In addition to nitric oxide (NO) and prostacyclin, endothelium-derived hyperpolarizing factor (EDHF) appears to be an important mediator of vasodilatation in various vascular beds, including the rat mesenteric artery. EDHF-mediated relaxation of these arteries is inhibited by the simultaneous presence of apamin and charybdotoxin, inhibitors of small (SK_Ca), and intermediate (IK_Ca) conductance calcium-sensitive potassium channels, respectively, but not by either toxin alone (Waldron & Garland, 1994). Subsequent work by Doughty et al. (1998) demonstrated that the site of action of apamin and charybdotoxin was on the endothelium, not the smooth muscle. Together, these findings have led some authors to speculate that the actions of the toxin inhibitors were exerted on two Ca²⁺-sensitive K⁺ channels located on the endothelium.

Although apamin is considered as a specific inhibitor of SK_Ca, it has been shown to influence charybdotoxin binding to voltage-gated K⁺ channels (K_V) channels (Zygmunt et al. 1997). Since K_V1.2 and K_V1.3 channels are also blocked by charybdotoxin, we investigated the involvement of these channels in EDHF-mediated relaxation in rat isolated mesenteric artery.

Male Wistar rats (225-250 g) were killed by cervical dislocation and mesenteric arteries of third and fourth order branches were dissected free. In isometrically mounted arteries (321 ± 9.8 µm), contracted with phenylephrine (0.5-2 µM), acetylcholine (ACh, 0.01-10 µM) elicited concentration-dependent relaxation in the presence of N-nitro-L-arginine methyl ester (100 µM) and indomethacin (2.8 µM), inhibitors of NO-synthase and cyclooxygenase, respectively.

The non-selective K_V inhibitor 4-aminopyridine (4-AP, 1 mM), and apamin (0.5 µM) alone had no significant effect on the EDHF-mediated relaxation by ACh (n = 6). In combination 4-AP and apamin had moderate effects on the EDHF-mediated relaxation (EC₅₀ 58 nM; mean E_max values ± S.E.M., 97.5 ± 2.2 % in the absence, and 64.8 ± 7.5 % in the presence of 4-AP plus apamin, respectively; n = 6).

Maurotoxin, shown to block cloned K_V1.2 and K_V1.3 channels (0.5 µM) was also without effect on vasorelaxation (n = 6). However, the combination of maurotoxin and apamin (0.5 µM) completely abolished EDHF-mediated relaxation in rat mesenteric arteries (n = 6). Immunocytochemistry using subtype-specific antibodies confirmed the expression of both K_V1.2 and K_V1.3 channels on endothelial cells in rat mesenteric arteries. However, this toxin may block potassium channels other than K_V1.2 and K_V1.3.

These data demonstrate for the first time since Waldron & Garland (1994) a novel toxin combination that abolishes EDHF-mediated relaxation in the rat mesenteric artery. We conclude that peptide toxin inhibition of EDHF-mediated vasorelaxation is best explained by the action of apamin and the peptide at a single site that may be structurally related to both K_V and SK_Ca. Further experiments will determine the selectivity of this toxin for K_V channels.We thank the BHF for financial support.

Doughty, J.M., Plane, F. & Langton, P.D. (1998). Am. J. Physiol. 276, 1107-1112.

Waldron, C.J. & Garland, C.J. (1994). Can. J. Physiol. Pharmacol. 72 (suppl. 1), 11.

Zygmunt, P.M., Edwards, G., Weston, A.H., Larsson, B. & Hogestatt, E.D. (1997). Br. J. Pharmacol. 121, 141-149.