7.5, 5.5, 4.0, 2.0, and 1.0 ml/min/g). Myocardial metabolism was monitored using 31P-NMR spectroscopy. At flows of 5.5 ml/min/g or higher epicardial adenosine levels were stable and comparable in ISO and non-ISO hearts (approximately 160 nM). At flows of 4.0 ml/min/g or higher venous adenosine release was stable and comparable in ISO and non-ISO hearts (approximately 30 pmol/min/g). At lower flows, epicardial adenosine and venous adenosine release both increased and were significantly higher in ISO hearts, compared to non-ISO hearts, at each flow rate. Whereas epicardial adenosine increased linearly in ISO and non-ISO hearts at low flows, venous adenosine release stabilized in ISO hearts perfused at 1.0 ml/min/g. Epicardial adenosine, venous adenosine release, and log [ATP]/[ADP] [Pi] all displayed significant correlations with the O2 supply:demand ratio which were comparable in ISO and non-ISO hearts. Elevated levels of epicardial adenosine were linearly related to log [ATP]/[ADP] [Pi] and cytosolic [AMP] and these relationships were comparable in ISO and non-ISO hearts. Alternatively, changes in venous adenosine release did not display simple relationships with log [ATP]/[ADP] [Pi] and cytosolic [AMP] and they were not comparable in ISO and non-ISO hearts. The data indicate that: (i) myocardial adenosine formation increases only below a metabolic threshold corresponding to log [ATP]/[ADP] [Pi] = 5.0 and O2 supply:demand = 1.5 in ISO and non-ISO guinea-pig hearts; (ii) stimulated epicardial adenosine levels appear to be consistently related to changes in cytosolic metabolism below this threshold in ISO and non-ISO hearts; (iii) more complex relationships exist between venous adenosine release and myocardial metabolism during graded perfusion, possibly reflecting the variety of factors modulating venous adenosine release.     

Adenine nucleotide release from isolated perfused guinea pig hearts and extracellular formation of adenosine.

The quantification of adenine nucleotides released from the heart is hampered by their rapid dephosphorylation to adenosine in the extracellular space catalyzed by highly active ectonucleotidases. To determine the total release of adenine nucleotides from isolated Langendorff-perfused guinea pig hearts, ecto 5'-nucleotidase was effectively blocked by infusion of alpha, beta-methylene-ADP (AOPCP, 50 microM). Adenine nucleotides were measured in the coronary venous effluent by the luciferin-luciferase method after enzymatic rephosphorylation to ATP. In hearts perfused at a constant flow rate (10 ml/min) with normoxic buffer (95% O2, 5% CO2) the release +/- SEM of adenine nucleotides and adenosine was 0.06 +/- 0.01 (n = 11) and 0.04 +/- 0.01 (n = 13) nmol/min. In the presence of AOPCP, the release of adenine nucleotides increased to 0.43 +/- 0.04 nmol/min (n = 9; p less than 0.05), whereas adenosine remained unchanged. Hypoxic perfusion (10% O2, 85% N2, 5% CO2) caused a threefold increase in adenine nucleotide release but a 40-fold increase in adenosine. In contrast, global ischemia (30 seconds) caused adenine nucleotide and adenosine release to rise to similar values of 1.06 +/- 0.10 and 0.80 +/- 0.14 nmol/min (n = 9). Stimulation of hearts with isoproterenol (4 nM) likewise increased the release of adenine nucleotides (0.50 +/- 0.04 nmol/min) and adenosine (0.87 +/- 0.21 nmol/min) (n = 6). To determine the cellular source of adenine nucleotides released from the heart, the coronary endothelial adenine nucleotide pool was selectively prelabeled by [3H]adenosine. Global ischemia increased the specific radioactivity of released adenine nucleotides by 57%. The findings indicate that 1) adenine nucleotides and adenosine are released at the same order of magnitude from the well-oxygenated heart; 2) beta-adrenergic stimulation and ischemia stimulate the release of adenine nucleotides and adenosine, both purines reaching vasoactive concentrations in the effluent perfusate; 3) during hypoxic perfusion only the release of adenosine is greatly enhanced; and 4) the coronary endothelium preferentially contributes to the ischemia-induced adenine nucleotide release.     

Adrenergic blockade blunts adenosine concentration and coronary vasodilation during hypoxia.

Myocardial hypoxia is thought to be an important stimulus for increasing interstitial adenosine concentration. The adenosine hypothesis of coronary control was investigated during steady-state hypoxia by making measurements of coronary venous and epicardial well adenosine concentrations in adrenergically intact dogs and in animals with alpha- and beta-receptor blockade. In the adrenergically intact group, hypoxia sufficient to lower coronary venous oxygen tension to 8 mm Hg increased coronary blood flow 243% from normoxic values. Both coronary venous and epicardial well adenosine concentrations were increased throughout the hypoxic period. In the adrenergically blocked group, hypoxia to a similar level of coronary venous oxygen tension produced an increase in coronary blood flow of only 75%, which was significantly less than in the adrenergically intact group (p less than 0.01). Coronary venous adenosine was only transiently elevated, and epicardial well adenosine was unchanged from control levels. In a separate group of alpha- and beta-receptor-blocked animals that received an infusion of L-homocysteine thiolactone during hypoxia, there was no difference in tissue S-adenosylhomocysteine levels compared with those of normoxic controls. It is concluded that much of the coronary vasodilation associated with systemic hypoxia is dependent on adrenergic activation and that adenosine may only play a role in sustained hypoxic vasodilation when adrenergic receptors are intact.     

Evidence that cytosolic and ecto 5'-nucleotidases contribute equally to increased interstitial adenosine concentration during porcine myocardial ischemia

The purpose of this study was to determine the roles of cytosolic and ecto 5'-nucleotidase in myocardial ischemia-induced increases in interstitial fluid (ISF) adenosine. Pentobarbital anesthetized, open chest pigs were instrumented with two microdialysis fibers in the distally perfused bed of the left anterior descending (LAD) coronary artery to estimate ISF metabolites. Fibers in control hearts were perfused with standard Krebs buffer. In two additional groups, after collecting one dialysate sample with normal Krebs, fibers were perfused with buffer supplemented with either L-homocysteine thiolactone (5 mM) or the ecto 5'-nucleotidase inhibitor α, β-methylene adenosine 5'-diphosphate (AOPCP, 5 mM). Hearts were then submitted to 60 minutes LAD occlusion and two hours reperfusion. Dialysate nucleosides and AMP were measured by high performance liquid chromatography. The local delivery of homocysteine did not alter preischemic dialysate adenosine concentration (0.30 ± 0.04 μM) compared to pre-homocysteine infusion (0.39 ± 0.04 μM) or control hearts (0.36 ± 0.04 μM), but AOPCP significantly decreased preischemic dialysate adenosine levels (from 0.36 ± 0.02 to 0.14 ± 0.03 μM). During LAD occlusion both homocysteine and AOPCP reduced dialysate levels by approximately 50 %. At 30 minutes ischemia dialysate adenosine concentrations were 19.47 ± 2.72, 11.41 ± 2.44, and 7.93 ± 1.01 μ:M in control, homocysteine, and AOPCP hearts, respectively. AOPCP significantly increased dialysate AMP levels; at 60 minutes ischemia AMP levels were 6.22 ± 2.97 μM in control hearts and 38.60 ± 5.69 μM in AOPCP treated hearts. These results suggest that both cytosolic and ecto 5'-nucleotidase contribute to ischemia-induced increases in ISF adenosine in porcine myocardium. Received: 13 October 1998, Returned for revision: 5 November 1998, Revision received: 17 December 1998, Accepted: 4 January 1999     

Ecto-5'-nucleotidase plays a role in the cardioprotective effects of heat shock protein 72 in ischemia-reperfusion injury in rat hearts

OBJECTIVE: Heat shock protein 72 (HSP72) is involved in the myocardial self-preservation system under several conditions such as ischemia-reperfusion injury or late preconditioning. However, its mechanism is not fully understood. Ecto-5'-nucleotidase is a key enzyme for synthesizing adenosine and plays an important role in ischemic preconditioning. In this study, we tested the hypothesis that ecto-5'-nucleotidase plays a role in the cardioprotection of HSP72. METHODS: Rat hearts (H group, n=6) were transfected with HSP72 gene by an intracoronary infusion of hemagglutinating virus of Japan (HVJ)-liposome complex. Control hearts (C group, n=6) were transfected with the beta-galactosidase gene. Following 30 min of normothermic ischemia, grafts were reperfused using Langendorff apparatus. RESULTS: The activity of ecto-5'-nucleotidase was significantly higher in H group than C group both before and after ischemia-reperfusion (H vs. C; 0.51+/-0.05 vs. 0.29+/-0.06, and 1.41+/-0.15 vs. 0.85+/-0.11 nmol/mg protein/min, P<0.05). H group also showed significant better functional recoveries than C group (P<0.05), as well as less creatine phosphokinase leakage (4.4+/-2.8 vs. 14.2+/-3.4 mU/min, P<0.05) and higher adenosine release (247.5+/-35.1 vs. 54.3+/-1.7 pmol/min, P<0.05). Administration of alpha,beta-methylene adenosine diphosphate (AMP-CP), an inhibitor of ecto-5'-nucleotidase, significantly diminished the tolerance to ischemia-reperfusion injury in H group (P<0.05). CONCLUSION: These results demonstrated that ecto-5'-nucleotidase activated by an overexpression of HSP72 attenuated ischemia-reperfusion injury in the rat myocardium. They suggest that ecto-5'-nucleotidase plays a role in the cardioprotective effects of HSP72 in rat hearts.     

Formation of S-adenosylhomocysteine in the heart. I: An index of free intracellular adenosine

To assess the concentration of free intracellular adenosine in the heart the kinetic properties of cytosolic S-adenosylhomocysteine (SAH) hydrolase were utilized at elevated levels of L-homocysteine (adenosine + L-homocysteine in equilibrium with SAH + H2O). Global hypoxia was induced in the isolated perfused guinea pig heart by graded reduction of perfusion medium PO2 in the presence of saturating concentrations of homocysteine (0.2-1.0 mM). Reduction of PO2 from 660 to 165 mm Hg increased the steady-state concentration of total tissue adenosine from 2.0 +/- 0.2 to 2.8 +/- 0.2 nmoles/g, while the rate of SAH formation increased linearly from 0.22 +/- 0.03 to 2.50 +/- 0.13 nmoles/min/g. When adenosine was exogenously applied at a concentration of 100 microM together with homocysteine (1 mM), SAH accumulation rates were much greater: 23.34 +/- 3.31 and 42.11 +/- 1.73 nmoles/min/g with normoxic (95% O2) and hypoxic (30% O2) perfusion, respectively. The apparent Km and Vmax values for SAH-hydrolase in vivo were estimated to be 20 microM and 59 nmoles/min/g wet wt, respectively. Since the relation between SAH formation and adenosine in the physiological concentration range is linear, the measured rate of SAH accumulation during normoxia and hypoxia permitted the calculation of the free intracellular adenosine level, which was 0.061 nmoles/g (0.08 microM) in the normoxic heart. With hypoxia (PO2 165 mm Hg), this value increased to 1.57 nmoles/g (2.0 microM). Free intracellular adenosine closely correlated with the hypoxia-induced changes in coronary flow. The data reveal that measurement of the rate of SAH accumulation during homocysteine infusion can be used for sensitive assessment of free intracellular adenosine levels. Assuming that the intracellular adenosine concentration equals that in the interstitial space, the results furthermore indicate that the degree of intracellular adenosine formation during hypoxic perfusion is quantitatively sufficient to account for most of the observed increases in coronary flow.     

Estimates of interstitial adenosine from surface exudates of isolated rat hearts

The adenosine concentration of exudate formed on the surface of isolated perfused rat hearts has been used to obtain estimates of interstitial values. At a constant perfusion of approximately 15 ml/min/g, exudate was collected from below ring seals that either fitted snugly (compressing seals) or that acted as wicks (wicking seals) to deflect venous effluent away from the apical surface. Steady state exudates flows obtained below each of these seals were 0.96 +/- 0.05 ml/min and 0.18 +/- 0.02 ml/min, respectively. Adenosine concentration of surface exudate and venous effluent from hearts with the compressing seal were 130 +/- 8 nM and 23 +/- 3 nM, respectively, and from those with the wicking seal were 770 +/- 93 nM and 36 +/- 9 nM, respectively. Interstitial adenosine concentration in a situation with no net filtration may be slightly higher than that achieved in the exudate from preparations with the wicking seal. Addition of exogenous adenosine to the perfusate (1.0 microM) decreased vascular resistance and automaticity of all preparations, increased the venous effluent adenosine concentration to 236 +/- 18 nM and 251 +/- 30 nM with the compressing and wicking seals, respectively, but did not significantly alter the exudate adenosine concentration with either of the seals. This finding suggests that increases in vascular adenosine may influence functional characteristics without altering interstitial levels. Perfusion with 10 microM adenosine increased adenosine concentration in both effluent and exudate in all preparations but the gradient was reversed so that effluent levels were significantly higher than exudate levels. We conclude that venous adenosine determinations significantly underestimate the interstitial adenosine concentration associated with endogenous adenosine production and significantly overestimate the interstitial levels achieved by infusion of exogenous adenosine.     

Inhibition of adenosine metabolism increases myocardial interstitial adenosine concentrations and coronary flow.

We employed an isolated guinea-pig heart model perfused at constant pressure (70 cmH2O) to test the hypothesis that inhibition of adenosine metabolism increases interstitial adenosine concentrations (as measured with epicardial discs) and coronary flow. Iodotubercidin (ITU, 1 microM) and EHNA (erythro-9-[2-hydroxy-3-nonyl] adenine, 5 microM) were used to inhibit adenosine kinase and deaminase, respectively during control conditions and during metabolic stimulation with 1 microM isoproterenol. The adenosine receptor blocker 8-phenyltheophylline (8-PT) was used during control conditions to assess whether the response seen was adenosine specific. ITU plus EHNA decreased heart rate (202 +/- 10 to 136 +/- 11 beats/min) and increased coronary flow (8.2 +/- 0.3 to 12.4 +/- 0.9 ml/min/g) without a change in MVO2, developed pressure or dP/dt. ITU plus EHNA increased adenosine concentrations in epicardial fluid (0.24 +/- 0.07 microM to 1.02 +/- 0.09 microM) and venous effluent (40 +/- 3 nM to 262 +/- 32 nM) during control conditions, and adenosine release increased from 389 +/- 96 pmols/min/g to 3480 +/- 365 pmols/min/g. 8-PT infusion reversed the effects on heart rate and coronary flow and resulted in a persistent elevation of epicardial fluid adenosine concentrations. During metabolic stimulation with 1 microM isoproterenol, ITU plus EHNA significantly limited the increase in heart rate and ventricular developed pressure and dP/dt while coronary flow increased to a significantly greater extent. Myocardial oxygen consumption was similar during metabolic stimulation between the two groups (vehicle vs. ITU plus EHNA). Epicardial fluid adenosine concentration in the vehicle-treated group increased from 0.17 +/- 0.3 microM to 0.34 +/- 0.02 microM at 15 min of isoproterenol stimulation whereas it increased from 1.10 +/- 0.02 microM to 2.90 +/- 0.46 microM in the ITU plus EHNA-treated group. Inhibition of adenosine metabolism during metabolic stimulation significantly increased venous adenosine concentrations and adenosine release and reduced inosine and hypoxanthine release proportionately. The release of adenosine+inosine+hypoxanthine was unchanged. Inhibition of adenosine metabolism provides evidence supporting the hypothesis that adenosine plays a role in regulating coronary vascular resistance as well as influencing heart rate and ventricular inotropy.     

Ventilatory responses to hyperkalemia and exercise in normoxic and hypoxic goats

The ventilatory response to moderate exercise is potentiated during hypoxia in goats, causing PaCO2 to decrease more from rest to exercise than in normoxia. We investigated the hypothesis that this response is due to the ventilatory stimulus provided by an interaction between exercise induced hyperkalemia and hypoxia. Plasma potassium concentration ([K+]), arterial blood gases and ventilation were measured in normoxia and hypoxia (PaO2 = 34-38 Torr) at rest and during steady-state exercise (5.6 kph; 5% grade) in seven goats. PaCO2 decreased during normoxic exercise (2.9 +/- 0.7 Tor; P less than 0.01), and decreased significantly more during hypoxic exercise (6.4 +/- 0.6 Torr; P less than 0.01). [K+] increased in both normoxic (1.0 +/- 0.1 mEq/L; P less than 0.01) and hypoxic (0.9 +/- 0.2 mEq/L; P less than 0.01) exercise, but these changes were not significantly different from each other. On a different day, resting goats were infused intravenously with 200 mM KCl for 5 min at a rate sufficient to obtain [K+] similar to exercise (8.6-12 ml/min) in normoxia and hypoxia. Hyperkalemia at rest caused similar PaCO2 decreases in normoxia (1.7 +/- 0.7 Torr; P less than 0.05) and hypoxia (1.7 +/- 0.5 Torr; P less than 0.01), but had no statistically significant effect on ventilation in either condition. These data indicate that hyperkalemia, at levels approximating those during moderate exercise, has a mild stimulatory effect on alveolar ventilation; however, hypoxia does not affect this response. We conclude that hyperkalemia does not provide sufficient ventilatory stimulation to account for exercise hyperpnea, nor does hypoxia potentiate the ventilatory stimulation from hyperkalemia at rest.     

Improvement by 5-amino-4-imidazole carboxamide riboside of the contractile dysfunction that follows brief periods of ischemia through increases in ecto-5-nucleotidase activity and adenosine release in canine hearts.

5-Amino-4-imidazole carboxamide (AICA) riboside increases adenosine release in ischemic myocardium, suggesting that AICA riboside improves contractile dysfunction. In 49 open-chest dogs, contractile function assessed by fractional shortening (FS) was observed 3 h after the onset of reperfusion following 15 min of occlusion of the left anterior descending coronary artery. During reperfusion, the treatment with AICA riboside increased adenosine concentration in the coronary venous blood (536+/-44 vs. 281+/-21 pmol/ml at 3 min of reperfusion, p<0.001) and peak coronary hyperemic flow (367+/-13 vs. 300+/-21 ml/100 g per min, p<0.001) when compared with the untreated group. FS at 3h of reperfusion increased in the AICA riboside group (21.1+/-2.3 vs. 12.8+/-0.6% in the untreated group, p<0.001). AICA riboside increased myocardial ecto-5'-nucleotidase activity. Administration of adenosine also augmented coronary hyperemic flow and increased FS to the levels of the AICA riboside group. Either 8-phenyltheophylline (an antagonist of adenosine receptors) or alpha,beta-methylene-adenosine 5'-diphosphate (an inhibitor of ecto-5'-nucleotidase) completely abolished the increased coronary hyperemic flow and improvements of myocardial contractile function due to AICA riboside. Thus it was concluded that AICA riboside improves the contractile dysfunction that follows a brief period of ischemia via adenosine-dependent mechanisms.     

Adenosine production and energy metabolism in ischaemic and metabolically stimulated rat heart

Adenosine may modulate blood flow and electrical activity in heart in response to changes in myocardial energy metabolism. In the present study, 31P NMR spectroscopy was used to examine the relation between cytosolic phosphate metabolite levels and release of adenosine into the venous effluent of isovolumic heart during graded low-flow ischaemia or metabolic stimulation with isoproterenol. When coronary flow rate was varied in steps between 1.6 and 12 ml/min/g, cytosolic ATP levels did not change significantly but the phosphorylation potential exhibited a linear correlation with flow rate below approximately 7 ml/min/g. Purine release (adenosine and inosine) correlated linearly with the cytosolic phosphorylation potential and free AMP concentration. Metabolic stimulation of hearts with isoproterenol (0.4, 3.0, and 60 nM), produced a significant fall in cytosolic ATP levels and decreased the cytosolic phosphorylation potential. Purine release in these hearts increased exponentially as the cytosolic phosphorylation potential dropped, and as cytosolic free AMP increased. These results support a link between the phosphorylation potential and the mechanism of adenosine production during ischaemia and metabolic stimulation. Presumably, this link is the activity of the enzyme 5'-nucleotidase, which is responsible for converting AMP to adenosine, together with the concentration of its substrate, AMP. In low-flow ischaemia, cytosolic AMP may control adenosine formation. With isoproterenol stimulation, a more complex relationship exists, indicating possible allosteric regulation of the enzyme(s) responsible for adenosine formation, in addition to changes in AMP concentration.     

Increased mitochondrial K(ATP) channel activity during chronic myocardial hypoxia: is cardioprotection mediated by improved bioenergetics?

Increased resistance to myocardial ischemia in chronically hypoxic immature rabbit hearts is associated with activation of ATP-sensitive K(+) (K(ATP)) channels. We determined whether chronic hypoxia from birth alters the function of the mitochondrial K(ATP) channel. The K(ATP) channel opener bimakalim (1 micromol/L) increased postischemic recovery of left ventricular developed pressure in isolated normoxic (FIO(2)=0.21) hearts to values (42+/-4% to 67+/-5% ) not different from those of hypoxic controls but did not alter postischemic recovery of developed pressure in isolated chronically hypoxic (FIO(2)=0.12) hearts (69+/-5% to 72+/-5%). Conversely, the K(ATP) channel blockers glibenclamide (1 micromol/L) and 5-hydroxydecanoate (5-HD, 300 micromol/L) attenuated the cardioprotective effect of hypoxia but had no effect on postischemic recovery of function in normoxic hearts. ATP synthesis rates in hypoxic heart mitochondria (3.92+/-0.23 micromol ATP. min(-1). mg mitochondrial protein(-1)) were significantly greater than rates in normoxic hearts (2.95+/-0.08 micromol ATP. min(-1). mg mitochondrial protein(-1)). Bimakalim (1 micromol/L) decreased the rate of ATP synthesis in normoxic heart mitochondria consistent with mitochondrial K(ATP) channel activation and mitochondrial depolarization. The effect of bimakalim on ATP synthesis was antagonized by the K(ATP) channel blockers glibenclamide (1 micromol/L) and 5-HD (300 micromol/L) in normoxic heart mitochondria, whereas glibenclamide and 5-HD alone had no effect. In hypoxic heart mitochondria, the rate of ATP synthesis was not affected by bimakalim but was attenuated by glibenclamide and 5-HD. We conclude that mitochondrial K(ATP) channels are activated in chronically hypoxic rabbit hearts and implicate activation of this channel in the improved mitochondrial bioenergetics and cardioprotection observed.     

Role of catecholamines and beta-receptors in ventilatory response during hypoxic exercise.

The ventilatory response to moderate exercise in hypoxia is potentiated in goats, decreasing PaCO2 more than in normoxic exercise. We investigated the hypothesis that this potentiation results from a ventilatory stimulus provided by increased levels of circulating catecholamines (norepinephrine and/or epinephrine), acting via beta-receptors. Plasma norepinephrine [NE] and epinephrine [E] concentrations, arterial blood gases and ventilation were measured in normoxia and hypoxia (PaO2 = 34-38 Torr) at rest and during moderate exercise (5.6 kph; 5% grade) in seven female goats. PaCO2 decreased from rest to exercise in normoxia (2.9 +/- 0.7 Torr; P less than 0.01), and decreased significantly more from rest during hypoxic exercise (6.4 +/- 0.6 Torr; P less than 0.01). [NE] increased in both normoxic (1.1 +/- 0.4 ng/ml; P less than 0.05) and hypoxic exercise (2.5 +/- 0.5 ng/ml; P less than 0.01); the [NE] increase in hypoxia was significantly greater (P less than 0.01). [E] increased in normoxic (0.3 +/- 0.1 ng/ml; P less than 0.05) but not hypoxic exercise (0.6 +/- 0.5 ng/ml; P greater than 0.2). Experiments were repeated following administration of the beta-adrenergic receptor blocker, propranolol (2 mg/kg, i.v.). After beta-blockade, PaCO2 decreases from rest to exercise in normoxia (3.2 +/- 0.7 Torr; P less than 0.01) and hypoxia (8.1 +/- 0.7 Torr; P less than 0.001) were not significantly different from control. The data indicate that beta-adrenergic receptor stimulation is not necessary for a greater decrease in PaCO2 during hypoxic versus normoxic exercise. The greater rise in [NE] suggests a possible role in ventilatory control during hypoxic exercise, perhaps via alpha-adrenergic receptors. However, recent evidence suggests that NE is inhibitory in goats, and that NE is unlikely to mediate extra ventilatory stimulation during hypoxic exercise.     

Protein tyrosine kinase is not involved in the infarct size-limiting effect of ischemic preconditioning in canine hearts

Protein kinase C (PKC) plays an important role in ischemic preconditioning (IP). Because (1) tyrosine kinase is located at the downstream of PKC for IP in the rabbit hearts and (2) we have reported that ecto-5'-nucleotidase is the substrate for PKC and plays a crucial role for the infarct size-limiting effect, we tested whether tyrosine kinase activation contributes to either activation of ecto-5'-nucleotidase or the infarct size-limiting effect of the early phase of IP in the canine heart. In dogs, the IP procedure (4 cycles of 5-minute occlusion of coronary artery) and exposure to 12, 13-phorbol myristate acetate (PMA) each activated myocardial ecto-5'-nucleotidase and Lck tyrosine kinase. Genistein (10, 30, and 100 microg. kg(-)(1). min(-)(1) IC), an inhibitor of tyrosine kinase, attenuated the activation of Lck tyrosine kinase but did not attenuate the activation of ecto-5'-nucleotidase due to either IP or PMA. In the other canine hearts, IP attenuated infarct size (49+/-5 versus 11+/-3 or 16+/-3%, P<0.01) due to 90 minutes of coronary occlusion followed by 6 hours of reperfusion, which was not blunted by 3 or 2 (30 and 100 microg. kg(-)(1). min(-)(1)) doses of genistein (infarct sizes, 15+/-4, 13+/-4, and 13+/-3%, respectively, and 17+/-3 and 15+/-4%, respectively) or lavendustin A. Tyrosine kinase does not activate ecto-5'-nucleotidase or trigger the infarct size-limiting effect of the early phase of IP in canine hearts.     

Interaction between hemorrhagic hypotension and hypoxia in regulation of renal vascular resistance in unanesthetized rabbits

Effects of interaction between hemorrhagic hypotension and hypoxia on the renal circulation were examined in awake rabbits. The hypothesis tested was that renal vasoconstriction during hemorrhagic hypotension is affected by arterial PO2 (PaO2). Awake rabbits were placed into an environmental chamber and exposed to either normoxia (PaO2 greater than 100 mmHg) or hypoxia (PaO2 less than or equal to 40 mmHg). Renal blood flow (RBF) was measured with 15 micron microspheres during normotension (mean arterial pressure = 86-97 mmHg), moderate hemorrhagic hypotension (mean arterial pressure = 62-65 mmHg), and severe hemorrhagic hypotension (mean arterial pressure = 49-50 mmHg). During normotension, RBF was 461 +/- 46 and 330 +/- 30 ml/min per 100 g (mean +/- SEM) in the normoxic and hypoxic groups, respectively (P less than 0.05), and renal vascular resistance (RVR) was 0.21 +/- 0.03 and 0.31 +/- 0.04 mmHg per ml/min per 100 g in the normoxic and hypoxic groups, respectively (NS). Renal blood flow fell progressively in each group during hypotension, but the magnitude of this response was unaffected by the level of PaO2. In contrast, RVR during severe hypotension was 0.42 +/- 0.11 and 2.08 +/- 0.57 mmHg per 100 g for normoxia and hypoxia, respectively (P less than 0.05), and the increase in RVR during severe hypotension compared to normotension was greater in the hypoxic than normoxic group (P less than 0.05). Thus, in unanesthetized rabbits, hypoxia reduces RBF but does not change RVR significantly during normotension, and hypoxia potentiates the increase in RVR during hemorrhagic hypotension.     

Endogenous adenosine produced during hypoxia attenuates neutrophil accumulation: coordination by extracellular nucleotide metabolism

Hypoxia is a well-documented inflammatory stimulus and results in tissue polymorphonuclear leukocyte (PMN) accumulation. Likewise, increased tissue adenosine levels are commonly associated with hypoxia, and given the anti-inflammatory properties of adenosine, we hypothesized that adenosine production via adenine nucleotide metabolism at the vascular surface triggers an endogenous anti-inflammatory response during hypoxia. Initial in vitro studies indicated that endogenously generated adenosine, through activation of PMN adenosine A(2A) and A(2B) receptors, functions as an antiadhesive signal for PMN binding to microvascular endothelia. Intravascular nucleotides released by inflammatory cells undergo phosphohydrolysis via hypoxia-induced CD39 ectoapyrase (CD39 converts adenosine triphosphate/adenosine diphosphate [ATP/ADP] to adenosine monophosphate [AMP]) and CD73 ecto-5'-nucleotidase (CD73 converts AMP to adenosine). Extensions of our in vitro findings using cd39- and cd73-null animals revealed that extracellular adenosine produced through adenine nucleotide metabolism during hypoxia is a potent anti-inflammatory signal for PMNs in vivo. These findings identify CD39 and CD73 as critical control points for endogenous adenosine generation and implicate this pathway as an innate mechanism to attenuate excessive tissue PMN accumulation.     

Superoxide dismutase enhances ischemia-induced reactive hyperemic flow and adenosine release in dogs. A role of 5'-nucleotidase activity.

To test the hypothesis that 5'-nucleotidase activity during ischemia is attenuated by oxygen-derived free radicals, we measured ischemia-induced reactive hyperemic flow, adenosine release, and 5'-nucleotidase activity in dogs (n = 62). A 1-minute occlusion of the coronary artery caused reactive hyperemic flow (307 +/- 5 versus 92 +/- 1 ml.100 g-1.min-1 at baseline) with increased release of adenosine (14.4 +/- 1.4 versus 0.4 +/- 0.1 nmol.100 g-1.min-1 at baseline). Superoxide dismutase augmented (p less than 0.001) both peak coronary blood flow (333 +/- 6 ml.100 g-1.min-1) and repayment (436 +/- 12 versus 320 +/- 7 ml/100 g in the untreated group). Adenosine release during reperfusion was augmented (22.7 +/- 1.9 nmol.100 g-1.min-1, p less than 0.001), and 8-phenyltheophylline completely abolished the enhanced reactive hyperemia. Enzymatic assay of 5'-nucleotidase activity revealed that the administration of superoxide dismutase increases ecto-5'-nucleotidase activity in ischemic myocardium. When an inhibitor of ecto-5'-nucleotidase, alpha, beta-methyleneadenosine 5'-diphosphate, was administered, the effects of superoxide dismutase were completely abolished. Thus, we conclude that 1) the augmentation of reactive hyperemic flow caused by superoxide dismutase is attributed to the enhanced release of adenosine and 2) the enhanced release of adenosine over the untreated controls is attributed to the protection of ecto-5'-nucleotidase activity during ischemia.     

Adaptation to high altitude hypoxia protects the rat heart against ischemia-induced arrhythmias. Involvement of mitochondrial K(ATP) channel.

The aim was to determine whether adaptation to chronic hypoxia protects the heart against ischemic arrhythmias and whether ATP-dependent potassium channels (K(ATP)) play a role in the antiarrhythmic mechanism. Adult male rats were adapted to intermittent high altitude hypoxia (5000 m, 4 h/day) and susceptibility to ischemia-induced ventricular arrhythmias was evaluated in the Langendorff-perfused hearts subjected to either an occlusion of the coronary artery for 30 min or pre-conditioning by brief occlusion of the same artery prior to 30-min reocclusion. In separate groups, either a K(ATP) blocker, glibenclamide (10 micromol/l), or a mitochondrial K(ATP) opener, diazoxide (50 micromol/l), were added to a perfusion medium 20 min before the occlusion. Adaptation to hypoxia reduced the total number of ventricular arrhythmias by 64% as compared with normoxic controls. Preconditioning by a single 3-min coronary artery occlusion was antiarrhythmic only in the normoxic group, while two occlusion periods of 5 min each were needed to pre-condition the hypoxic hearts. Glibenclamide increased the number of arrhythmias in the normoxic hearts from 1316+/-215 to 2091+/-187 (by 59%) and in the hypoxic group from 636+/-103 to 1777+/-186 (by 179%). In contrast, diazoxide decreased the number of arrhythmias only in the normoxic group from 1374+/-96 to 582+/-149 (by 58%), while its effect in the hypoxic group was not significant. It is concluded that long-term adaptation of rats to high altitude hypoxia decreases the susceptibility of their hearts to ischemic arrhythmias and increases an antiarrhythmic threshold of pre-conditioning. The mitochondrial K(ATP) channel, rather than the sarcolemmal K(ATP) channel, appears to be involved in the protective mechanism afforded by adaptation.     

Thyroid hormone upregulates ecto-5'-nucleotidase/CD73 in C6 rat glioma cells

Thyroid hormones have profound effects on the central nervous system, such as proliferation, secretion of growth factors and gene expression regulation. Ecto-NTPDases and ecto-5'-nucleotidase can control the extracellular ATP/adenosine levels, which have been described as proliferation factors. Here, we investigated the influence of T(3) on the enzyme cascade which catalyzes interconversion of purine nucleotides in rat C6 glioma cells. Exposure of C6 cells to T(3) caused a dose dependent increase of 30% in the AMP hydrolysis up to 0.25 nM, which was suppressed by actinomycin. No significant alteration was observed on ATP/ADP hydrolysis and T(4) at higher concentrations (10-1000 nM) promoted an increase in AMP hydrolysis that was not dose dependent. T(3) treatment also increased the expression of CD73 mRNA. Besides the importance of the ecto-5'-NT in the cell proliferation and differentiation, its overexpression can enhance extracellular adenosine levels, which could also be an important proliferation signal.