Potassium channelopathy-like defect underlies early-stage cerebrovascular dysfunction in a genetic model of small vessel disease.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), caused by dominant mutations in the NOTCH3 receptor in vascular smooth muscle, is a genetic paradigm of small vessel disease (SVD) of the brain. Recent studies using transgenic (Tg)Notch3(R169C) mice, a genetic model of CADASIL, revealed functional defects in cerebral (pial) arteries on the surface of the brain at an early stage of disease progression. Here, using parenchymal arterioles (PAs) from within the brain, we determined the molecular mechanism underlying the early functional deficits associated with this Notch3 mutation. At physiological pressure (40 mmHg), smooth muscle membrane potential depolarization and constriction to pressure (myogenic tone) were blunted in PAs from TgNotch3(R169C) mice. This effect was associated with an ∼ 60% increase in the number of voltage-gated potassium (KV) channels, which oppose pressure-induced depolarization. Inhibition of KV1 channels with 4-aminopyridine (4-AP) or treatment with the epidermal growth factor receptor agonist heparin-binding EGF (HB-EGF), which promotes KV1 channel endocytosis, reduced KV current density and restored myogenic responses in PAs from TgNotch3(R169C) mice, whereas pharmacological inhibition of other major vasodilatory influences had no effect. KV1 currents and myogenic responses were similarly altered in pial arteries from TgNotch3(R169C) mice, but not in mesenteric arteries. Interestingly, HB-EGF had no effect on mesenteric arteries, suggesting a possible mechanistic basis for the exclusive cerebrovascular manifestation of CADASIL. Collectively, our results indicate that increasing the number of KV1 channels in cerebral smooth muscle produces a mutant vascular phenotype akin to a channelopathy in a genetic model of SVD.

K(Ca)3.1 channel downregulation and impaired endothelium-derived hyperpolarization-type relaxation in pulmonary arteries from chronically hypoxic rats.

Calcium-activated potassium channels of small (K(Ca)2, SK) and intermediate (K(Ca)3.1, IK) conductance are involved in endothelium-dependent relaxation of pulmonary arteries. We hypothesized that the function and expression of K(Ca)2 and K(Ca)3.1 increase as a compensatory mechanism to counteract hypoxia-induced pulmonary hypertension in rats. For functional studies, pulmonary arteries were mounted in microvascular myographs for isometric tension recordings. The K(Ca) channel expression was evaluated by immunoblotting and quantitative PCR. Although ACh induced similar relaxations, the ACh-induced relaxations were abolished by the combined inhibition of nitric oxide synthase (by L-nitro-arginine, L-NOARG), cyclo-oxygenase (by indomethacin) and soluble guanylate cyclase (by ODQ) in pulmonary arteries from hypoxic rats, whereas 20 ± 6% (n = 8) maximal relaxation in response to ACh persisted in arteries from normoxic rats. Inhibiting Na(+),K(+)-ATPase with ouabain or blocking K(Ca)2 and K(Ca)3.1 channels reduced the persisting ACh-induced relaxation. In the presence of L-NOARG and indomethacin, a novel K(Ca)2 and K(Ca)3.1 channel activator, NS4591, induced concentration- and endothelium-dependent relaxations, which were markedly reduced in arteries from chronically hypoxic rats compared with arteries from normoxic rats. The mRNA levels of K(Ca)2.3 and K(Ca)3.1 were unaltered, whereas K(Ca)2.3 protein expression was upregulated and K(Ca)3.1 protein expression downregulated in pulmonary arteries from rats exposed to hypoxia. In conclusion, endothelium-dependent relaxation was conserved in pulmonary arteries from chronically hypoxic rats, while endothelium-derived hyperpolarization (EDH)-type relaxation was impaired in chronically hypoxic pulmonary small arteries despite upregulation of K(Ca)2.3 channels. Since impaired EDH-type relaxation was accompanied by K(Ca)3.1 channel protein downregulation, these findings suggest that K(Ca)3.1 channels are important for the maintenance of EDH-type relaxation.

Opening of small and intermediate calcium-activated potassium channels induces relaxation mainly mediated by nitric-oxide release in large arteries and endothelium-derived hyperpolarizing factor in small arteries from rat.

This study was designed to investigate whether calcium-activated potassium channels of small (SK(Ca) or K(Ca)2) and intermediate (IK(Ca) or K(Ca)3.1) conductance activated by 6,7-dichloro-1H-indole-2,3-dione 3-oxime (NS309) are involved in both nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF)-type relaxation in large and small rat mesenteric arteries. Segments of rat superior and small mesenteric arteries were mounted in myographs for functional studies. NO was recorded using NO microsensors. SK(Ca) and IK(Ca) channel currents and mRNA expression were investigated in human umbilical vein endothelial cells (HUVECs), and calcium concentrations were investigated in both HUVECs and mesenteric arterial endothelial cells. In both superior (∼1093 µm) and small mesenteric (∼300 µm) arteries, NS309 evoked endothelium- and concentration-dependent relaxations. In superior mesenteric arteries, NS309 relaxations and NO release were inhibited by both N(G),N(G)-asymmetric dimethyl-l-arginine (ADMA) (300 µM), an inhibitor of NO synthase, and apamin (0.5 µM) plus 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34) (1 µM), blockers of SK(Ca) and IK(Ca) channels, respectively. In small mesenteric arteries, NS309 relaxations were reduced slightly by ADMA, whereas apamin plus an IK(Ca) channel blocker almost abolished relaxation. Iberiotoxin did not change NS309 relaxation. HUVECs expressed mRNA for SK(Ca) and IK(Ca) channels, and NS309 induced increases in calcium, outward current, and NO release that were blocked by apamin and TRAM-34 or charybdotoxin. These findings suggest that opening of SK(Ca) and IK(Ca) channels leads to endothelium-dependent relaxation that is mediated mainly by NO in large mesenteric arteries and by EDHF-type relaxation in small mesenteric arteries. NS309-induced calcium influx appears to contribute to the formation of NO.

Mechanisms underlying epithelium-dependent relaxation in rat bronchioles: analogy to EDHF-type relaxation in rat pulmonary arteries.

This study investigated the mechanisms underlying epithelium-derived hyperpolarizing factor (EpDHF)-type relaxation in rat bronchioles. Immunohistochemistry was performed, and rat bronchioles and pulmonary arteries were mounted in microvascular myographs for functional studies. An opener of small (SK(Ca)) and intermediate (IK(Ca))-conductance calcium-activated potassium channels, NS309 (6,7-dichloro-1H-indole-2,3-dione 3-oxime) was used to induce EpDHF-type relaxation. IK(Ca) and SK(Ca)3 positive immunoreactions were observed mainly in the epithelium and endothelium of bronchioles and arteries, respectively. In 5-hydroxytryptamine (1 microM)-contracted bronchioles (828 +/- 20 microm, n = 84) and U46619 (0.03 microM)-contracted arteries (720 +/- 24 microm, n = 68), NS309 (0.001-10 microM) induced concentration-dependent relaxations that were reduced by epithelium/endothelium removal and by blocking IK(Ca) channels with charybdotoxin and in bronchioles also by blocking SK(Ca) channels with apamin. Inhibition of cyclooxygenase, nitric oxide synthase, and cytochrome 2C isoenzymes, or blockade of large (BK(Ca))-conductance calcium-activated potassium channels with iberiotoxin, failed to reduce NS309 relaxation. In contrast to the pulmonary arteries, relaxations to a beta(2)-adrenoceptor agonist, salbutamol, were reduced in bronchioles by removing the epithelium or blocking IK(Ca) and/or SK(Ca) channels. Extracellular K(+) (2-20 mM) induced relaxation in both bronchioles and arteries. An inhibitor of Na(+)-K(+)-ATPase, ouabain, abolished relaxations to NS309, salbutamol, and K(+). These results suggest that IK(Ca) and SK(Ca)3 channels are located in the epithelium of bronchioles and endothelium of pulmonary arteries. Analog to the endothelium-derived hyperpolarizing factor (EDHF)-type relaxation in pulmonary arteries, these channels may be involved in EpDHF-type relaxation of bronchioles caused by epithelial K(+) efflux followed by activation of Na(+)-K(+)-ATPase in the underlying smooth muscle layer.

Activation of endothelial and epithelial K(Ca) 2.3 calcium-activated potassium channels by NS309 relaxes human small pulmonary arteries and bronchioles.

BACKGROUND AND PURPOSE: Small (K(Ca) 2) and intermediate (K(Ca) 3.1) conductance calcium-activated potassium channels (K(Ca) ) may contribute to both epithelium- and endothelium-dependent relaxations, but this has not been established in human pulmonary arteries and bronchioles. Therefore, we investigated the expression of K(Ca) 2.3 and K(Ca) 3.1 channels, and hypothesized that activation of these channels would produce relaxation of human bronchioles and pulmonary arteries. EXPERIMENTAL APPROACH: Channel expression and functional studies were conducted in human isolated small pulmonary arteries and bronchioles. K(Ca) 2 and K(Ca) 3.1 currents were examined in human small airways epithelial (HSAEpi) cells by whole-cell patch clamp techniques. RESULTS: While K(Ca) 2.3 expression was similar, K(Ca) 3.1 protein was more highly expressed in pulmonary arteries than bronchioles. Immunoreactive K(Ca) 2.3 and K(Ca) 3.1 proteins were found in both endothelium and epithelium. K(Ca) currents were present in HSAEpi cells and sensitive to the K(Ca) 2.3 blocker UCL1684 and the K(Ca) 3.1 blocker TRAM-34. In pulmonary arteries contracted by U46619 and in bronchioles contracted by histamine, the K(Ca) 2.3/ K(Ca) 3.1 activator, NS309, induced concentration-dependent relaxations. NS309 was equally potent in relaxing pulmonary arteries, but less potent in bronchioles, than salbutamol. NS309 relaxations were blocked by the K(Ca) 2 channel blocker apamin, while the K(Ca) 3.1 channel blocker, charybdotoxin failed to reduce relaxation to NS309 (0.01-1 µM). CONCLUSIONS AND IMPLICATIONS: K(Ca) 2.3 and K(Ca) 3.1 channels are expressed in the endothelium of human pulmonary arteries and epithelium of bronchioles. K(Ca) 2.3 channels contributed to endo- and epithelium-dependent relaxations suggesting that these channels are potential targets for treatment of pulmonary hypertension and chronic obstructive pulmonary disease.

Calcium-activated potassium channels - a therapeutic target for modulating nitric oxide in cardiovascular disease?

IMPORTANCE OF THE FIELD: Cardiovascular risk factors are often associated with endothelial dysfunction, which is also prognostic for occurrence of cardiovascular events. Endothelial dysfunction is reflected by blunted vasodilatation and reduced nitric oxide (NO) bioavailability. Endothelium-dependent vasodilatation is mediated by NO, prostacyclin, and an endothelium-derived hyperpolarising factor (EDHF), and involves small (SK) and intermediate (IK) conductance Ca(2+)-activated K(+) channels. Therefore, SK and IK channels may be drug targets for the treatment of endothelial dysfunction in cardiovascular disease. AREAS COVERED IN THIS REVIEW: SK and IK channels are involved in EDHF-type vasodilatation, but recent studies suggest that these channels are also involved in the regulation of NO bioavailability. Here we review how SK and IK channels may regulate NO bioavailability. WHAT THE READER WILL GAIN: Opening of SK and IK channels is associated with EDHF-type vasodilatation, but, through increased endothelial cell Ca(2+) influx, L-arginine uptake, and decreased ROS production, it may also lead to increased NO bioavailability and endothelium-dependent vasodilatation. TAKE HOME MESSAGE: Opening of SK and IK channels can increase both EDHF and NO-mediated vasodilatation. Therefore, openers of SK and IK channels may have the potential of improving endothelial cell function in cardiovascular disease.

Role of calcium-activated potassium channels with small conductance in bradykinin-induced vasodilation of porcine retinal arterioles.

PURPOSE: Endothelial dysfunction and impaired vasodilation may be involved in the pathogenesis of retinal vascular diseases. In the present study, the mechanisms underlying bradykinin vasodilation were examined and whether calcium-activated potassium channels of small (SK(Ca)) and intermediate (IK(Ca)) conductance are involved in regulation of endothelium-dependent vasodilation in retinal arterioles was investigated. METHODS: Porcine retinal arterioles (diameter approximately 112 microm, N = 119) were mounted in microvascular myographs for isometric tension recordings. The arterioles were contracted with the thromboxane analogue, U46619, and concentration-response curves were constructed for bradykinin and a novel opener of SK(Ca) and IK(Ca) channels, NS309. RESULTS: In U46619-contracted arterioles, bradykinin and NS309 induced concentration-dependent relaxations. In vessels without endothelium, bradykinin relaxation was abolished and NS309 relaxation was attenuated. Inhibition of NO synthase with asymmetric dimethylarginine and/or cyclooxygenase with indomethacin markedly reduced bradykinin and NS309 relaxation. NO synthase and cyclooxygenase inhibition together with oxyhemoglobin abolished bradykinin relaxation and attenuated NS309 relaxation. Blocking of SK(Ca) and IK(Ca) channels with apamin plus charybdotoxin or blocking of SK(Ca) channels alone in the absence and the presence of indomethacin markedly reduced bradykinin and NS309 relaxation, whereas blocking of IK(Ca) channels had no significant effect. In vessels without endothelium, blocking of SK(Ca) channels alone had no effect on sodium nitroprusside-induced relaxation. CONCLUSIONS: In porcine retinal arterioles, NO and prostaglandins mediate endothelium-dependent relaxation to bradykinin and NS309. Moreover, these findings suggest that SK(Ca) channels contribute to NO-mediated relaxation induced by bradykinin and NS309 and, hence, may play an important role in retinal arterial endothelial function.