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 Experiments were performed to elucidate the mechanism by which alterations of extracellular pH (pH o ) change membrane potential (E M ) in rat mesenteric and pulmonary arteries. 2 Changing pH o from 7.4 to 6.4 or 8.4 produced a depolarisation or hyperpolarisation, respectively, in mesenteric and pulmonary arteries. Anandamide (10 mM) or bupivacaine (100 mM) reversed the hyperpolarisation associated with alkaline pH o , shifting the E M of both vessels to levels comparable to that at pH 6.4. In pulmonary arteries, clofilium (100 mM) caused a significant reversal of hyperpolarisation seen at pH 8.4 but was without effect at pH 7.4. 3 K þ channel blockade by 4-aminopyridine (4-AP) (5 mM), tetraethylammonium (TEA) (10 mM), Ba 2 þ (30 mM) and glibenclamide (10 mM) depolarised the pulmonary artery. However, shifts in E M with changes in pH o remained and were sensitive to anandamide (10 mM), bupivacaine (100 mM) or Zn 2 þ (200 mM). 4 Anandamide (0.3-60 mM) or bupivacaine (0.3-300 mM) caused a concentration-dependent increase in basal tone in pulmonary arteries. 5 RT-PCR demonstrated the expression of TASK-1, TASK-2, THIK-1, TRAAK, TREK-1, TWIK-1 and TWIK-2 in mesenteric arteries and TASK-1, TASK-2, THIK-1, TREK-2 and TWIK-2 in pulmonary arteries. TASK-1, TASK-2, TREK-1 and TWIK-2 protein was demonstrated in both arteries by immunostaining. 6 These experiments provide evidence for the presence of two-pore domain K þ channels in rat mesenteric and pulmonary arteries. Collectively, they strongly suggest that modulation of TASK-1 channels is most likely to have mediated the pH-induced changes in membrane potential observed in these vessels, and that blockade of these channels by anandamide or bupivacaine generates a small increase in pulmonary artery tone.

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.

Background and purpose: The small and intermediate conductance, Ca 2 þ -sensitive K þ channels (SK Ca and IK Ca , respectively) which are pivotal in the EDHF pathway may be differentially activated. The importance of caveolae in the functioning of IK Ca and SK Ca channels was investigated. Experimental approach: The effect of the caveolae-disrupting agent methyl-b-cyclodextrin (MbCD) on IK Ca and SK Ca localization and function was determined. Key results: EDHF-mediated, SK Ca -dependent myocyte hyperpolarizations evoked by acetylcholine in rat mesenteric arteries (following blockade of IK Ca with TRAM-34) were inhibited by MbCD. Hyperpolarizations evoked by direct SK Ca channel activation (using NS309 in the presence of TRAM-34) were also inhibited by MbCD, an effect reversed by cholesterol. In contrast, IK Ca -dependent hyperpolarizations (in the presence of apamin) were unaffected by MbCD. Similarly, in porcine coronary arteries, EDHF-mediated, SK Ca -dependent (but not IK Ca -dependent) endothelial cell hyperpolarizations evoked by substance P were inhibited by MbCD. In mesenteric artery homogenates subjected to sucrose-density centrifugation, caveolin-1 and SK3 (SK Ca ) proteins but not IK1 (IK Ca ) protein migrated to the buoyant, caveolin-rich fraction. MbCD pretreatment redistributed caveolin-1 and SK3 proteins into more dense fractions. In immunofluorescence images of porcine coronary artery endothelium, SK3 (but not IK1) and caveolin-1 were co-localized. Furthermore, caveolin-1 immunoprecipitates prepared from native porcine coronary artery endothelium contained SK3 but not IK1 protein.Conclusions and Implications: These data provide strong evidence that endothelial cell SK Ca channels are located in caveolae while the IK Ca channels reside in a different membrane compartment. These studies reveal cellular organisation as a further complexity in the EDHF pathway signalling cascade.

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: