1 The apamin-sensitive small-conductance Ca 2+ -activated K + channel (SK Ca ) was characterized in porcine coronary arteries. 2 In intact arteries, 100 nM substance P and 600 mM 1-ethyl-2-benzimidazolinone (1-EBIO) produced endothelial cell hyperpolarizations (27.8+0.8 mV and 24.1+1.0 mV, respectively). Charybdotoxin (100 nM) abolished the 1-EBIO response but substance P continued to induce a hyperpolarization (25.8+0.3 mV).3 In freshly-isolated endothelial cells, outside-out patch recordings revealed a unitary K + conductance of 6.8+0.04 pS. The open-probability was increased by Ca 2+ and reduced by apamin (100 nM). Substance P activated an outward current under whole-cell perforated-patch conditions and a component of this current (38%) was inhibited by apamin. A second conductance of 2.7+0.03 pS inhibited by d-tubocurarine was observed infrequently. 4 Messenger RNA encoding the SK2 and SK3, but not the SK1, subunits of SK Ca was detected by RT ± PCR in samples of endothelium. Western blotting indicated that SK3 protein was abundant in samples of endothelium compared to whole arteries. SK2 protein was present in whole artery nuclear fractions. 5 Immuno¯uorescent labelling con®rmed that SK3 was highly expressed at the plasmalemma of endothelial cells and was not expressed in smooth muscle. SK2 was restricted to the peri-nuclear regions of both endothelial and smooth muscle cells. 6 In conclusion, the porcine coronary artery endothelium expresses an apamin-sensitive SK Ca containing the SK3 subunit. These channels are likely to confer all or part of the apamin-sensitive component of the endothelium-derived hyperpolarizing factor (EDHF) response.

1 This study characterizes the K + channel(s) underlying charybdotoxin-sensitive hyperpolarization of porcine coronary artery endothelium.2 Two forms of current-voltage (I/V) relationship were evident in whole-cell patch-clamp recordings of freshly-isolated endothelial cells. In both cell types, iberiotoxin (100 nM) inhibited a current active only at potentials over +50 mV. In the presence of iberiotoxin, charybdotoxin (100 nM) produced a large inhibition in 38% of cells and altered the form of the I/V relationship. In the remaining cells, charybdotoxin also inhibited a current but did not alter the form. 3 Single-channel, outside-out patch recordings revealed a 17.1+0.4 pS conductance. Pipette solutions containing 100, 250 and 500 nM free Ca 2+ demonstrated that the open probability was increased by Ca 2+. This channel was blocked by charybdotoxin but not by iberiotoxin or apamin. 4 Hyperpolarizations of intact endothelium elicited by substance P (100 nM; 26.1+0.7 mV) were reduced by apamin (100 nM; 17.0+1.8 mV) whereas those to 1-ethyl-2-benzimidazolinone (1-EBIO, 600 mM, 21.0+0.3 mV) were unaected (21.7+0.8 mV). Substance P, bradykinin (100 nM) and 1-EBIO evoked charybdotoxin-sensitive, iberiotoxin-insensitive whole-cell perforated-patch currents.

Mechanisms underlying K+‐induced hyperpolarizations in the presence and absence of phenylephrine were investigated in endothelium‐denuded rat mesenteric arteries (for all mean values, n=4). Myocyte resting membrane potential (m.p.) was −58.8±0.8 mV. Application of 5 mM KCl produced similar hyperpolarizations in the absence (17.6±0.7 mV) or presence (15.8±1.0 mV) of 500 nM ouabain. In the presence of ouabain +30 μM barium, hyperpolarization to 5 mM KCl was essentially abolished. In the presence of 10 μM phenylephrine (m.p. −33.7±3 mV), repolarization to 5 mM KCl did not occur in the presence or absence of 4‐aminopyridine but was restored (−26.9±1.8 mV) on addition of iberiotoxin (100 nM). Under these conditions the K+‐induced repolarization was insensitive to barium (30 μM) but abolished by 500 nM ouabain alone. In the presence of phenylephrine + iberiotoxin the hyperpolarization to 5 mM K+ was inhibited in the additional presence of 300 nM levcromakalim, an action which was reversed by 10 μM glibenclamide. RT–PCR, Western blotting and immunohistochemical techniques collectively showed the presence of α1‐, α2‐ and α3‐subunits of Na+/K+‐ATPase in the myocytes. In K+‐free solution, re‐introduction of K+ (to 4.6 mM) hyperpolarized myocytes by 20.9±0.5 mV, an effect unchanged by 500 nM ouabain but abolished by 500 μM ouabain. We conclude that under basal conditions, Na+/K+‐ATPases containing α2‐ and/or α3‐subunits are partially responsible for the observed K+‐induced effects. The opening of myocyte K+ channels (by levcromakalim or phenylephrine) creates a ‘K+ cloud’ around the cells which fully activates Na+/K+‐ATPase and thereby abolishes further responses to [K+]o elevation. British Journal of Pharmacology (2002) 136, 918–926. doi:

In intact mesenteric arteries, increasing [K + ] o by 5 mM hyperpolarized both endothelial and smooth muscle cells. Subsequent exposure to 10 mM phenylephrine depolarized both cell types which were then repolarized by a 5 mM increase in [K + ] o . In endothelium-denuded vessels, increasing [K + ] o by 5 mM hyperpolarized the smooth muscle but K + had no e ect after depolarization by 10 mM phenylephrine. On subsequent exposure to iberiotoxin plus 4-aminopyridine, the repolarizing action of 5 mM K + was restored. In endothelium-intact vessels exposed to phenylephrine, pretreatment with a gap junction inhibitor (gap 27) reduced K + -mediated smooth muscle repolarization without a ecting the endothelial cell response. It is concluded that phenylephrine-induced e ux of K + via smooth muscle K + channels produces a local increase in [K + ] o which impairs repolarization to added K + . Thus, studies involving vessels precontracted with agonists which increase [K + ] o maximize the role of gap junctions and minimize any contribution to the EDHF pathway from endothelium-derived K + .

1 In porcine coronary arteries, smooth muscle hyperpolarizations produced by the nitric oxide donor, NOR-1, and the prostacyclin analogue, iloprost, were compared with those induced by substance P and bradykinin and attributed to the endothelium-derived hyperpolarizing factor (EDHF). 2 In the presence of 300 mM L-nitroarginine and 10 mM indomethacin, iloprost-induced hyperpolarizations were partially inhibited by 10 mM glibenclamide whereas those to NOR-1, substance P and bradykinin were una ected. 3 Hyperpolarizations produced by maximally-e ective concentrations of NOR-1 and NS1619 were identical (to 765 mV). They were signi®cantly less than those generated by either substance P or bradykinin (to approximately 780 mV) and were abolished by iberiotoxin 100 nM, a concentration which had essentially no e ect on responses to substance P or bradykinin. 4 Incubation of segments of intact arteries for 16 ± 22 h in bicarbonate-bu ered Krebs solution had little e ect on EDHF responses to substance P or bradykinin. In contrast, after incubation for this period of time in HEPES-bu ered Tyrode solution or Krebs containing 10 mM HEPES the EDHF response to substance P was abolished and that to bradykinin was markedly reduced. The residual bradykinin-induced hyperpolarization following incubation in Tyrode solution was inhibited by iberiotoxin and by 10 mM 17-octadecynoic acid. 5 We conclude that substance P activates only the EDHF pathway in the presence of nitric oxide synthase and cyclo-oxygenase inhibitors. Incubation in HEPES-bu ered Tyrode solution abolishes the EDHF responses to substance P and bradykinin to reveal an additional hyperpolarizing mechanism, associated with the opening of K + channels, activated only by bradykinin. British Journal of Pharmacology (2001) 133, 1145 ± 1153 Keywords: EDHF; iberiotoxin; porcine coronary artery; gap junctions; HEPES; bradykinin; prostacyclin; substance P; nitric oxide donor; hyperpolarization Abbreviations: BK Ca , large conductance calcium-sensitive K + channel; 1-EBIO, 1-ethyl-2-benzimidazolinone; EDHF, endotheliumderived hyperpolarizing factor; HEPES, N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulphonic acid); IntroductionIn small arteries and arterioles it is now well established that agonists which interact with vascular endothelial cell receptors can hyperpolarize and relax the underlying smooth muscle. It has long been assumed that the endothelium is stimulated to release an endothelium-derived hyperpolarizing factor (EDHF) which di uses across the myo-endothelial space to exert its e ects by opening smooth muscle K + channels (see reviews by Edwards & Weston, 1998; Fe le tou & Vanhoutte, 1999). However, the identity of this`factor' remains unknown and in view of the dissimilar pharmacology of the EDHF responses in di erent species and blood vessels, the possibility that several endogenous substances are involved cannot be discounted.In most vascular preparations, a characteristic feature of the EDHF response is its full inhibition by charybdotoxin+apamin but only slig...