Role of potassium channels in endothelium‐dependent relaxation resistant to nitroarginine in the rat hepatic artery

Abstract

1 In the presence of indomethacin (IM, 1OiM) and Nw-nitro-L-arginine (L-NOARG, 0.3 mM), acetylcholine (ACh) induces an endothelium-dependent smooth muscle hyperpolarization and relaxation in the rat isolated hepatic artery. The potassium (K) channel inhibitors, tetrabutylammonium (TBA, 1 mM) and to a lesser extent 4-aminopyridine (4-AP, 1 mM) inhibited the L-NOARG/IM-resistant relaxation induced by ACh, whereas apamin (0.1-0.3 gM), charybdotoxin (0.1-0.3MM), iberiotoxin (0.1 MM) and dendrotoxin (0.1 Mm) each had no effect. TBA also inhibited the relaxation induced by the receptor-independent endothelial cell activator, A23187. 2 When combined, apamin (0.1 MM)+charybdotoxin (0.1 uM), but not apamin (0.1 MM)+iberiotoxin (0.1 Mm) or a triple combination of 4-AP (1 mM) + apamin (0.1 MM) + iberiotoxin (0.1 Mm), inhibited the L-NOARG/IM-resistant relaxation induced by ACh. At a concentration of 0.3 gM, apamin + charybdotoxin completely inhibited the relaxation. This toxin combination also abolished the L-NOARG/ IM-resistant relaxation induced by A23187. 3 In the absence of L-NOARG, TBA (1 mM) inhibited the ACh-induced relaxation, whereas charybdotoxin (0.3 MM)+ apamin (0.3 gM) had no effect, indicating that the toxin combination did not interfere with the L-arginine/NO pathway. 4 The gap junction inhibitors halothane (2 mM) and 1-heptanol (2 mM), or replacement of NaCl with sodium propionate did not affect the L-NOARG/IM-resistant relaxation induced by ACh.5 Inhibition of Na+/K+-ATPase by ouabain (1 mM) had no effect on the L-NOARG/IM-resistant relaxation induced by ACh. Exposure to a K+-free Krebs solution, however, reduced the maximal relaxation by 13% without affecting the sensitivity to ACh. 6 The results suggest that the L-NOARG/IM-resistant relaxation induced by ACh in the rat hepatic artery is mediated by activation of K-channels sensitive to TBA and a combination of apamin+charybdotoxin. Chloride channels, Na+/K+-ATPase and gap junctions are probably not involved in the response. It is proposed that endothelial cell activation induces secretion of an endothelium-derived hyperpolarizing factor(s) (EDHF), distinct from NO and cyclo-oxygenase products, which activates more than one type of K-channel on the smooth muscle cells. Alternatively, a single type of K-channel, to which both apamin and charybdotoxin must bind for inhibition to occur, may be the target for EDHF.

Dominant role of an endothelium‐derived hyperpolarizing factor (EDHF)‐like vasodilator in the ciliary vascular bed of the bovine isolated perfused eye

“…In contrast, charybdotoxin (50 nM) alone signi®cantly attenuated acetylcholine-induced vasodilatation, and uncovered a vasoconstrictor response. In previous studies (Zygmunt & Hogestatt, 1996;Edwards et al, 1998;1999) charybdotoxin alone did not attenuate acetylcholineinduced vasodilatation as powerfully as was seen in the bovine eye, and this may re¯ect the absence of the unusual acetylcholine-induced vasoconstriction observed in our preparation. Full characterization of this vasoconstrictor response was beyond the scope of this study, but it appears to be endothelium-dependent, since it was not observed when the endothelium was functionally impaired with CHAPS.…”

“…The vasodilatation was, however, inhibited by TEA, which is a relatively non-selective calcium-sensitive K + (K + Ca ) channel inhibitor (Cook & Quast, 1990), although it does not block SK + Ca , even at concentrations up to 4 mM (Zima et al, 2000). Our ability to block acetylcholine-induced vasodilatation in the bovine eye with TEA is in keeping with its ability to inhibit EDHF-mediated vasodilatation in human forearm resistance vessels (Honing et al, 2000) and in rat mesenteric arteries (Chen & Cheung, 1997 (Zygmunt & Hogestatt, 1996;Edwards et al, 1998;1999). On the basis of these results, the EDHF pathway is thought to involve the opening of SK + Ca channels, and IK + Ca channels but not BK + Ca channels on the endothelial cell (Edwards et al, 1998;Ohashi et al, 1999).…”

Evidence that potassium channels make a major contribution to SIN‐1‐evoked relaxation of rat isolated mesenteric artery

“…In tissues pre-incubated with L-arginine and NO synthase inhibitors together, SIN-i-evoked relaxations showed a similar sensitivity to charybdotoxin as intact arterial segments, presumably because the L-arginine prevented the total inhibition of NO synthase. Furthermore, in intact tissues in which NO synthesis was inhibited and charybdotoxin had little effect on SIN-i-evoked relaxation, the addition of L-arginine for 60 min partially restored the sensitivity of SIN-i-evoked Zygmunt & Hogestatt (1996), to explain the finding that, in rat scle relaxation.…”

Endothelium-Derived Epoxyeicosatrienoic Acids and Vascular Function

“…[7][8][9] Part of the reason for some of the controversy was that EETs were initially reported to initiate smooth muscle hyperpolarization by activating iberiotoxin-sensitive large conductance Ca 2ϩ -activated K ϩ (BK Ca ) channels, 10 whereas EDHFdependent relaxation was more universally sensitive to the combination of charybdotoxin and apamin (inhibitors of small and intermediate conductance K Ca channels) than to iberiotoxin. 11 Another point that argued against the involvement of EETs in the regulation of vascular tone was that, whereas several of the initial studies aimed at characterizing EDHF used BK Ca -expressing cultured smooth muscle cells as a detector, smooth muscle cells from many freshly isolated arteries do not express BK Ca channels. 12 It is now clear, however, that the decisive charybdotoxin and apaminsensitive hyperpolarizing event in EDHF-mediated responses is the hyperpolarization of the vascular endothelium and not of smooth muscle cells.…”