Endogenous adenosine inhibits P-selectin-dependent formation of coronary thromboemboli during hypoperfusion in dogs.

The activation of platelets and the formation of neutrophil- platelet conjugates may lead to the development of thromboemboli. We studied whether blockade of adenosine receptors during coronary hypoperfusion may cause thromboemboli via P-selectin-dependent mechanisms in 30 open-chest dogs. When coronary blood flow was reduced to 20% of the control, it was stable at low levels with increases in adenosine levels. When 8-p-sulfophenyltheophylline, an adenosine receptor antagonist, was infused during coronary hypoperfusion, coronary blood flow decreased gradually and approached almost zero 20 min after its administration. Histological examination revealed thromboemboli in the small coronary vessels. During hypoperfusion in the presence of 8-p-sulfophenyltheophylline, the mAb against P-selectin attenuated both the reduction in coronary blood flow and the formation of thromboemboli, and improved contractile and metabolic dysfunction of the myocardium. Flow cytometric analysis indicated that the expression of P-selectin on platelet and neutrophil-platelet adhesion were increased during coronary hypoperfusion, and that both were further augmented by 8-p-sulfophenyltheophylline. Immunohistochemical examination showed no staining of P-selectin in the ischemic myocardium. Adenosine inhibited the thrombin-induced expression of P-selectin on platelet and neutrophil- platelet adhesion via adenosine A2 receptors. Adenosine appears to inhibit the formation of thromboemboli during coronary hypoperfusion by suppressing the expression of P-selectin on platelets and neutrophil-platelet adhesion.

Impact of coronary risk factors on contribution of nitric oxide and adenosine to metabolic coronary vasodilation in humans.

OBJECTIVES: The contribution of nitric oxide (NO) and adenosine to the increase in coronary blood flow (CBF) induced by cardiac pacing was investigated in 28 subjects with angiographically normal coronary arteries with and without one or more risk factors for atherosclerosis. BACKGROUND: NO and adenosine are important in the regulation of coronary circulation, and the inhibition of NO synthesis increases adenosine production during cardiac pacing in experimental models. METHODS: Coronary artery diameters and CBF were assessed by quantitative coronary arteriography and Doppler flow velocity measurement. Plasma levels of nitrites and nitrates (NOx) (stable end products of NO), adenosine and lactate were measured, and blood gas analysis was performed. RESULTS: The extent of CBF response to cardiac pacing did not differ between the 14 subjects with and the 8 subjects without risk factors for atherosclerosis. NOx (12.0+/-0.9 vs. 14.9+/-1.1 ,amol/liter [mean+/-SD], p < 0.05), but not adenosine (50.8+/-7.2 vs. 50.8+/-6.5 nmol/liter), levels in coronary sinus blood increased in the subjects without risk factors. In contrast, adenosine (58.9+/-7.5 vs. 77.4+/-9.8 nmol/liter, p < 0.05), but not NOx (11.1+/-1.1 vs. 12.2+/-1.1 micromol/liter), levels increased in subjects with risk factors. Aminophylline, an antagonist of adenosine receptors, blunted CBF response to cardiac pacing in six subjects with risk factors. The number of risk factors showed a negative correlation (p < 0.05) with NOx production and a positive correlation (p < 0.05) with adenosine production during cardiac pacing, respectively. CONCLUSIONS: NO and adenosine are increased during metabolic coronary vasodilation induced by cardiac pacing. Adenosine production may be a compensatory mechanism when NO production is reduced.

Vesnarinone limits infarct size via adenosine-dependent mechanisms in the canine heart.

BACKGROUND: Recently, vesnarinone, a synthetic inotropic agent, was reported to inhibit adenosine transport into cells, which may increase adenosine levels in the heart and in turn mediate cardioprotection. Thus, vesnarinone may also have protective effects in sustained ischemia-reperfusion, because adenosine limits infarct size. METHODS AND RESULTS: In open-chest dogs, the left anterior descending coronary arteries were occluded for 90 minutes followed by 6 hours of reperfusion. Vesnarinone limited infarct size compared with controls (6.8+/-2.2% versus 44.7+/-3.9%), which was completely reversed by a nonselective adenosine receptor antagonist, 8-sulfophenyltheophylline (44.1+/-6.8%), and partially blunted by an inhibitor of ecto-5'-nucleotidase, alpha,beta-methyleneadenosine 5'-diphosphate (AMP-CP, 28.9+/-4.7%). Dipyridamole, an inhibitor of adenosine uptake into cells, only modestly limited infarct size (27.4+/-5.5%). Furthermore, vesnarinone increased adenosine release during coronary hypoperfusion, which was attenuated by AMP-CP. In vitro, vesnarinone increased the activity of ecto-5'-nucleotidase of the myocardium. CONCLUSIONS: We conclude that vesnarinone potently limits infarct size via adenosine-dependent mechanisms, mainly through activation of ecto-5'-nucleotidase.

Plasma adenosine levels increase in patients with chronic heart failure.

BACKGROUND: Adenosine is believed to be cardioprotective; however, it has not been elucidated whether the plasma adenosine level is increased in chronic heart failure. METHODS AND RESULTS: Seventy-one patients attending a specialized heart failure clinic during a 6-month period were grouped according to the cause of chronic heart failure and the New York Heart Association function class. There were 40 patients with chronic heart failure due to ischemic heart diseases and 31 patients with valvular heart diseases and dilated cardiomyopathy. Control subjects consisted of 64 healthy laboratory staff members and patients without chronic heart failure. We found that the plasma adenosine levels were increased in patients with ischemic and nonischemic heart failure (218 +/- 23 and 211 +/- 21 nmol/L, respectively, versus 62 +/- 3 nmol/L for healthy subjects) and that the extent of increases in adenosine levels correlated well with the severity of chronic heart failure. CONCLUSIONS: We conclude that adenosine levels in the systemic blood increase in patients in ischemic and nonischemic chronic heart failure. Because adenosine counteracts catecholamine-, renin-angiotensin-, and cytokine-induced cellular injury, increased adenosine levels may be endogenous compensatory mechanisms for heart failure.

Adenosine and cardioprotection in the diseased heart.

Biological and mechanical stressors such as ischemia, hypoxia, cellular ATP depletion, Ca2+ overload, free radicals, pressure and volume overload, catecholamines, cytokines, and renin-angiotensin may independently cause reversible and/or irreversible cardiac dysfunction. As a defense against these forms of stress, several endogenous self-protective mechanisms are exerted to avoid cellular injury. Adenosine, a degradative substance of ATP, may act as an endogenous cardioprotective substance in pathophysiological conditions of the heart, such as myocardial ischemia and chronic heart failure. For example, when brief periods of myocardial ischemia precede sustained ischemia, infarct size is markedly limited, a phenomenon known as ischemic preconditioning. We found that ischemic preconditioning activates the enzyme responsible for adenosine release, ie, ecto-5'-nucleotidase. Furthermore, the inhibitor of ecto-5'-nucleotidase reduced the infarct size-limiting effect of ischemic preconditioning, which establishes the cause-effect relationship between activation of ecto-5'-nucleotidase and the infarct size-limiting effect. We also found that protein kinase C is responsible for the activation of ecto-5'-nucleotidase. Protein kinase C phosphorylated the serine and threonine residues of ecto-5'-nucleotidase. Therefore, we suggest that adenosine produced via ecto-5'-nucleotidase gives cardioprotection against ischemia and reperfusion injury. Also, we found that plasma adenosine levels are increased in patients with chronic heart failure. Ecto-5'-nucleotidase activity increased in the blood and the myocardium in patients with chronic heart failure, which may explain the increases in adenosine levels in the plasma and the myocardium. In addition, we found that further elevation of plasma adenosine levels due to either dipyridamole or dilazep reduces the severity of chronic heart failure. Thus, we suggest that endogenous adenosine is also beneficial in chronic heart failure. We propose potential mechanisms for cardioprotection attributable to adenosine in pathophysiological states in heart diseases. The establishment of adenosine therapy may be useful for the treatment of either ischemic heart diseases or chronic heart failure.

Activated polymorphonuclear leukocytes induce constriction of canine coronary artery via Mac-1, but not LFA-1, and ICAM-1.

Polymorphonuclear leukocytes (PMNs) can induce endothelium-dependent constriction of the vascular ring. We evaluated the roles of adhesion molecules in PMNs-induced coronary vasoconstriction. We measured changes in the isometric tension of isolated rings of canine coronary arteries with and without endothelium suspended in an organ chamber after the addition of autologous PMNs. In concentration of 5 x 10(5) to 1 x 10(7) cells/ml, fMLP-stimulated PMNs increased the tension of coronary artery with endothelium in a dose-dependent manner (23 +/- 3 to 102 +/- 11% of KCl-induced constriction). PMNs did not damage the endothelial function of coronary arteries as determined by an acetylcholine exposure. Mechanical rubbing of the endothelial cells abolished the PMN-induced vasoconstriction. The PMN-induced vasoconstriction (5 x 10(6) cells/ml: 98 +/- 8% of KCl-induced constriction) was inhibited when PMNs were pretreated with the monoclonal antibody against Mac-1 (54 +/- 4% of KCl-induced constriction) but not against LFA-1 against ICAM-1 (31 +/- 2% of KCl-induced constriction). The PMN-induced vasoconstriction was inhibited when PMNs were pretreated with the monoclonal antibody against CD18, and the extent of inhibition was comparable with CD11b/CD18. The combination of pretreatments of PMNs with CD11b/CD18 and of endothelium with ICAM-1 did not inhibit the vasoconstriction more than ICAM-1 alone. These results suggest that activated PMNs may mediate adherent-dependent constriction of canine coronary artery via Mac-1 (CD11b/CD18), but not LFA-1 (CD11a/CD18), with ICAM-1.

Adenosine inhibits leukocyte-induced vasoconstriction.

Polymorphonuclear leukocytes (PMNs) can induce endothelium-dependent constriction of vascular rings. Because adenosine inhibits the function of PMNs, we examined the effects of adenosine on the PMN-induced coronary vasoconstriction. We measured changes in the isometric tension of isolated rings of canine coronary arteries suspended in an organ chamber filled with Krebs-Henseleit solution after the addition of autologous PMNs. N-formyl-L-methionyl-leucyl-phenylalanine (FMLP)-stimulated PMNs increased the tension of the coronary artery with the endothelium in a concentration-dependent manner. Treatment of FMLP-stimulated PMNs with adenosine inhibited both the adhesion of PMNs to the endothelium and the PMN-induced vasoconstriction. Stimulation of PMNs with CGS-21680C, but not with cyclohexyladenosine, inhibited both the adhesion of PMNs to the endothelium and the PMN-induced vasoconstriction. However, treatment of coronary arteries with adenosine had no effect on the adherence of PMNs to the endothelium and the PMN-induced constriction. These results suggest that stimulation of adenosine A2a receptors on PMNs may inhibit the PMN-induced vasoconstriction by inhibiting the adhesion of PMNs to the endothelium.

Amelioration of ischemia- and reperfusion-induced myocardial injury by 17beta-estradiol: role of nitric oxide and calcium-activated potassium channels.

BACKGROUND: 17Beta-estradiol increases the production of nitric oxide (NO) and prostacyclin and opens Ca2+-activated K+ (K(Ca)) channels. Whether these effects of 17beta-estradiol reduce infarct size and the incidence of ventricular arrhythmia was investigated in dogs subjected to myocardial ischemia and reperfusion. METHODS AND RESULTS: Infarct size was measured in open-chest dogs after 90 minutes' occlusion of the left anterior descending coronary artery and a subsequent 6 hours of reperfusion. Infusion of 17beta-estradiol into the coronary artery was initiated 10 minutes before coronary occlusion and continued until after 1 hour of reperfusion, with the exception of the occlusion period. The difference in NO concentration between coronary venous and arterial blood 10 minutes after the onset of reperfusion was significantly greater in dogs treated with 17beta-estradiol (10 ng x kg(-1) x min(-1)) than in control animals. Infarct size (13.1+/-3.0% versus 43.7+/-5.4% of the area at risk) and the incidence of ventricular arrhythmia during ischemia and reperfusion periods were significantly reduced in the 17beta-estradiol group. Both N(G)-nitro-L-arginine methyl ester (an inhibitor of NO synthase) and iberiotoxin (a blocker of K(Ca) channels) reduced both the infarct size-limiting effect (infarct size, 29.3+/-3.0% and 31.7+/-2.1%, respectively) and the antiarrhythmic effect of 17beta-estradiol; indomethacin (an inhibitor of cyclooxygenase) did not attenuate the beneficial effects of 17beta-estradiol. CONCLUSIONS: 17Beta-estradiol reduced both myocardial infarct size and the occurrence of ischemia- and reperfusion-induced ventricular arrhythmias, which appear to be mediated by NO and the opening of K(Ca) channels in canine hearts.

Inhibition of nitric oxide synthesis increases adenosine production via an extracellular pathway through activation of protein kinase C.

BACKGROUND: NO is known to deactivate protein kinase C (PKC). Because we have reported that the activation of PKC activates ecto-5'-nucleotidase, we examined whether the inhibition of NO synthesis increases ecto-5'-nucleotidase activity through the activation of PKC. METHODS AND RESULTS: The left anterior descending coronary artery (LAD) was cannulated and perfused with blood through a bypass tube from the left carotid artery in 65 open-chest dogs. The intracoronary administration of NG-nitro-L-arginine methyl ester (L-NAME, 10 microg x kg[-1] x min[-1]), an NO synthase inhibitor, for 30 minutes increased (P<.05) adenosine levels in coronary venous blood (123+/-10 versus 21+/-3 pmol/mL) and ecto-5'-nucleotidase activity (64+/-6 versus 41+/-4 nmol x mg[-1] x min[-1]) in the LAD-perfused myocardium. The intracoronary administration of alpha,beta-methyleneadenosine 5'-diphosphate, an inhibitor of ecto-5'-nucleotidase, or GF109203X or calphostin C, both of which are PKC inhibitors, attenuated the L-NAME-induced increases in adenosine levels and ecto-5'-nucleotidase activity. Treatment of cultured human coronary arterial endothelial cells (HCAECs) with L-NAME for 30 minutes increased ecto-5'-nucleotidase activity, which was inhibited by either GF109203X or calphostin C. NO releasers decreased both ecto-5'-nucleotidase and PKC activities in HCAECs. Treatment of HCAECs with zaprinast, a selective inhibitor of cGMP-specific phosphodiesterase, with or without atrial natriuretic peptide, increased intracellular cGMP concentrations but did not change ecto-5'-nucleotidase activity. CONCLUSIONS: These results indicate that the inhibition of NO synthesis increases both adenosine production and ecto5'-nucleotidase activity through the activation of PKC and that NO modulates ecto-5'-nucleotidase via cGMP-independent mechanisms.

Plasma levels of nitrite/nitrate and platelet cGMP levels are decreased in patients with atrial fibrillation.

Patients with atrial fibrillation have been reported to exhibit abnormal hemostasis. Since nitric oxide (NO) exerts antithrombotic effects and attenuates platelet function, we evaluated two indicators of plasma NO levels, the plasma levels of nitrite and nitrate (NOx), and the levels of cGMP in platelets. We also examined whether indicators of plasma NO levels were associated with abnormalities in parameters related to platelet function, blood coagulation, and fibrinolysis. We evaluated 45 patients with chronic sustained atrial fibrillation (33 men and 12 women, age range 63 +/- 2 years) compared with 45 sex- and age- (+/- 2 years) matched nonhospitalized subjects with sinus rhythm. There were no significant differences between the two groups in the incidence of risk factors for stroke except for ischemic heart disease or in echocardiographic parameters. Plasma levels of NOx measured using the Greiss reagent (mean [interquartile range]: 15.6 [9.5 to 25.7] versus 24.1 [14.2 to 40.8] mumol/L, n = 45) and the platelet cGMP levels (0.33 [0.16 to 0.67] versus 0.63 [0.31 to 1.29] pmol/10(9) platelets, n = 9) were significantly (P < .05) lower in the patients with atrial fibrillation than in the control subjects. Plasma levels of D-dimer, beta-thromboglobulin, and fibrinogen were significantly (P < .05) higher in the patients with atrial fibrillation. The two groups did not differ as to the plasma levels of tissue plasminogen activator or plasminogen activator inhibitor-1. Our findings suggest that a decrease in plasma NO levels may account for the hemostatic abnormalities observed in patients with atrial fibrillation.

Ecto-5'-nucleotidase mediates infarct size-limiting effect by ischemic preconditioning in the rabbit heart.

We examined whether ecto-5'-nucleotidase mediates infarct limitation by ischemic preconditioning in the rabbit heart. Ecto-5'-nucleotidase activity in ischemic region after ischemic preconditioning was greater than that in nonischemic regions (23.6 +/- 2.5 vs. 13.6 +/- 1.0 nmol/mg protein/min; p < 0.01). With an inhibitor of 5'-nucleotidase, alpha,beta-methylene adenosine 5'-diphosphate (AMP-CP), ecto-5'-nucleotidase activity in the ischemic region was comparable to that in the nonischemic region. Mean blood pressure was reduced from 73 +/- 2 to 62 +/- 3 mm Hg with intravenous AMP, whereas it did not change with coperfusion of AMP and AMP-CP, suggesting effective inhibition of ecto-5'-nucleotidase. Separately, myocardial infarction was created by 30-min coronary occlusion and 3 h of reperfusion. Infarct size expressed as percentage volume in risk area was reduced by ischemic preconditioning compared with that in the control (7.8 +/- 2.5% vs. 38.1 +/- 4.0%; p < 0.01). However, infarct size in the group given AMP-CP plus ischemic preconditioning was similar to that in the control (36.2 +/- 2.8% vs. 38.1 +/- 4.0%; NS), suggesting that ecto-5'-nucleotidase mediates infarct limitation by ischemic preconditioning in the rabbit.

Temporary acidosis during reperfusion limits myocardial infarct size in dogs.

We tested the hypothesis that myocardial extracellular acidosis during early reperfusion limits infarct size. The left anterior descending coronary artery was perfused with blood through a bypass tube in dogs. We occluded the bypass tube for 40 (protocol I; n = 24 hearts) and 90 min (protocol II; n = 36 hearts). In protocols I and II, we infused one group of hearts with HCl (60 micrograms.kg-1.min-1) for 60 min after the onset of reperfusion (the metabolic acidosis group), and another group of hearts were ventilated with 3 liters of 70% O2-30% CO2 mixed with room air 10 min before the onset of reperfusion for 70 min (the respiratory acidosis group). pH in the coronary venous blood and myocardial pH during reperfusion in the metabolic and respiratory acidosis groups were lower than those in the control groups. Infarct sizes in the metabolic (16.4 +/- 2.5 and 22.3 +/- 2.5%) and respiratory (16.7 +/- 2.6 and 22.3 +/- 2.5%) acidosis groups in protocols I and II, respectively, were smaller than those in the control groups (33.1 +/- 3.0 and 40.6 +/- 4.1%, respectively). Thus we conclude that temporary acidosis during reperfusion limits infarct size.

Effects of ischemic preconditioning on contractile and metabolic function during hypoperfusion in dogs.

We examined the effects of ischemic preconditioning (i.p.) on metabolic and contractile function during coronary hypoperfusion in dogs. After the left anterior descending coronary artery (LAD) was occluded for 5 min (i.p.) and reperfused for 10 min, coronary blood flow (CBF) of the LAD was decreased to 33% of the control. I.p. increased (P < 0.05) lactate extraction ratio and the pH of coronary venous blood and decreased (P < 0.05) myocardial oxygen consumption and fractional shortening during hypoperfusion compared with those in the control group, although i.p. did not change the endocardial-to-epicardial blood flow ratio of the regional myocardium during hypoperfusion. I.p. increased (P < 0.05) the adenosine levels in coronary venous blood during hypoperfusion. I.p. increased (P < 0.05) myocardial ecto-5'-nucleotidase activity. Administration of 8-sulfophenyltheophylline or alpha, beta-methyleneadenosine 5'-diphosphate blunted the i.p.-induced changes in metabolic and contractile parameters during hypoperfusion. These results suggest that i.p. reduced the severity of anaerobic myocardial metabolism of ischemic hearts by increasing the adenosine levels via an extracellular pathway.

Elevation of plasma adenosine levels may attenuate the severity of chronic heart failure.

Adenosine is known to be an endogenous cardioprotective substance. Since we have reported that adenosine levels increase in patients with chronic heart failure, we tested whether further elevation of the adenosine levels due to dipyridamole or dilazep for 6 months modulates the pathophysiology of chronic heart failure. In patients with chronic heart failure, either dipyridamole (300 mg/d n = 17) or dilazep (300 mg/d n = 5) were administered for 6 months. Twenty-two patients (mean +/- SE age 58 +/- 4 years old) attending a specialized chronic heart failure (CHF) clinic over 6 months and judged as in New York Heart Association (NYHA) function class II or III were examined. The other drugs used for the treatment of CHF were not altered during the study. There were 5 patients with CHF caused by ischemic heart diseases, and 17 patients with either valvular heart diseases or dilated cardiomyopathy. We found that increases in the plasma adenosine levels (202 +/- 34 and 372 +/- 74) nmol/L before and after dipyridamole administration, P < 0.005 ameliorate the severity of CHF (NYHA: 2.1 +/- 0.5 to 1.7 +/- 0.2). Both ejection fraction and maximal oxygen consumption increased. These improvements in the severity of chronic heart failure returned to baseline levels 6 months after discontinuation of dipyridamole. Comparable results were obtained in the dilazep protocol. We suggest that the elevation of plasma adenosine levels improves the pathophysiology of CHF.

Nisoldipine selectively induces coronary vasodilation and improves mild myocardial ischemia in dogs: a potential role of cellular acidosis.

We examined whether nisoldipine, a calcium (Ca) channel blocker, increases coronary blood flow (CBF) without decreasing aortic blood pressure (AoP) with ischemic and nonischemic hearts, and whether the presence of cellular acidosis in ischemic myocardium contributes to the augmentation of coronary vasodilation due to nisoldipine. In 42 dogs, coronary perfusion pressure (CPP) was reduced so that CBF decreased to 60% of the baseline, and CPP was maintained constant thereafter. First, we administered nisoldipine into a systemic vein in the ischemic and nonischemic hearts. Second, nisoldipine was administered into the canine coronary artery of the ischemic myocardium, with and without administration of either sodium bicarbonate (NaHCO3), sodium hydroxide (NaOH), or amiloride. Nisoldipine (0.25-4.0 mg/kg, i.v.) increased CBF by 59% in the ischemic myocardium more than the nonischemic myocardium (by 34%) without reducing AoP. The infusion of nisoldipine (40 ng/kg/min, IC) increased CBF markedly by about 55% in the ischemic myocardium with increases in fractional shortening (FS; 11 +/- 2% to 21 +/- 2%) and lactate extraction ratio (LER; -19 +/- 4% to 15 +/- 2%). Increases in CBF, FS, and LER were markedly attenuated during administration of nisoldipine with concomitant administration of either NaHCO3 or NaOH. Furthermore, the extent of increases in CBF (54 +/- 2 mL/100 g/min), FS (13 +/- 2%), and LER (-17 +/- 4%) were also markedly attenuated due to the concomitant treatment with amiloride. We conclude that myocardial cellular acidosis plays an important role in mediating coronary vasodilation affected by nisoldipine in the ischemic myocardium. H+ may modulate the property of voltage-dependent Ca channels via Na(+)-H+ exchange.

Role of activation of protein kinase C in the infarct size-limiting effect of ischemic preconditioning through activation of ecto-5'-nucleotidase.

BACKGROUND: We have reported previously that ischemic preconditioning limits infarct size by increasing ecto-5'-nucleotidase activity. Since we have also reported that protein kinase C activation increases ecto-5'-nucleotidase activity in rat cardiomyocytes, we tested whether activation of protein kinase C during ischemic preconditioning contributes to the infarct size-limiting effect through augmentation of ecto-5'-nucleotidase activity in the canine heart. METHODS AND RESULTS: The coronary artery was occluded four times for 5 minutes with alternating 5-minute periods of reperfusion (ischemic preconditioning). Then the coronary artery was occluded for 90 minutes followed by 6 hours of reperfusion. Infarct size, normalized by the risk area, in the ischemic preconditioning group was smaller than in the control group (42.6 +/- 3.6% in the control group versus 7.9 +/- 1.8% in the ischemic preconditioning group, P < .001). Myocardial ecto-5'-nucleotidase activity was increased after the ischemic preconditioning procedure but the increase in ecto-5'-nucleotidase was attenuated by inhibitors of protein kinase C (polymyxin B and GF109203X). Both polymyxin B and GF109203X blunted the infarct size-limiting effect of ischemic preconditioning (infarct size 33.1 +/- 6.9% and 35.1 +/- 6.4%, respectively). The infarct size-limiting effect was also blunted by an inhibitor of ecto-5'-nucleotidase. Transient administration of methoxamine mimicked the increase in ecto-5'-nucleotidase activity and the infarct size-limiting effect, both of which were abolished by inhibitors of protein kinase C. CONCLUSIONS: We conclude that activation of ecto-5'-nucleotidase and protein kinase C contributes to the infarct size-limiting effect of ischemic preconditioning.

Role of protein kinase C-alpha in activation of ecto-5'-nucleotidase in the preconditioned canine myocardium.

We have reported that activation of protein kinase C (PKC) increases ecto-5'-nucleotidase activity, which may contribute to the infarct size-limiting effect of ischemic preconditioning. Since we have reported that Ca(2+)- and phospholipid-sensitive PKC is activated due to ischemic preconditioning, we further tested 1) whether PKC-alpha or -beta is translocated to the cellular membrane of the preconditioned canine myocardium, and 2) whether activation of PKC contributes to the increase in ecto-5'-nucleotidase activity via phosphorylation-dependent mechanisms. Four times of 5 minutes coronary occlusion separated by 5 minutes of reperfusion (ischemic preconditioning) translocated PKC-alpha to the cellular membrane in the canine hearts, although PKC-beta, -delta, -epsilon, and -zeta were not translocated. The activity of Ca(2+)- and phospholipid-sensitive PKC increased, which was attenuated by the removal of either Ca2+ or phosphatidylserine. Ecto-5'-nucleotidase was also activated in the preconditioned myocardium compared with control. Inhibition of PKC due to GF109203X blunted the activation of myocardial ecto-5'-nucleotidase. Okadaic acid (an inhibitor of phosphatase) enhanced the increases in ecto-5'-nucleotidase activity due to preconditioning, and this enhancement was blunted by GF109203X. We conclude that ischemic preconditioning activates PKC-alpha, and thus ecto-5'-nucleotidase.

Nifedipine limits infarct size via NO-dependent mechanisms in dogs.

OBJECTIVES: Amlodipine increases NO levels in coronary vessels and aorta via bradykinin-dependent mechanisms in vitro. We have previously reported that nifedipine increases cardiac NO levels in the ischemic canine hearts, suggesting that nifedipine may also have protective effects against ischemia and reperfusion injury, because the enhancement of NO production limits infarct size. We tested whether nifedipine limits infarct size via NO-dependent mechanisms. METHODS: In open chest dogs, the left anterior descending coronary artery was perfused with blood through a bypass tube and occluded for 90 min followed by 6 hours of reperfusion. Infarct size was assessed at 6 hours of reperfusion. Nifedipine of 3 or 6 microg/kg/min was infused into the bypass tube between 10 min prior to the onset of ischemia and 60 min of reperfusion. RESULTS: Neither systemic blood pressure nor heart rate changed during infusion of nifedipine. Infarct size was reduced by the administration of nifedipine (3 or 6 microg/kg/min) compared with the untreated condition (25.6+/-2.6 and 19.1+/-3.5 vs. 43.4+/-5.6%, respectively), which was completely blunted by L-NAME (45.0+/-3.6 and 45.4+/-4.2 vs. 47.9+/-3.9% in the nifedipine (3 or 6 microg/kg/min) with L-NAME groups vs. the L-NAME group). Myeloperoxidase activity of the myocardium increased after 6 hours of reperfusion, which was attenuated by nifedipine. The limitation of infarct size and the attenuation in myeloperoxidase actiivity were completely blunted by L-NAME. There were no significant differences in collateral blood flow at 45 min of ischemia between each group. CONCLUSIONS: We conclude that the Ca channel blocker, nifedipine, limits infarct size via NO-dependent mechanisms.