Characterization of acetylcholine-induced membrane hyperpolarization in endothelial cells.

Abstract

The characteristics of the hyperpolarization response to acetylcholine (ACh) in endothelial cells from the guinea pig coronary artery were studied by microelectrode recording technique. ACh (30 nM to 3 ,uM) induced membrane hyperpolarization in a dose-dependent manner. The sustenance of the response required the presence of external calcium. The hyperpolarization was not affected by nifedipine (1 ,uM) but was inhibited by the potassium channel blockers charybdotoxin (10 nM), tetraethylammonium (1 mM), and 4-aminopyridine (0.5 mM).Glibenclamide (10 ,M) and apamin (1 ,uM) were not effective. The inhibitors of endotheliumderived relaxing factor/nitric oxide synthesis Nw-nitro L-arginine (50 ,M) and NG-monomethyl L-arginine (30 ,uM) had no effect on the resting membrane potential or the ACh-induced responses. No hyperpolarization was observed with application of sodium nitroprusside (10 ixM) or 8-bromo-cGMP (0.1 ,uM). Ouabain (10 tM) depolarized the membrane significantly by 5 mV, but the ACh hyperpolarization was not affected. Indomethacin (10 JAM) was without effect on the resting membrane potential or the hyperpolarization to ACh. These results show that ACh-induced hyperpolarization is dependent on external calcium and can be inhibited by certain potassium channel blockers. The hyperpolarization response is not mediated by endothelium-derived relaxing factor/nitric oxide, cGMP, a cyclooxygenase product, or stimulation of the Na-K pump. (Circulation Research 1992;70:257-263) T he relaxation response to acetylcholine (ACh) in vascular tissues is dependent on the presence of an endothelium. In many cases, the relaxation can be attributed to the release of an endothelium-derived relaxing factor (EDRF).' It is now generally acknowledged that nitric oxide (NO) or a nitrosothiol compound is the EDRF and L-arginine is the precursor.2 The heme moiety of soluble guanylate cyclase is the NO receptor, and vasorelaxation is associated with an increase in the cGMP level.2Another endothelium-dependent response to ACh in vascular smooth muscle cells is membrane hyperpolarization.3 The cellular mechanism mediating endothelium-dependent hyperpolarization is distinct from that of EDRF/NO.3 For example, vasorelaxation and production of cGMP induced by EDRF/NO are inhibited by methylene blue and hemoglobin.2 However, the hyperpolarization in re-

Endothelium‐dependent hyperpolarization and intercellular electrical coupling in guinea‐pig mesenteric arterioles

“…This is not the case in arterioles where the media consists of only one or two cell layers and both smooth muscle and endothelial cells have comparable cell membrane capacitance of around 10 pF (Yamamoto et al 1998). Ca¥-activated K¤ channels appear to be involved in AChinduced hyperpolarization in endothelial cells (Olesen et al 1988;Chen & Cheung, 1992;Marchenko & Sage, 1996). In the present experiments, ACh induced a two-phase hyperpolarization, an initial rapid and a slower secondary phase.…”

Mechanisms of L‐NG nitroarginine/indomethacin‐resistant relaxation in bovine and porcine coronary arteries

“…In Tyrode solution, ouabain and a decrease in [Na+le had much less effect on L-N' nitroarginineresistant relaxation of BCA; this has been also shown in guineapig coronary arteries (Chen & Cheung, 1992), rat mesenteric arteries (Ayotunde et al, 1991) and human coronary arteries (Nakashima et al, 1993). Since the inhibitory properties of ouabain and a reduced [Nal]e critically depend on the solutions used, the effect observed with PBS is more likely due to some depolarizing effect of ouabain and reduced [Nal]e rather than interference with EDHF directly.…”

Modulation of blood flow and tissue perfusion by endothelium‐derived relaxing factor

“…In the case of substance P this follows Ca2" release from internal stores, with secondary opening of KCa channels (Sharma & Davis, 1994). The same mechanism could also explain attenuated relaxation of guinea-pig coronary artery to acetylcholine in the presence of K'a blockade (Chen & Cheung, 1992;Eckman, Frankovitch & Keef, 1992). However, in cultured porcine endothelium and isolated rabbit aorta K'a channels contribute to the EDRF response to flow but not that to agonists (Cooke et al 1991 b;.…”

“…Recent evidence suggests that electrotonic transmission of information between the two cell types is unidirectional, passing only from smooth muscle to endothelium via 'rectifying' gap junctions, so that the resulting smooth muscle hyperpolarization is probably entirely mediated by diffusible factors originating from the endothelium (Beny & Pacicca, 1994). Several groups have postulated the existence of an endothelium-derived hyperpolarizing factor (EDHF) on the basis that smooth muscle hyperpolarization may only be partially attenuated by inhibition of EDRF or prostaglandin synthesis (Feletou & Vanhoutte, 1988;Tare et al 1990;Chen & Cheung, 1992;Garland & McPherson, 1992). Furthermore, acetylcholine appears to stimulate release of the putative EDHF via M1 muscarinic receptors, and EDRF activity via M2 receptors (Komori & Suzuki, 1987).…”