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

Abstract

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 (SK_Ca) and large (BK_Ca) conductance calcium-sensitive potassium (K)-channels, respectively, but not by each toxin alone. The selective BK_Ca 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 μM), kaliotoxin (0.3 μM), β-dendrotoxin (0.3 μM), dofetilide (1 μM) and terikalant (10 μM), each in combination with apamin (0.3 μM) had no effect on the EDHF-mediated relaxation induced by acetylcholine in the presence of N^ω-nitro-L-arginine (0.3 mM) and indomethacin (10 μM), inhibitors of nitric oxide (NO) synthase and cyclo-oxygenase, respectively (n=2–3). Although the K_V inhibitor margatoxin (0.3 μM) was also without effect (n=5), the combination of margatoxin and apamin produced a small inhibition of the response (pEC₅₀ and 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 μM) 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 μM; 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 μM; n=3) nor ciclazindol (10 μM; n=5), alone or in combination with apamin (0.3 μM; 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 μM; n=2).

5. By use of membranes prepared from rat brain cortex, [¹²⁵I]-charybdotoxin binding was consistent with an interaction at a single site with a K_D of approximately 25 pM. [¹²⁵I]-charybdotoxin binding was unaffected by iberiotoxin (0.1 μM, n=6), but was increased by apamin in a concentration-dependent manner (E_max 43±10%, P<0.05 and pEC₅₀ 7.1±0.2; n=7–8). Agitoxin-2 (10 nM) displaced [¹²⁵I]-charybdotoxin binding by 91±3% (n=6) and prevented the effect of apamin (1 μM; 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 BK_Ca. Instead, the target K-channels for EDHF seem to be structurally related to both K_V and BK_Ca. The possibility that a subtype of SK_Ca may be the target for EDHF is discussed.

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