The combination of the two toxins, apamin (a specific inhibitor of small conductance calcium-activated potassium channels: SK_Ca) and charybdotoxin (a non specific inhibitor of large and intermediate conductance calcium-activated potassium channels: BK_Ca and IK_Ca, respectively) is required fully to block endothelium-dependent hyperpolarization (EDHF; Busse et al., 2002). The release of an endothelial cytochrome P450 monooxygenase (P450) metabolite, which hyperpolarizes and relaxes the vascular smooth muscle cells, by activating BK_Ca could account for some EDHF-mediated responses (Campbell et al., 1996; Fisslthaler et al., 1999; Weston et al., 2003). Since charybdotoxin inhibits both BK_Ca and IK_Ca, a P450 metabolite could partially contribute to the EDHF-mediated responses in some arteries. Experiments were designed, in the guinea-pig isolated carotid artery, to assess the effects of new specific and non-peptide inhibitors of SK_Ca and IK_Ca on the EDHF-mediated responses and secondly to determine whether the production of a P450 metabolite and the activation of BK_Ca might contribute to endothelium-dependent hyperpolarizations.

Guinea-pigs were euthanized with a lethal dose of pentobarbital and the carotid arteries were dissected free and mounted in an organ bath. Membrane potential was recorded in the vascular smooth muscle cells of the guinea-pig isolated carotid artery with sharp microelectrodes implanted from the adventitial side. All the experiments were performed in the presence of inhibitors of NO-synthases and cyclooxygenases, N-L-nitro arginine (100 µM) and indomethacin (5 µM), respectively.

Acetylcholine (10 nM to 10 µM) induced a concentration-dependent and endothelium-dependent hyperpolarization (-23 ± 1 mV, n = 7, for acetylcholine 10 µM). Two blockers of SK_Ca, apamin (0.5 µM) or 6,10-diaza-3(1,3),8(1,4)-dibenzena-1,5(1,4)-diquinolinacyclodecaphane (UCL 1684, Rosa et al., 1998) at 10 µM (but not at 1 µM) produced a partial but significant inhibition of the hyperpolarization produced by acetylcholine (for instance, at 10 µM acetylcholine: -15 ± 2, and -12 ± 2 mV, n = 5, for apamin and UCL 1684, respectively; p < 0.05 vs control). In contrast, two blockers of IK_Ca, charybdotoxin (0.1 µM) or a clotrimazole derivative, 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34, Wulff et al., 2000) at both 5 and 10 µM were completely ineffective. The combination of apamin + charybdotoxin, apamin + TRAM-34 (10 µM) or UCL 1684 (10 µM) + TRAM-34 (10 µM) very significantly inhibited the EDHF-mediated responses induced by acetylcholine (for instance, at 10 µM acetylcholine: -6 ± 2, -7 ± 0, and -6 ± 1 mV, n = 5, 3 and 4, respectively for each combination; p < 0.05 vs control). The effect of the combination of apamin + the lower concentration of TRAM-34 (5 µM) was not significantly different from that of apamin alone. In the presence of this latter combination of blockers, the residual EDHF-mediated response produced by acetylcholine (0.1 to 10 µM) was not inhibited further by the addition of either a specific blocker of BK_Ca, iberiotoxin (0.1 µM), or by an epoxyeicosatrienoic acid (EET)-specific antagonist, 14,15-epoxyeicosa-5(Z)-enoic acid (14,15 EEZE: 10 µM; Gauthier et al., 2002).

In the quiescent carotid artery of the guinea pig, it is concluded that the activation of SK_Ca plays a predominant role in the acetylcholne-induced endothelium-dependent hyperpolarization of the smooth muscle cells. Small molecular weight, non-peptide blockers of SK_Ca and IK_Ca mimic the effects of apamin and charybdotoxin, respectively, further substantiating the involvement of these two calcium-sensitive potassium channels (Busse et al., 2002). The results provide no evidence for the production of a cytochrome P450 metabolite and the subsequent activation of BK_Ca in EDHF-mediated responses in the guinea-pig carotid artery.

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