Valery I. Kozlovski

Compensation of endothelium-dependent responses in coronary circulation of eNOS-deficient mice

Nitric oxide plays a fundamental role in the regulation of blood flow. Here we analyzed compensatory mechanisms for the genetic eNOS deficiency in aorta and in coronary circulation. Vasodilation induced by acetylcholine, bradykinin, adenosine, and ADP as well as by S-nitroso-penicillamine (SNAP) was assessed in isolated aorta and in isolated mouse hearts from eNOS−/− and age-matched eNOS+/+ mice. In aorta from eNOS+/+ mice acetylcholine-induced vasodilation was entirely dependent on NO, and this response was absent in aorta from eNOS−/− mice. In eNOS+/+ mouse hearts responses induced by bradykinin, adenosine and ADP were partially dependent on NO, but not on PGI2, cytochrome P450-dependent metabolites, or H2O2. On the other hand, vasodilation induced by acetylcholine involved NO, but not PGI2, in its immediate, short-lasting phase, whereas PGI2 and NO mediated delayed, longer-lasting phase of this response. In eNOS−/− mouse hearts coronary vasodilator function was compensated. Responses induced by acetylcholine and adenosine, but not by bradykinin or ADP, were in part compensated by NO, most likely derived from nNOS. However, the major mechanisms compensating for the loss of eNOS in the coronary circulation did not rely on NO, PGI2, cytochrome P450-derived metabolites of arachidonic acid or on H2O2. Deficiency of eNOS is largely compensated in coronary circulation but not in aorta.

Large-conductance K+ channel opener CGS7184 as a regulator of endothelial cell function.

Large-conductance Ca(2+)-activated potassium (BK(Ca)) channels are present in endothelium, but their regulatory role remains uncharacterized. The aim of the present study was to investigate the pharmacological effects of the BK(Ca) channel opener ethyl-1-[[(4-chlorophenyl)amino]oxo]-2-hydroxy-6-trifluoromethyl-1H-indole-3-carboxylate (CGS7184) on endothelium in the aorta and coronary circulation, particularly with regard to nitric oxide (NO)-dependent regulation of vascular tone, as well as effects of CGS7184 on NO production, calcium homeostasis, and mitochondrial function in cultured endothelial cells. The vasorelaxant action of CGS7184 was studied in coronary circulation and in the aorta using isolated perfused guinea pig heart and rat aortic rings, respectively. The effects of CGS7184 on calcium homeostasis, mitochondrial membrane potential, NO production, and mitochondrial respiration were tested in cultures of EA.hy 926 endothelial cells. The BK(Ca) channel opener CGS7184 caused a concentration-dependent (0.03-3 microM) relaxation of the rat aorta and coronary vasodilatation in the isolated guinea pig heart. Both responses were profoundly inhibited by the nitric oxide (NO) synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) (100 microM). CGS7184 (5 microM) also increased basal NO production in EA.hy 926 cells by approximately two-fold. Moreover, CGS7184 induced a concentration-dependent (0.1-10 microM) elevation in intracellular calcium concentration. Interestingly, CGS7184 affected mitochondrial function by causing mitochondrial potential depolarization and an increase in oxygen consumption in EA.hy 926 endothelial cells. The BK(Ca) channel opener CGS7184 activates NOS pathways and modulates mitochondrial function in the endothelium. Both effects may be triggered by the CGS7184-induced modulation of intracellular Ca(2+) homeostasis in EA.hy 926 endothelial cells.

Effects of two β3-Agonists, CGP 12177A and BRL 37344, on coronary flow and contractility in isolated Guinea pig heart

The functional role of beta(3)-adrenergic receptors in the heart is still not clear. The actions of two widely used beta(3)-adrenoceptor agonists, such as BRL 37344 and CGP 12177, were studied in the isolated guinea pig heart, perfused at constant pressure according to the Langendorff technique. Heart contractility (dP/dt, first derivative of pressure measured over time) and coronary flow (CF) were assessed simultaneously. BRL 37344 and CGP 12177A at a concentration range of 10-8-10-5 M increased dP/dt and CF. The selective beta(3)-antagonist L-748337 (10-6 M) did not significantly influence either BRL 37344 or CGP 12177A-induced responses. However, both dP/dt and CF responses to BRL 37344 and CGP 12177A at a concentration of 10-7 M were abolished in the presence of the beta(1)/beta(2)-antagonist nadolol (10-5 M). In contrast, cardiovascular responses to CGP 12177A at a higher concentration of 10-5 M were hardly inhibited by nadolol (10-5 M). In addition, BRL 37344 and CGP 12177A at concentrations as low as 10-8 M almost completely abolished an isoprenaline-induced increase in contractility, suggesting that both BRL 37344 and CGP 12177A display beta(1)-antagonistic properties. These data suggest that the stimulatory cardiovascular responses to BRL 37344 at a full range of concentrations, and CGP 12177A at a low concentration of 10-7 M, are not mediated by beta(3)-adrenergic receptors, but rather by activation of beta(1)- or beta(2)-adrenergic receptors. Cardiovascular effects of CGP 12177A at a high concentration of 10-5 M are independent of beta(1)/beta(2)/beta(3)-adrenergic receptors. Summing up, it seems that in the isolated guinea pig heart the functional role of beta(3)-adrenoceptors is not significant. Nonetheless, BRL 37344 and CGP 12177A are not ideal tools for investigation of beta(3)-adrenergic receptor-dependent effects, because these compounds interact with other types of beta-adrenergic receptors.

Anti-thrombotic effects of nebivolol and carvedilol: Involvement of β2 receptors and COX-2/PGI2 pathways

BACKGROUND Third generation β-adrenolytics, such as selective β1 adrenoceptor antagonist nebivolol and non-selective β1/β2 and α1 adrenoceptor antagonist carvedilol, display beneficial nitric oxide (NO)-dependent vasodilator activities that contribute to their therapeutic efficacy. In the present work, we analyzed whether nebivolol and carvedilol, as well as other β-adrenolytics with similar pharmacological profiles (selective β1 adrenoceptor antagonist - atenolol and non-selective α/β adrenoceptor antagonist - labetalol), possess the ability to induce PGI2-dependent anti-thrombotic activity in vivo in normotensive rats. METHODS Anti-thrombotic effects of nebivolol and carvedilol were studied in vivo in anaesthetized rats with extracorporeal circulation superfusing collagen strips. We also assessed vasodilation induced by these drugs in isolated perfused guinea pig hearts according to Langendorffs procedures. RESULTS Nebivolol (both d- and l-isomers) (0.1-1mgkg(-1)) and carvedilol (1-3mgkg(-1)), but not atenolol (1mgkg(-1)) or labetalol (3mgkg(-1)), induced a dose-dependent and sustained anti-thrombotic response in rat model of thrombosis with extracorporeal circulation. The cyclooxygenase (COX)-2 inhibitors, rofecoxib (1mgkg(-1)) and indomethacin (5mgkg(-1)) abrogated this response, while l-NAME (5mgkg(-1)) had no significant effect. In the presence of β1/β2 adrenoceptor antagonist nadolol (1mgkg(-1)), but not in the presence of selective β1 adrenoceptor antagonist atenolol (4mgkg(-1)), anti-thrombotic responses to nebivolol, as well as carvedilol, were lost. Neither nebivolol nor carvedilol affected platelet aggregation in vitro, however both nebivolol and carvedilol induced NO-dependent vasodilation in guinea pig coronary circulation that was not dependent on β2 adrenoceptors. CONCLUSIONS We demonstrated for the first time that nebivolol and carvedilol, independently of their adrenergic receptor blocking activities, induced anti-thrombotic effects in vivo that involved β2 adrenoceptors and the activation of the COX-2/PGI2 pathway.