Prolongation of the cardiac QRS complex is linked to increased mortality and may result from drug‐induced inhibition of cardiac sodium channels (hNaV1.5). There has been no systematic evaluation of preclinical and marketed drugs for their additional potential to cause QRS prolongation via gap junction uncoupling.

Background and purpose: The small and intermediate conductance, Ca 2+ ‐sensitive K + 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‐β‐cyclodextrin (MβCD) 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 MβCD. Hyperpolarizations evoked by direct SK Ca channel activation (using NS309 in the presence of TRAM‐34) were also inhibited by MβCD, an effect reversed by cholesterol. In contrast, IK Ca ‐dependent hyperpolarizations (in the presence of apamin) were unaffected by MβCD. 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 MβCD. 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. MβCD 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. British Journal of Pharmacology (2007) 151 , 332–340; doi: 10.1038/sj.bjp.0707222

We have examined the relative contributions of small‐ and intermediate‐conductance Ca2+‐activated K+ channels (SKCa and IKCa) to the endothelium‐derived hyperpolarizing factor (EDHF) pathway response in small mesenteric arteries of Zucker Diabetic Fatty (ZDF) rats, before and after the development of Type II diabetes, together with Lean controls. Smooth muscle membrane potential was recorded using sharp microelectrodes in the presence of 10 μM indomethacin plus 100 μM Nω‐nitro‐L‐arginine. SKCa was selectively inhibited with 100 nM apamin, whereas IKCa was blocked with 10 μM TRAM‐39 (2‐(2‐chlorophenyl)‐2,2‐diphenylacetonitrile). Resting membrane potentials were similar in arteries from 17‐ to 20‐week‐old control and diabetic rats (approximately −54 mV). Responses elicited by 1 and 10 μM acetylcholine (ACh) were significantly smaller in the diabetic group (e.g. hyperpolarizations to −69.5±0.8 mV (ZDF; n=12) and −73.2±0.6 mV (Lean; n=12; P<0.05) evoked by 10 μM ACh). The IKCa‐mediated components of the ACh responses were comparable between groups (hyperpolarizations to approximately −65 mV on exposure to 10 μM ACh). However, SKCa‐mediated responses were significantly reduced in the diabetic group (hyperpolarizations to −63.1±1.0 mV (ZDF; n=6) and −71.5±1.2 mV (Lean; n=6; P<0.05) on exposure to 10 μM ACh. Impaired ACh responses were not observed in arteries from 5‐ to 6‐week‐old (pre‐diabetic) animals. SKCa subunit mRNA expression was increased in the diabetic group. The EDHF pathway, especially the SKCa‐mediated response, is impaired in Type II diabetic ZDF rats without a reduction in channel gene expression. These results may be particularly relevant to the microvascular complications of diabetes. The functional separation of SKCa and IKCa pathways is discussed. British Journal of Pharmacology (2006) 148, 434–441. doi:10.1038/sj.bjp.0706748

Although it is well established that diabetes impairs endothelium-dependent vasodilation, including those pathways involving vascular myocyte large-conductance Ca(2+)-activated K(+) channels (BK(Ca)), little is known about the effects of diabetes on BK(Ca) activation as an intrinsic response to contractile stimulation. We have investigated this mechanism in a model of Type 2 diabetes, the male Zucker diabetic fatty (ZDF) rat. BK(Ca) function in prediabetic (5-7 wk) and diabetic (17-20 wk) ZDF and lean control animals was assessed in whole arteries using myograph and electrophysiology techniques and in freshly dissociated myocytes by patch clamping. Log EC(25) values for phenylephrine concentration-tension curves were shifted significantly to the left by blockade of BK(Ca) with iberiotoxin (IBTX) in arteries from non- and prediabetic animals but not from diabetic animals. Smooth muscle hyperpolarizations of arteries evoked by the BK(Ca) opener NS-1619 were significantly reduced in the diabetic group. Voltage-clamp recordings indicated that IBTX-sensitive currents were not enhanced to the extent observed in nondiabetic controls by increasing the Ca(2+) concentration in the pipette solution or the application of NS-1619 in myocytes from diabetic animals. An alteration in the expression of BK(Ca) beta(1) subunits was not evident at either the mRNA or protein level in arteries from diabetic animals. Collectively, these results suggest that myocyte BK(Ca) of diabetic animals does not significantly oppose vasoconstriction, unlike that of prediabetic and control animals. This altered function was related to a reduced Ca(2+)-dependent activation of the channel not involving beta(1) subunits.

Experiments were performed to elucidate the mechanism by which alterations of extracellular pH (pHo) change membrane potential (EM) in rat mesenteric and pulmonary arteries. Changing pHo from 7.4 to 6.4 or 8.4 produced a depolarisation or hyperpolarisation, respectively, in mesenteric and pulmonary arteries. Anandamide (10 μM) or bupivacaine (100 μM) reversed the hyperpolarisation associated with alkaline pHo, shifting the EM of both vessels to levels comparable to that at pH 6.4. In pulmonary arteries, clofilium (100 μM) caused a significant reversal of hyperpolarisation seen at pH 8.4 but was without effect at pH 7.4. K+ channel blockade by 4‐aminopyridine (4‐AP) (5 mM), tetraethylammonium (TEA) (10 mM), Ba2+ (30 μM) and glibenclamide (10 μM) depolarised the pulmonary artery. However, shifts in EM with changes in pHo remained and were sensitive to anandamide (10 μM), bupivacaine (100 μM) or Zn2+ (200 μM). Anandamide (0.3–60 μM) or bupivacaine (0.3–300 μM) caused a concentration‐dependent increase in basal tone in pulmonary arteries. 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. These experiments provide evidence for the presence of two‐pore domain K+ 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. British Journal of Pharmacology (2004) 142, 192–202. doi:10.1038/sj.bjp.0705691

The aims of the study were to compare the myogenic and structural properties of middle cerebral arteries (MCAs) from the stroke-prone spontaneously hypertensive rat (SHRSP) with MCAs from the spontaneously hypertensive rat (SHR) before stroke development in SHRSP. Rats were fed a "Japanese" diet (low-protein rat chow and 1% NaCl in drinking water) for 8 wk, and cerebral arteries were studied in vitro at 12 wk using a pressure arteriograph. Systolic pressure was significantly increased in SHRSP compared with SHR at 12 wk. Between 60 and 180 mmHg, MCAs from SHR maintained an essentially constant diameter, i.e., displayed a "myogenic range," whereas the diameter of MCAs from SHRSP progressively increased as a function of pressure. Passive lumen diameter of MCAs from SHRSP was reduced at high pressure, and wall thickness and wall/lumen were increased, compared with SHR. Wall cross-sectional area was also increased in MCAs from SHRSP compared with the SHR, indicating growth. The stress-strain relationship was shifted to the left in MCAs from SHRSP, indicating decreased MCA distensibility compared with SHR. However, collagen staining with picrosirius red revealed a redistribution of collagen to the outer half of the MCA wall in SHRSP compared with SHR. These data demonstrate impaired myogenic properties in prestroke SHRSP compared with SHR, which may explain stroke development. The structural differences in MCAs from SHRSP compared with SHR were a consequence of both growth and a reduced distensibility.

The apamin‐sensitive small‐conductance Ca2+‐activated K+ channel (SKCa) was characterized in porcine coronary arteries. In intact arteries, 100 nM substance P and 600 μM 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). 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 Ca2+ 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. Messenger RNA encoding the SK2 and SK3, but not the SK1, subunits of SKCa 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. Immunofluorescent labelling confirmed 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. In conclusion, the porcine coronary artery endothelium expresses an apamin‐sensitive SKCa 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. British Journal of Pharmacology (2002) 135, 1133–1143; doi:10.1038/sj.bjp.0704551

This study characterizes the K+ channel(s) underlying charybdotoxin‐sensitive hyperpolarization of porcine coronary artery endothelium. 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. Single‐channel, outside‐out patch recordings revealed a 17.1±0.4 pS conductance. Pipette solutions containing 100, 250 and 500 nM free Ca2+ demonstrated that the open probability was increased by Ca2+. This channel was blocked by charybdotoxin but not by iberiotoxin or apamin. 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 μM, 21.0±0.3 mV) were unaffected (21.7±0.8 mV). Substance P, bradykinin (100 nM) and 1‐EBIO evoked charybdotoxin‐sensitive, iberiotoxin‐insensitive whole‐cell perforated‐patch currents. A porcine homologue of the intermediate‐conductance Ca2+‐activated K+ channel (IK1) was identified in endothelial cells. In conclusion, porcine coronary artery endothelial cells express an intermediate‐conductance Ca2+‐activated K+ channel and the IK1 gene product. This channel is opened by activation of the EDHF pathway and likely mediates the charybdotoxin‐sensitive component of the EDHF response. British Journal of Pharmacology (2002) 137, 1346–1354. doi:10.1038/sj.bjp.0705057

1. 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). 2. 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 microM barium, hyperpolarization to 5 mM KCl was essentially abolished. 3. In the presence of 10 microM 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 microM) but abolished by 500 nM ouabain alone. 4. 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 microM glibenclamide. 5. RT-PCR, Western blotting and immunohistochemical techniques collectively showed the presence of alpha(1)-, alpha(2)- and alpha(3)-subunits of Na(+)/K(+)-ATPase in the myocytes. 6. 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 microM ouabain. 7. We conclude that under basal conditions, Na(+)/K(+)-ATPases containing alpha(2)- and/or alpha(3)-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.

In intact mesenteric arteries, increasing [K+]o by 5 mM hyperpolarized both endothelial and smooth muscle cells. Subsequent exposure to 10 μM 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 effect after depolarization by 10 μM 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 affecting the endothelial cell response. It is concluded that phenylephrine‐induced efflux 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+.British Journal of Pharmacology (2001) 134, 1–5; doi:10.1038/sj.bjp.0704256