Anti-thrombotic effects of nebivolol and carvedilol: Involvement of ß2 receptors and COX-2/PGI2 pathways.

BACKGROUND: Third generation ß-adrenolytics, such as selective ß1 adrenoceptor antagonist nebivolol and non-selective ß1/ß2 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 ß-adrenolytics with similar pharmacological profiles (selective ß1 adrenoceptor antagonist - atenolol and non-selective α/ß 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 Langendorff's 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 ß1/ß2 adrenoceptor antagonist nadolol (1mgkg(-1)), but not in the presence of selective ß1 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 ß2 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 ß2 adrenoceptors and the activation of the COX-2/PGI2 pathway.

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.

Prostacyclin, but not nitric oxide, is the major mediator of acetylcholine-induced vasodilatation in the isolated mouse heart.

In many species, acetylcholine (Ach) induces coronary vasodilatation via endothelium-derived nitric oxide (NO). The aim of the present study was to examine if this rule pertains also to the coronary circulation of the mouse. We examined the involvement of NO and prostacyclin (PGI2) in the coronary flow response to Ach as compared to response to bradykinin (Bk) in hearts isolated from FVB or C57Bl/6 mice and perfused according to the Langendorff technique. In the isolated mouse heart, response to Ach consisted of two distinct phases: immediate, transient vasodilatation/vasoconstriction (less than 1 min) that differed between FVB and C57Bl/6 mice; and delayed sustained vasodilatation (up to 8 min) that was similar in FVB and C57Bl/6 mice. In FVB mice, the immediate phase of the Ach response consisted of a short-lasting vasodilatation followed by a vasoconstriction. In contrast, in C57Bl/6 mice, the immediate phase of the Ach response consisted exclusively of a short-lasting vasoconstriction. However, both in FVB and C57Bl/6 mice, the delayed vasodilatation was a major part of the coronary flow response to Ach and it was associated with an increase in 6-keto-PGF(1 alpha) concentration in the effluent. L-NAME (5 x 10(-4) M) displayed a minor effect on the delayed phase of the Ach response in either mice strain. In turn, indomethacin (10(-6) M), but not rofecoxib (5 x 10(-6)M), completely inhibited the delayed phase of the Ach response and the concomitant PGI2 release. On the other hand, vasodilatation induced by Bk was markedly inhibited by L-NAME, while it was unaffected by indomethacin in FVB as well as in C57Bl/6 mice. In summary, in the isolated mouse heart, Ach-induced coronary flow response displays an unusual biphasic nature and is mediated in major part by PGI2, but not by NO. Thus, in the isolated mouse heart, in parallel to Bk or other agents that are suited for the functional assessment of NO-dependent endothelial function, Ach should be used to assess PGI2-dependent endothelial function.

Inhibition of neutral endopeptidase by thiorphan does not modify coronary vascular responses to angiotensin I, angiotensin II and bradykinin in the isolated guinea pig heart.

Both angiotensin-converting enzyme (ACE) and neutral endopeptidase (NEP) are involved in the regulation of renin-angiotensin and kallikrein-kinin systems. The aim of the present study was to assess the role of NEPand ACE in the regulation of vascular responses to angiotensin I (Ang I), angiotensin II (Ang II) and bradykinin (Bk) in the coronary circulation. For this purpose we used typical inhibitors of ACE and NEP, perindoprilate (1 microM) and thiorphan (1 micromM and 10 microM), respectively, and analyzed their effects on the coronary vasoconstrictor responses to Ang I and Ang II and coronary vasodilator responses to Bk in the isolated guinea pig heart. Perindoprilate abolished coronary vasoconstriction induced by Ang I and potentiated coronary vasodilation evoked by Bk. Thiorphan at a concentration of 1 muM slightly reduced response to Ang I without a significant effect on the responses to Ang II and Bk. However, thiorphan at a concentration of 10 muM abolished coronary vasoconstrictor response to Ang I and enhanced Bk-induced vasodilation. Importantly, in the presence of perindoprilate, addition of thiorphan (10 microM) did not modify further either responses to Ang I, Ang II or to Bk. In conclusion, vascular responses induced by Ang I, Ang II and Bk in the isolated guinea pig heart are regulated by ACE but not by NEP. Moreover, thiorphan is not a perfect tool to asses functional role of NEP as it displays ACE inhibitory activity.

On the mechanism of coronary vasodilation induced by angiotensin-(1-7) in the isolated guinea pig heart.

Various mechanisms have been postulated to be involved in angiotensin-(1-7)-induced endothelium-dependent vasodilation. Here, we characterized the vasodilator action of angiotensin-(1-7) in the isolated guinea pig heart. Angiotensin-(1-7) (1-10 nmol, bolus) induced dose-dependent increase in the coronary flow. The coronary vasodilation induced by angiotensin-(1-7) was significantly reduced by the nitric oxide synthase inhibitor, L-N(G)-nitroarginine methyl ester (L-NAME) (100 microM) and abolished by a B(2) receptor antagonist, icatibant (100 nM). Coronary vasodilation induced by bradykinin (3 pmol, bolus) was inhibited by L-NAME and icatibant to similar extent as that induced by angiotensin-(1-7). Neither the selective AT(2) angiotensin receptor antagonist, PD123319 (1 microM), nor the antagonist of a putative angiotensin-(1-7) receptors, [D-alanine-7]-angiotensin-(1-7) (A-779, 1 microM), influenced the response to angiotensin-(1-7). In conclusion, in the isolated guinea pig heart angiotensin-(1-7) induces coronary vasodilation that is mediated by endogenous bradykinin and subsequent stimulation of nitric oxide release through endothelial B(2) receptors. In contrast to other vascular beds, AT(2) angiotensin receptors and specific angiotensin-(1-7) receptors do not appear involved in angiotensin-(1-7)-induced coronary vasodilation in the isolated guinea pig heart.

Radical scavenging and NO-releasing properties of selected beta-adrenoreceptor antagonists.

It is claimed that novel beta-adrenolytic drugs possess superior antioxidant properties as compared to classical selective or non-selective beta-adrenoceptor antagonists. Here we tested this notion by analyzing radical scavenging properties of selected beta-adrenolytic drugs and their ability to release nitric oxide in biological preparations. Selective beta1-adrenolytics such as nebivolol, atenolol, metoprolol and non-selective beta-adrenolytics with alpha1-receptor blocking properties such as carvedilol and labetalol were chosen for analysis. NO-releasing properties of nebivolol and carvedilol distinguished third generation beta-adrenolytics from their older counterparts while the reactivity towards hydroxyl and peroxyl radicals discerns only carvedilol but not nebivolol. Thus, superior clinical efficacy of third generation beta-adrenolytics may be related to their ability to release NO rather then to their direct antioxidant properties.

Free radicals generated by xanthine/xanthine oxidase system augment nitric oxide synthase (NOS) and cyclooxygenase (COX)-independent component of bradykinin-induced vasodilatation in the isolated guinea pig heart.

Recently, we have reported that bradykinin (Bk)-induced vasodilation was selectively potentiated by a low concentration of reactive oxygen species (ROS) generated by xanthine/xanthine oxidase system (XOX) in the coronary circulation of the isolated guinea pig heart. In an attempt to identify a mechanism of Bk response that is amplified by XOX, we analyze here the involvement of B1/ B2 receptors and the participation of NOS/COX pathways in the Bk responses before and after intracoronary infusion of XOX in the isolated guinea pig heart. Bk (0.3-3 pmoles) and acetylcholine (Ach) (100-300 pmoles) induced a dose-dependent coronary vasodilation. In the presence of a non-selective nitric oxide synthase (NOS) inhibitor L-NAME (10(-4) M) and non-selective cyclooxygenase inhibitor indomethacin (5 x 10(-5)M), vasodilation induced by Bk or Ach was inhibited. XOX infusion into the coronary circulation augmented Bk-induced vasodilation by approximately 100-300%. This effect was sustained and was observed at least 1h after XOX infusion. In contrast to Bk response, vasodilation induced by Ach was not modified by XOX infusion. Surprisingly, in the presence of L-NAME+indomethacin, Bk-induced response was still amplified by XOX. In relative terms, this effect was even more pronounced. Again, under these experimental conditions, the response to Ach remained largely unchanged. In the presence of B2 receptor antagonist, icatibant (100 nM), Ach-induced vasodilation was unaffected, while Bk-induced vasodilation was abolished before and after XOX. In conclusion, in the isolated guinea pig heart low concentration of exogenous ROS generated by XOXsystem resulted in a sustained augmentation of Bk-induced coronary vasodilatation that cannot be explained by the up-regulation of B1 receptors, or the amplification of activity of NOS-cGMP or COX pathways. The chemical identity of NOS/COX-independent component of Bk response that is up-regulated by XOX remains to be determined. EDHF is the most likely candidate.

Nebivovol and carvedilol induce NO-dependent coronary vasodilatation that is unlikely to be mediated by extracellular ATP in the isolated guinea pig heart.

In contrast to classical beta-adrenoreceptor antagonists, nebivolol and carvedilol possess endothelium-dependent vasorelaxant properties. It has been proposed that nebivolol and carvedilol activate microvascular endothelium into producing NO by the release of extracellular ATP and subsequent stimulation of endothelial P(2) receptors. Here we tested this hypothesis in the coronary circulation of the isolated guinea pig heart. We analyzed the role of NO in the coronary vasodilatation induced by nebivolol and carvedilol as well as a possible involvement of extracellular ATP in these responses. Nebivolol and carvedilol (3-30 x 10(-6) M) induced a concentration-dependent coronary vasodilatation that was inhibited by NO-synthase inhibitor, L-NAME (10(-4) M). In contrast to nebivolol and carvedilol, neither atenolol nor labetalol acted as a coronary vasodilator. Vasodilatation induced by nebivolol and carvedilol was affected neither by the P(1) receptor antagonist, 8-sulfophenyl theophylline (8-SPT, 10(-5) M), nor by the P(2) receptor antagonist, suramin (10(-5) M). On the other hand, ATP-induced coronary vasodilatation (0.3-10 x 10(-6) M) was strongly inhibited by L-NAME (10(-4) M), partially inhibited by 8-SPT (10(-5) M), while suramin (10(-5) M) had a minor effect. In conclusion, in the isolated guinea pig heart nebivolol and carvedilol, but not their classical counterparts (atenolol, labelatol), act as NO-dependent coronary vasodilators. It seems unlikely that this response is mediated by the release of extracellular ATP.

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.

The methylester of gamma-butyrobetaine, but not gamma-butyrobetaine itself, induces muscarinic receptor-dependent vasodilatation.

Gamma-butyrobetaine (GBB) is known mostly as a bio-precursor of carnitine, a key molecule in the regulation of myocardial energy metabolism. The metabolites of carnitine and GBB were investigated for acetylcholine-like activity decades ago. The present study shows that the methylester of GBB (GBB-ME) exerts its biological activity by binding to muscarinic acetylcholine receptors. GBB-ME dose-dependently decreased the blood pressure in anaesthetised rats and also produced endothelium-dependent vasodilation in the isolated guinea-pig heart. The biological effects of GBB-ME were inhibited partially by the NOS inhibitor N(omega)-nitro-L-arginine methylester (L-NAME) and abolished by the acetylcholine receptor antagonist atropine, thus supporting the hypothesis that GBB-ME acts as muscarinic agonist. Moreover, we have shown here for the first time that GBB-ME binds directly to transfected human muscarinic (m) acetylcholine receptors, the potency order being m2>m5> or =m4> or =m1>m3. GBB itself showed neither biological activity nor significant affinity for the m1-5 receptors. We conclude that GBB-ME, but not the parent GBB, possesses acetylcholine-like activity in vivo and in vitro.

Antiarrhythmic profile and endothelial action of novel decahydroquinoline derivatives.

We tested antiarrhythmic and endothelial action of novel decahydroquinoline derivatives. Antiarrhythmic activity was analyzed using models of aconitine-, calcium chloride-, and adrenaline-induced arrhythmias in rats. Potency to induce nitric oxide (NO)-dependent coronary vasodilation was assessed in isolated guinea pig heart perfused according to Langendorff technique. Among 15 novel decahydroquinoline derivatives (D1-15), four of them displayed antiarrhythmic activity (D12-D15). D12-D15 compounds were more active in the model of aconitine-induced arrhythmias than in calcium chloride-induced arrhythmias and were inactive in the model of adrenaline-induced arrhythmias. Profile of antiarrhythmic activity of D12-D15 compounds was similar to that of quinidine and procainamide. Interestingly, in the isolated guinea pig heart D14 and D15 (10(-5) M) induced coronary vasodilation, that was mediated by endothelium-derived NO. In conclusion, novel decahydroquinoline derivatives described here (D12-D15) show antiarrhythmic activity typical of antiarrhythmic drugs of class I. Importantly, some of these compounds (D14, D15) release NO from coronary endothelium, which may provide an additional therapeutic benefit.

Effects of two beta3-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.