Role of gap junctions and EETs in endothelium-dependent hyperpolarization of porcine coronary artery

The effects of endothelium-derived hyperpolarizing factor (EDHF: elicited using substance P or bradykinin) were compared with those of 11,12-EET in pig coronary artery. Smooth muscle cells were usually impaled with microelectrodes through the adventitial surface. Substance P (100 nM) and 11,12-EET (11,12-epoxyeicosatrienoic acid; 3 μM) hyperpolarized endothelial cells in intact arteries. These actions were unaffected by 100 nM iberiotoxin but were abolished by charybdotoxin plus apamin (each 100 nM). Substance P (100 nM) and bradykinin (30 nM) hyperpolarized intact artery smooth muscle; Substance P had no effect after endothelium removal. 11,12-EET hyperpolarized de-endothelialized vessels by 12.6±0.3 mV, an effect abolished by 100 nM iberiotoxin. 11,12-EET hyperpolarized intact arteries by 18.6±0.8 mV, an action reduced by iberiotoxin, which was ineffective against substance P. Hyperpolarizations to 11,12-EET and substance P were partially inhibited by 100 nM charybdotoxin and abolished by further addition of 100 nM apamin. 30 μM barium plus 500 nM ouabain depolarized intact artery smooth muscle but responses to substance P and bradykinin were unchanged. 500 μM gap 27 markedly reduced hyperpolarizations to substance P and bradykinin which were abolished in the additional presence of barium plus ouabain. Substance P-induced hyperpolarizations of smooth muscle cells immediately below the internal elastic lamina were unaffected by gap 27, even in the presence of barium plus ouabain. In pig coronary artery, 11,12-EET is not EDHF. Smooth muscle hyperpolarizations attributed to ‘EDHF' are initiated by endothelial cell hyperpolarization involving charybdotoxin- (but not iberiotoxin) and apamin-sensitive K+ channels. This may spread electrotonically via myoendothelial gap junctions but the involvement of an unknown endothelial factor cannot be excluded. Keywords: Epoxyeicosatrienoic acid, EDHF, porcine coronary artery, hyperpolarization, substance P, bradykinin, gap junctions, gap 27, endothelium, 11,12-EET Introduction In the coronary circulation, the identity of endothelium-derived hyperpolarizing factor (EDHF) remains to be determined. Based on studies in rat hepatic and mesenteric arteries, Edwards et al. (1998) first showed the critical importance of endothelial cell hyperpolarization in the EDHF pathway. These workers concluded that ‘EDHF' was likely to be K+ released as a consequence of endothelial cell K+ channel opening and hyperpolarization. In other vessels such as rabbit aorta, it has been proposed that EDHF might not even be a ‘factor' per se. Instead, the EDHF phenomenon might result from the transfer of endothelial cell hyperpolarization to the smooth muscle through gap junctions (Chaytor et al., 1998). In bovine and porcine coronary arteries, an alternative current view is that EDHF may be a cytochrome P450 metabolite, such as an epoxyeicosatrienoic acid (EET) (Hecker et al., 1994; Popp et al., 1996; Li & Campbell, 1997; Fisslthaler et al., 1999). Indeed, EETs are produced from coronary artery endothelial cells (Rosolowsky et al., 1990; Campbell et al., 1996; Rosolowsky & Campbell, 1996) and are capable of hyperpolarizing vascular smooth muscle (Campbell et al., 1996; Eckman et al., 1998). In bovine coronary artery myocytes, EETs open the large-conductance calcium-sensitive K+ channel (BKCa: Gebremedhin et al., 1998), which is selectively blocked by iberiotoxin (Kaczorowski et al., 1996). Similarly, in segments of guinea-pig coronary artery 11,12-EET induces an iberiotoxin-sensitive hyperpolarization (Eckman et al., 1998). However, iberiotoxin has little, if any, effect on the electrical or mechanical responses to EDHF in vessels including the pig and guinea-pig coronary arteries (Zygmunt & Hogestatt, 1996; Corriu et al., 1996; Chataigneau et al., 1998; Eckman et al., 1998; Quignard et al., 1999). In the porcine coronary artery it seems unlikely that EDHF is simply K+ derived from endothelial cells since small increases in extracellular K+ do not hyperpolarize its smooth muscle (Quignard et al., 1999). Gap junctions may be involved in the EDHF response in this artery, but whether these structures play an important role remains to be established. Additionally, a role for EETs cannot be excluded. Thus the aim of the present study was to characterize the electrophysiological aspects of the EDHF response in the porcine coronary artery paying particular attention to the contribution of gap junctions and of EETs in this phenomenon.