G Edwards

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 mMKCl produced similar hyperpolarizations in the absence (17.6±0.7 mV) or presence (15.8±1.0 mV) of 500 nMouabain. In the presence of ouabain +30 μMbarium, hyperpolarization to 5 mMKCl was essentially abolished. In the presence of 10 μMphenylephrine (m.p. −33.7±3 mV), repolarization to 5 mMKCl 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 nMouabain alone. In the presence of phenylephrine + iberiotoxin the hyperpolarization to 5 mMK⁺was inhibited in the additional presence of 300 nMlevcromakalim, an action which was reversed by 10 μMglibenclamide. 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 nMouabain but abolished by 500 μMouabain. 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⁺]_oelevation. British Journal of Pharmacology(2002)136, 918–926. doi:10.1038/sj.bjp.0704787

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). In the presence of 300 μM L‐nitroarginine and 10 μM indomethacin, iloprost‐induced hyperpolarizations were partially inhibited by 10 μM glibenclamide whereas those to NOR‐1, substance P and bradykinin were unaffected. Hyperpolarizations produced by maximally‐effective concentrations of NOR‐1 and NS1619 were identical (to −65 mV). They were significantly less than those generated by either substance P or bradykinin (to approximately −80 mV) and were abolished by iberiotoxin 100 nM, a concentration which had essentially no effect on responses to substance P or bradykinin. Incubation of segments of intact arteries for 16–22 h in bicarbonate‐buffered Krebs solution had little effect on EDHF responses to substance P or bradykinin. In contrast, after incubation for this period of time in HEPES‐buffered 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 μM 17‐octadecynoic acid. We conclude that substance P activates only the EDHF pathway in the presence of nitric oxide synthase and cyclo‐oxygenase inhibitors. Incubation in HEPES‐buffered 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; doi:10.1038/sj.bjp.0704157

The characteristics of endothelium‐dependent hyperpolarization in rat hepatic artery have been further investigated in the presence of inhibitors of cyclo‐oxygenase and nitric oxide synthase. Using sharp micro‐electrodes, the smooth muscle hyperpolarization induced by acetylcholine, KCl or 1‐ethyl‐2‐benzimidazolinone (1‐EBIO) in intact hepatic arteries was abolished by 30 μM barium plus 500 nM ouabain. In vessels without endothelium, the smooth muscle hyperpolarization induced by KCl was not reduced by 30 μM barium alone. However, in the presence of barium the effects of KCl were partially inhibited by 100 nM ouabain and essentially abolished by 500 nM ouabain. Using sharp micro‐electrodes, the hyperpolarization of both the smooth muscle and the endothelium induced by 1‐EBIO or by acetylcholine was unaffected by 100 nM iberiotoxin. However, in the presence of 100 nM charybdotoxin, the effects of 1‐EBIO were abolished whereas those of acetylcholine were only partially reduced. The hyperpolarization induced by levcromakalim was unaffected by either charybdotoxin or iberiotoxin. Under whole‐cell patch‐clamp recording conditions, 1‐EBIO induced a voltage‐insensitive, charybdotoxin‐sensitive K⁺ current in cultured endothelial cells but was without effect on K⁺ currents in smooth muscle cells isolated from hepatic arteries. It is concluded that the endothelium‐dependent hyperpolarization of smooth muscle induced by either acetylcholine or by 1‐EBIO in rat hepatic artery is initially associated with the opening of endothelial calcium‐sensitive K⁺‐channels insensitive to iberiotoxin. The resulting accumulation of K⁺ in the myoendothelial space activates an isoform of Na⁺/K⁺‐ATPase which is sensitive to low concentrations of ouabain. British Journal of Pharmacology (1999) 128, 1064–1070; doi:10.1038/sj.bjp.0702916