Mechanism of Acetylcholine-induced Endothelium-dependent Relaxation in the Rabbit Carotid Artery by M3-receptor Activation

The present study were designed to characterize the action mechanisms of acetylcholine (ACh) -induced endothelium-dependent relaxation in arteries precontracted with high K (70 mM). For this, we simultaneously measured both muscle tension and cytosolic free Ca2 concentration ([Ca2 ]i), using fura-2, in endothelium-intact, rabbit carotid arterial strips. In the artery with endothelium, high K increased both [Ca2 ]i and muscle tension whereas ACh (10microM) significantly relaxed the muscle and increased [Ca2 ]i. In the presence of NG-nitro-L-arginine (L-NAME, 0.1 mM), ACh increased [Ca2 ]i without relaxing the muscle. In the artery without endothelium, high K increased both [Ca2 ]i and muscle tension although ACh was ineffective. 4-DAMP (10 nM) or atropine (0.1microM) abolished ACh-induced increase in [Ca2 ]i and relaxation. The increase of [Ca2 ]i and vasorelaxation by ACh was siginificantly reduced by either 3microM gadolinium, 10microM lanthanum, or by 10microM SKF 96365. These results suggest that in rabbit carotid artery, ACh-evoked relaxation of 70 mM K -induced contractions appears to be mediated by the release of NO. ACh-evoked vasorelaxation is mediated via the M3 subtype, and activation of the M3 subtype is suggested to stimulate nonselective cation channels, leading to increase of [Ca2 ]i in endothelial cells.

Phorbol Esters Attenuate The Action of Isoproterenol on Vascular Smooth Muscle

The effects of phorbol esters were studied in rabbit carotid artery to evaluate the action of protein kinase C on the regulation of vascular tone by isoproterenol. The vascular rings, 2 mm in width, were myographied isometrically in an isolated organ bath and the effects of phorbol 12,13-dibutyrate(PDBu) and phorbol 12-myristate 13-acetate(PMA) were determined. Isoproterenol, a beta adrenergic agonist, relaxed the vessel which was precontracted by phenylephrine, but not that by phorbol esters. The action of isoproterenol was attenuated by removal of endothelium or pretreatment with methylene blue or nitro-L-arginine. The pretreatment with phorbol esters at concentrations which did not induce contraction, decreased isoproterenol-induced relaxation of vascular rings with or without endothelium. The action of PDBu on isoproterenol-induced relaxation was less effective than that of PMA, unlike those observed in contractile response, but the contractile effect of the former was more potent than that of the latter. PMA did not affect relaxant effect of forskolin, an activator of adenyl cyclase. Staurosporine, a protein kinase C inhibitor, inhibited the action of these drugs on both isoproterenol-induced relaxation and the contractile response. These results suggest that the relaxation induced by isoproterenol was reducd by the activation of protein kinase C, which may be isozyme different from that involved in contractile response.

Effect of Verapamil on the Cyclic Flow Reductions of Rabbit Carotid Stenosis

Previous studies have shown that experimental canine coronary or rabbit cardtid qrtery stenosis that is associated with endothelial injury results in a typical pattern of blood flow characterized by gradual decreases in arterial flow followed by restorations of flow to normal values. This pattern of flow, called cyclic flow reduction(CFR), is the consequence of recurrent platelet aggregation at the site of the stenosis and endothelial injury and subsequent dislodgement of the thrombus. This study was designed to test the efficacy of verapamil in ingibiting in vivo platelet aggregation in a rabbit model of cardotid artery stenosis and ecdothelial injury. Carotid blood flow was measured continuously with a electromagnetic flowmeter probe that is positioned proximal to the constrictor. During placement of constrictor and angioplasty balloon, CFR developed in 8 of 20 rabbits with a mean frequency of 10.0+/-2.2 cycles/h. CFRs were observed for 30min, and IV verapamil was administered till declining of blood pressure(up to 100ug/kg). After intravenous verapamil, the mean frequency of CFR insignificantly decreased to 8.7+/-2.1 cycles/h(p=ns). After 20mg/kg of aspirin were given intravenously, the CFR were abolished in 5 rabbits, the mean frequency of CFR decreased in 1 rabbit, and no significant change was observed in 2 rabbits. It is concluded that verapamil is relatively ineffective in inhibiting in vivo platelet aggregation at doses that don't change hemodynamics significantly.