Involvement of voltage-dependent potassium channels in the EDHF-mediated relaxation of rat hepatic artery

1 In the rat hepatic artery, the acetylcholine-induced relaxation mediated by endothelium-derived hyperpolarizing factor (EDHF) is abolished by a combination of apamin and charybdotoxin, inhibitors of small (SKCa) and large (BKCa) conductance calcium-sensitive potassium (K)-channels, respectively, but not by each toxin alone. The selective BKCa inhibitor iberiotoxin cannot replace charybdotoxin in this combination. Since delayed rectifier K-channels (K-V) represent another target for charybdotoxin, we explored the possible involvement of K-V in EDHF-mediated relaxation in this artery. 2 The K-V inhibitors, agitoxin-2 (0.3 mu M), kaliotoxin (0.3 mu M), beta-dendrotoxin (0.3 mu M), dofetilde (1 mu M) and terikalant (10 mu M), each in combination with apamin (0.3 mu M) had no effect on the EDHF-mediated relaxation induced by acetylcholine in the presence of N-omega-nitro-L-arginine (0.3 mM) and indomethacin (10 mu M), inhibitors of nitric oxide (NO) synthase and cyclo-oxygenase, respectively (n=2-3). Although the K-V inhibitor margatoxin (0.3 mu M) was also without effect (n=5), the combination of margatoxin and apamin produced a small inhibition of the response (pEC(50) anti E-max values were 7.5+/-0.0 and 95+/-1% in the absence and 7.0+/-0.1 and 81+/-6% in the presence of margatoxin plus apamin, respectively; n=6; P<0.05). 3 Ciclazindol (10 mu M) partially inhibited the EDHF-mediated relaxation by shifting the acetylcholine-concentration-response curve 12 fold to the right (n=6; P<0.05) and abolished the response when combined with apamin (0.3 mu M; n=6). This combination did not inhibit acetylcholine-induced relaxations mediated by endothelium-derived NO (n=5). 4 A 4-aminopyridine-sensitive delayed rectifier current (I-K(V)) was identified in freshly-isolated single smooth muscle cells from rat hepatic artery. None of the cells displayed a rapidly-activating and -inactivating A-type current. Neither charybdotoxin (0.3 mu M; n=3) nor ciclazindol (10 mu M; n=5), alone or in combination with apamin (0.3 mu M; n=4-5), had an effect on I-K(V). A tenfold higher concentration of ciclazindol (0.1 mM, n=4) markedly inhibited I-K(V), but this effect was not increased in the additional presence of apamin (0.3 mu M; n=2). 5 By use of membranes prepared from rat brain cortex, [I-125]-charybdotoxin binding was consistent with an interaction at a single site with a K-D of approximately 25 pM. [I-125]-charybdotoxin binding was unaffected by iberiotoxin (0.1 mu M, n=6), but was increased by apamin in a concentration-dependent manner (E-max 43+/-10%, P<0.05 and pEC(50) 7.1+/-0.2; n=7-8). Agitoxin-2 (10 nM) displaced [I-125]-charybdotoxin binding by 91+/-3% (n=6) and prevented the effect of apamin (1 mu M; n=6). 6 It is concluded that the EDHF-mediated relaxation in the rat hepatic artery is not mediated by the opening of either K-V or BKCa. Instead, the target K-channels for EDHF seem to be structurally related to both K-V and BKCa. The possibility that a subtype of SKCa may be the target for EDHF is discussed.