title: 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 (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 (KV) represent another target for charybdotoxin, we explored the possible involvement of KV in EDHF-mediated relaxation in this artery. 2. The KV inhibitors, agitoxin-2 (0.3 microM), kaliotoxin (0.3 microM), beta-dendrotoxin (0.3 microM), dofetilide (1 microM) and terikalant (10 microM), each in combination with apamin (0.3 microM) 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 microM), inhibitors of nitric oxide (NO) synthase and cyclo-oxygenase, respectively (n = 2-3). Although the KV inhibitor margatoxin (0.3 microM) was also without effect (n = 5), the combination of margatoxin and apamin produced a small inhibition of the response (pEC50 and Emax 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 microM) 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 microM; 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 (IK(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 microM; n = 3) nor ciclazindol (10 microM; n = 5), alone or in combination with apamin (0.3 microM; n = 4-5), had an effect on IK(V). A tenfold higher concentration of ciclazindol (0.1 mM, n = 4) markedly inhibited IK(V), but this effect was not increased in the additional presence of apamin (0.3 microM; 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 KD of approximately 25 pM. [125I]-charybdotoxin binding was unaffected by iberiotoxin (0.1 microM, n = 6), but was increased by apamin in a concentration-dependent manner (Emax 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 microM; n = 6). 6. It is concluded that the EDHF-mediated relaxation in the rat hepatic artery is not mediated by the opening of either KV or BKCa. Instead, the target K-channels for EDHF seem to be structurally related to both KV and BKCa. The possibility that a subtype of SKCa may be the target for EDHF is discussed.
authors.affiliation: School of Biological Sciences, University of Manchester, U.K.
authors.forename: P M
authors.initials: PM
authors.lastname: Zygmunt
authors.forename: G
authors.initials: G
authors.lastname: Edwards
authors.forename: A H
authors.initials: AH
authors.lastname: Weston
authors.forename: B
authors.initials: B
authors.lastname: Larsson
authors.forename: E D
authors.initials: ED
authors.lastname: Högestätt
mesh_terms: D015761:4-Aminopyridine / Q000494:pharmacology; D000109:Acetylcholine / Q000494:pharmacology; D000818:Animals; D000889:Anti-Arrhythmia Agents / Q000378:metabolism / Q000494:pharmacology; D001030:Apamin / Q000378:metabolism / Q000494:pharmacology; D001667:Binding, Competitive; D001685:Biological Factors / Q000494:pharmacology / Q000502:physiology*; D002540:Cerebral Cortex / Q000187:drug effects / Q000378:metabolism; D018999:Charybdotoxin / Q000378:metabolism / Q000494:pharmacology; D002839:Chromans / Q000378:metabolism / Q000494:pharmacology; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology*; D004347:Drug Interactions; D005260:Female; D006499:Hepatic Artery / Q000187:drug effects* / Q000378:metabolism; D007211:Indoles / Q000378:metabolism / Q000494:pharmacology; D007213:Indomethacin / Q000494:pharmacology; D009126:Muscle Relaxation / Q000187:drug effects / Q000502:physiology; D009131:Muscle, Smooth, Vascular / Q000187:drug effects* / Q000502:physiology; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors*; D019335:Nitroarginine / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D010627:Phenethylamines / Q000378:metabolism / Q000494:pharmacology; D010880:Piperidines / Q000378:metabolism / Q000494:pharmacology; D026902:Potassium Channel Blockers; D015221:Potassium Channels / Q000187:drug effects / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013449:Sulfonamides / Q000378:metabolism / Q000494:pharmacology
chemical_list: D000889:Anti-Arrhythmia Agents; D001685:Biological Factors; D002839:Chromans; D016861:Cyclooxygenase Inhibitors; D007211:Indoles; D010627:Phenethylamines; D010880:Piperidines; D026902:Potassium Channel Blockers; D015221:Potassium Channels; D013449:Sulfonamides; C067410:endothelium-dependent hyperpolarization factor; D018999:Charybdotoxin; D019335:Nitroarginine; D001030:Apamin; D015761:4-Aminopyridine; D019001:Nitric Oxide Synthase; C063899:terikalant; D000109:Acetylcholine; C063533:dofetilide; D007213:Indomethacin; C009990:ciclazindol