Marie-Jeanne Mathy

Involvement of the β3 adrenoceptor in nebivolol-induced vasorelaxation in the rat aorta

Nebivolol is a highly selective &bgr;1 adrenoceptor blocker with additional vasodilating properties. Although it has been shown that the nebivolol-induced vasorelaxation is nitric oxide (NO) and cGMP dependent, the receptor that mediates these actions remains controversial, and serotonergic as well as &bgr;-adrenergic pathways may be involved. Therefore, functional experiments investigating the receptor involved in nebivolol-induced vasorelaxation were performed in the rat aorta. Isolated aortic rings were exposed to cumulative concentrations of nebivolol. Nebivolol concentrations of 3 &mgr;mol/L and higher caused vasorelaxation, which was inhibited by the presence of the NO synthase inhibitor l-NNA (100 &mgr;mol/L), or by mechanical removal of the endothelium. Exposure of the vessel rings to the selective 5-HT1A antagonist NAN-190 (1 &mgr;mol/L) or the 5-HT1/2 antagonist methysergide (1 &mgr;mol/L) did not influence nebivolol-induced vasorelaxation. Similarly, the incubation with the &bgr;2-adrenoceptor antagonist butoxamine (50 &mgr;mol/L) did not prevent vasorelaxation. The selective &bgr;3-adrenoceptor antagonist S-(−)-cyanopindolol (1 &mgr;mol/L), however, significantly counteracted the nebivolol-induced vasorelaxation. Furthermore, exposure of the aortic rings to cumulative concentrations of the &bgr;3 selective adrenoceptor agonist BRL37344 caused, like nebivolol, NO-dependent vasorelaxation that was antagonized by S-(−)-cyanopindolol. The results suggest that nebivolol-induced NO-dependent vasorelaxation is, at least in part, caused by a &bgr;3-adrenoceptor agonistic effect.

Antioxidant activity of nebivolol in the rat aorta.

The &bgr;-blocker nebivolol is a racemic mixture of d- and l- enantiomers that displays negative inotropic as well as direct vasorelaxant activity. In addition, it has been proposed that nebivolol exerts endothelium-protective effects caused by its antioxidant properties. In the present study we investigated the effect of d-, l-, and d/l-nebivolol on reactive oxygen species (ROS)-induced endothelial damage and compared it with carvedilol and metoprolol. Isolated rat aortic rings were exposed to ROS by electrolysis of the organ bath medium. Before and after electrolysis, endothelial function was measured by preconstricting the vessels with phenylephrine followed by the addition of methacholine. Carvedilol and nebivolol protected against ROS-induced endothelial damage, whereas metoprolol did not. The protective effect of nebivolol proved not to be stereoselective. Furthermore, we attempted to determine whether nebivolol acts a scavenger itself or whether another mechanism is involved. By means of HPLC measurements it was shown that nebivolol concentrations were decreased after exposure to electrolysis-induced ROS, thus indicating that nebivolol is degraded by its reaction with ROS. Functional experiments, in the rat aorta, demonstrated that exposure of nebivolol to ROS also affects its vasodilator activity. In conclusion, the present study demonstrates that nebivolol alleviates ROS-induced impairment of endothelium-dependent vasorelaxation. This protective effect is very likely the result of a direct ROS-scavenging action by the nebivolol molecule itself.

Does Cyclic AMP Mediate Rat Urinary Bladder Relaxation by Isoproterenol

Cyclic AMP is the prototypical second messenger of β-adrenergic receptors, but recent findings have questioned its role in mediating smooth muscle relaxation upon β-adrenergic receptor stimulation. We have investigated the signaling mechanisms underlying β-adrenergic receptor-mediated relaxation of rat urinary bladder. Concentration-response curves for isoproterenol-induced bladder relaxation were generated in the presence or absence of inhibitors, with concomitant experiments using passive tension and KCl-induced precontraction. The adenylyl cyclase inhibitor 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ 22,536; 1 μM), the protein kinase A inhibitors 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7; 10 μM), N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89; 1 μM), and Rp-adenosine 3′,5′-cyclic monophosphorothioate (Rp-cAMPS; 30 μM), and the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ; 3 μM) produced only minor if any inhibition of relaxation against passive tension or KCl-induced precontraction. Among various potassium channel inhibitors, BaCl2 (10 μM), tetraethylammonium (3 μM), apamin (300 nM), and glibenclamide (10 μM) did not inhibit isoproterenol-induced relaxation. Some inhibition of the isoproterenol effects against KCl-induced tone but not against passive tension was seen with inhibitors of calcium-dependent potassium channels such as charybdotoxin and iberiotoxin (30 nM each). A combination of SQ 22,536 and ODQ significantly inhibited relaxation against passive tension by about half, but not that against KCl-induced tone. Moreover, the combination failed to enhance inhibition by charybdotoxin against KCl-induced tone. We conclude that cAMP and cGMP each play a minor role in β-adrenergic receptor-mediated relaxation against passive tension, and calcium-dependent potassium channels play a minor role against active tension.

Sphingosine Kinase–Dependent Activation of Endothelial Nitric Oxide Synthase by Angiotensin II

Objective—In addition to their role in programmed cell death, cell survival, and cell growth, sphingolipid metabolites such as ceramide, sphingosine, and sphingosine-1-phosphate have vasoactive properties. Besides their occurrence in blood, they can also be formed locally in the vascular wall itself in response to external stimuli. This study was performed to investigate whether vasoactive compounds modulate sphingolipid metabolism in the vascular wall and how this might contribute to the vascular responses. Methods and Results—In isolated rat carotid arteries, the contractile responses to angiotensin II are enhanced by the sphingosine kinase inhibitor dimethylsphingosine. Endothelium removal or NO synthase inhibition by N&ohgr;-nitro-l-arginine results in a similar enhancement. Angiotensin II concentration-dependently induces NO production in an endothelial cell line, which can be diminished by dimethylsphingosine. Using immunoblotting and intracellular calcium measurements, we demonstrate that this sphingosine kinase–dependent endothelial NO synthase activation is mediated via both phosphatidylinositol 3-kinase/Akt and calcium-dependent pathways. Conclusions—Angiotensin II induces a sphingosine kinase–dependent activation of endothelial NO synthase, which partially counteracts the contractile responses in isolated artery preparations. This pathway may be of importance under pathological circumstances with reduced NO bioavailability. Moreover, a disturbed sphingolipid metabolism in the vascular wall may lead to reduced NO bioavailability and endothelial dysfunction.

SGD 101-75 IS DISTINGUISHED FROM OTHER SELECTIVE ALPHA-1-ADRENOCEPTOR AGONISTS BY THE INHIBITION OF ITS PRESSOR-RESPONSES BY CALCIUM ENTRY BLOCKADE AND VASODILATATION IN PITHED RATS AND CATS

The vasopressor effects of the selective alpha 1-adrenoceptor agonist Sgd 101/75 (2-[2-methylindazol-4-imino]-imidazolidine HCl) were analyzed in pithed rats and cats. Vasodilatation by the beta 2-adrenoceptor agonist salbutamol (1 mg/kg i.v.) or by the converting enzyme inhibitor captopril (5 mg/kg i.v.) antagonized the vasoconstriction by Sgd 101/75 in pithed rats. The effect of salbutamol was abolished by restoration of the baseline diastolic pressure by infusion of vasopressin. Calcium entry blockade by nifedipine (0.1-3 mg/kg i.v.) and (-)-verapamil (0.3 and 1 mg/kg i.v.) dose dependently inhibited the rise in the diastolic pressure induced by Sgd 101/75 pithed rats. This inhibition could not be attenuated by an infusion of vasopressin. In pithed cats, nifedipine most effectively antagonized the pressor effects of Sgd 101/75. In this respect, Sgd 101/75 is different from other alpha 1-adrenoceptor agonists, which are known to elicit a vasoconstriction which is virtually insensitive to vasodilatory measures and calcium entry blockade. These findings may be explained on the basis of a further subdivision of vascular postjunctional alpha 1-adrenoceptors.

Vascular responsiveness in isolated perfused kidneys of diabetic hypertensive rats

Objective The aim of this study was to investigate whether diabetes and hypertension cause additive effects in the responses to various vasoconstrictor and vasodilator agents, in isolated perfused kidneys obtained from streptozotocin (STZ)-diabetic Wistar-Kyoto (WKY) rats and from diabetic spontaneously hypertensive rats (SHR). Methods SHR and WKY rats were administered STZ 55 mg/kg by intravenous injection into a lateral tail vein at age 12 weeks. Eight weeks later the left kidneys were isolated and perfused via the left renal artery with a physiological salt solution. Renal perfusion pressure was measured continuously. Concentration response curves were plotted for various vasoconstrictor and vasodilator agents. Results Both the diabetic and the hypertensive state were associated with an increased wet kidney weight. The contractile responses of the renal arterial system to phenylephrine (PhE), serotonin (5-HT) and angiotensin II (Ang II) in terms both of the maximal rise in perfusion pressure (mmHg) and of the sensitivity (log EC50) were the same in preparations from diabetic WKY rats and in those from normoglycaemic WKY rats. The maximal contractile responses both to PhE and to Ang II were enhanced in kidneys from SHR compared with those in kidneys from their normotensive controls, whereas simultaneously occurring diabetes impaired this sensitization. After precontraction with 3x10-6mol/l PhE both endotheliumdependent (methacholine) and endothelium-independent (sodium nitroprusside) vasodilator drugs caused the same vasodilator response in the preparations taken from the four groups of animals. Conclusion In isolated perfused kidneys obtained from STZ-diabetic WKY rats and SHR, the isolated diabetic state did not influence the vasocon strict ion caused by various agonists. However, the enhanced vascular reactivity in the hypertensive state was blunted by simultaneously occurring diabetes mellitus. Endothelium-dependent and -independent vasorelaxation in this model was not affected neither by the hypertensive nor by the diabetic state.

Prejunctional and postjunctional inhibitory actions of eprosartan and candesartan in the isolated rabbit mesenteric artery.

Effects of angiotensin II type 1 (AT1) receptor antagonists eprosartan and candesartan and AT2 receptor antagonist PD123319 on Ang II-induced facilitation of noradrenergic neurotransmission were investigated in isolated rabbit mesenteric artery under isometric conditions. Sympathoinhibitory potency of AT1 blockers was compared with their potency concerning inhibition of direct vasoconstrictor effect of Ang II. To investigate blockade of presynaptic AT1 and AT2 receptors, effects of Ang II on electrical field stimulation (EFS)–induced contractions in presence or absence of eprosartan, candesartan, or PD123319 were studied. To investigate blockade of postsynaptic AT1 receptors, effects of either eprosartan or candesartan on concentration-response curves of Ang II were studied. In addition, effect of Ang II on postsynaptic &agr;-adrenoceptor-mediated responses was studied using noradrenaline. EFS (1, 2, and 4 Hz) caused an increase of contractile force. At stimulation frequencies of 1, 2, and 4 Hz, a subpressor concentration of Ang II (0.5 n M) increased stimulation-induced vasoconstrictor responses by 2.8 ± 0.5, 2.4 ± 0.4, and 1.6 ± 0.1 of control values, respectively (p < 0.05 compared with control for all frequencies). The enhancement could be antagonized by eprosartan (1 n M–0.1 &mgr;M) and candesartan (1 n M–0.1 &mgr;M). The AT2 antagonist PD123319 (10 n M) did not influence Ang II-induced facilitation of stimulation-induced contractions. Contractile responses to exogenous noradrenaline were unaltered in presence of Ang II 0.5 n M. Ang II (1 n M–0.3 &mgr;M) caused a concentration-dependent increase in contractile force, which could be antagonized by eprosartan (pD2´ 8.8 ± 0.19) and candesartan (pD2´ 11.3 ± 0.23). Thus, the facilitating effect of Ang II on noradrenergic neurotransmission is mediated by presynaptically located AT1 receptors and not by AT2 receptors. For eprosartan, sympathoinhibition was achieved at concentrations that also block AT1 receptors on vascular smooth muscle. In contrast, for candesartan, presynaptic inhibitory concentrations were considerably higher than those required for postsynaptic inhibition.

Sympatho‐inhibitory actions of irbesartan in pithed spontaneously hypertensive and Wistar–Kyoto rats

Angiotensin II (Ang II) can enhance sympathetic neurotransmission by acting on (AT1) receptors that are located on sympathetic nerve terminals. We investigated presynaptic blockade by the selective AT1‐receptor antagonist irbesartan in pithed spontaneously hypertensive rats and normotensive Wistar–Kyoto rats (WKY). We compared the presynaptic inhibitory dose with that required for the blockade of AT1‐receptors on vascular smooth muscle in both strains.

Sgd 101/75: cardiovascular effects in various animal preparations; interactions with vascular postjunctional α1- and α2-adrenoceptors

1 The effect of the imidazolidine Sgd 101/75 (2‐[2‐methylindazol‐4‐imino]‐imidazolidine HCl) on blood pressure, as well as its α‐adrenoceptor agonist activity and affinity for these receptors, were examined in various animal preparations. 2 After both intravenous administration to conscious spontaneously hypertensive rats and intravenous injection or infusion via the vertebral artery in chloralose‐anaesthetized cats, Sgd 101/75 (1–10 mg kg−1) elicited pressor responses. Intracisternal application of Sgd 101/75 (1 mg kg−1) to chloralose‐anaesthetized cats did not affect blood pressure. 3 In the pithed rat and pithed cat the vasopressor responses to i.v. Sgd 101/75 were effectively antagonized by prazosin (0.1–1.0 mg kg−1, i.v.) but much less by yohimbine (1 mg kg−1, i.v.). 4 Sgd 101/75 proved a less potent and less selective displacing agent of [3H]‐clonidine‐ and [3H]‐prazosin‐binding in rat brain membranes than clonidine. 5 The results suggest that Sgd 101/75 is a selective α1‐adrenoceptor agonist, devoid of any centrally or peripherally mediated hypotensive activity; this is probably caused by the low capacity of Sgd 101/75 for stimulating α2‐adrenoceptors.

Different AT1 receptor subtypes at pre- and postjunctional sites: AT1A versus AT1B receptors.

Angiotensin (AT) II is known to enhance responses to electrical field stimulation (EFS) via AT1 receptors located on sympathetic nerve terminals. Differences in potency exist between AT1 receptor antagonists regarding the inhibition of the prejunctional and postjunctional AT1 receptors. It is hypothesized that prejunctional AT1 receptors might belong to the AT1B receptor subtype. Accordingly, the authors investigated whether AT1B receptor inhibition by high concentrations of PD123319 could suppress ATII-augmented noradrenergic transmission (prejunctional) in the rabbit thoracic aorta by means of a noradrenaline spillover model. Additionally, the influence of PD123319 on ATII-enhanced constrictor responses to electrical field stimulation was investigated in the isolated rabbit mesenteric artery. Furthermore, the authors investigated whether PD123319 could influence the constrictor responses (postjunctional) to ATII in both preparations. In the thoracic aorta, ATII (10 n M) caused a significant enhancement of EFS-evoked [3H]-noradrenaline release by a factor of 2.0 ± 0.1. This reinforcement could be inhibited by PD123319 (0.1, 1, and 10 &mgr;M). The constrictor response to ATII was unaffected by PD123319. In the mesenteric artery, ATII (0.5 n M) caused a significant enhancement of constrictor responses to EFS by factors of 2.9 ± 0.3, 2.3 ± 0.3, and 1.6 ± 0.1 at 1, 2, and 4 Hz, respectively. This enhancement could be attenuated by PD123319 (1 and 10 &mgr;M). The constrictor response to ATII was unaffected by PD123319. It is concluded that the prejunctional AT1 receptors belong to the AT1B subtype whereas postjunctional AT1 receptors do not.