The expression and function of Ca(2+)-sensing receptors in rat mesenteric artery; comparative studies using a model of type II diabetes.

BACKGROUND AND PURPOSE: The extracellular calcium-sensing receptor (CaR) in vascular endothelial cells activates endothelial intermediate-conductance, calcium-sensitive K(+) channels (IK(Ca)) indirectly leading to myocyte hyperpolarization. We determined whether CaR expression and function was modified in a rat model of type II diabetes. EXPERIMENTAL APPROACH: Pressure myography, western blotting, sharp microelectrode and K(+)-selective electrode recordings were used to investigate the functional expression of the CaR and IK(Ca) in rat mesenteric arteries. KEY RESULTS: Myocyte hyperpolarization to the CaR activator calindol was inhibited by Calhex 231. U46619-induced vessel contraction elevated the extracellular [K(+)] around the myocytes, and inhibition of this 'K(+) cloud' by iberiotoxin was needed to reveal calindol-induced vasodilatations. These were antagonized by Calhex 231 and significantly smaller in Zucker diabetic fatty rat (ZDF) vessels than in Zucker lean (ZL) controls. Myocyte hyperpolarizations to calindol were also smaller in ZDF than in ZL arteries. In ZDF vessels, endothelial cell CaR protein expression was reduced; IK(Ca) expression was also diminished, but IK(Ca)-generated hyperpolarizations mediated by 1-EBIO were unaffected. CONCLUSIONS AND IMPLICATIONS: The reduced CaR-mediated hyperpolarizing and vasodilator responses in ZDF arteries result from a decrease in CaR expression, rather than from a modification of IK(Ca) channels. Detection of CaR-mediated vasodilatation required the presence of iberiotoxin, suggesting a CaR contribution to vascular diameter, that is, inversely related to the degree of vasoconstriction. Compromise of the CaR pathway would favour the long-term development of a higher basal vascular tone and could contribute to the vascular complications associated with type II diabetes.

Effects of methyl beta-cyclodextrin on EDHF responses in pig and rat arteries; association between SK(Ca) channels and caveolin-rich domains.

BACKGROUND AND PURPOSE: The small and intermediate conductance, Ca2+-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-beta-cyclodextrin (MbetaCD) 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 MbetaCD. Hyperpolarizations evoked by direct SK(Ca) channel activation (using NS309 in the presence of TRAM-34) were also inhibited by MbetaCD, an effect reversed by cholesterol. In contrast, IK(Ca)-dependent hyperpolarizations (in the presence of apamin) were unaffected by MbetaCD. 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 MbetaCD. 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. MbetaCD 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.

Impairment of endothelial SK(Ca) channels and of downstream hyperpolarizing pathways in mesenteric arteries from spontaneously hypertensive rats.

BACKGROUND AND PURPOSE: Previous studies have shown that endothelium-dependent hyperpolarization of myocytes is reduced in resistance arteries from spontaneously hypertensive rats (SHRs). The aim of the present study was to determine whether this reflects down-regulation of endothelial K(+) channels or their associated pathways. EXPERIMENTAL APPROACH: Changes in vascular K(+) channel responses and expression were determined by a combination of membrane potential recordings and Western blotting. KEY RESULTS: Endothelium-dependent myocyte hyperpolarizations induced by acetylcholine, 6,7-dichloro-1H-indole-2,3-dione 3-oxime (NS309) (opens small- and intermediate-conductance calcium-sensitive K(+) channels, SK(Ca) and IK(Ca), respectively) or cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine (SK(Ca) opener) were reduced in mesenteric arteries from SHRs. After blocking SK(Ca) channels with apamin, hyperpolarizations to acetylcholine and NS309 in SHR arteries were similar to those of controls. Hyperpolarization to 5 mM KCl was reduced in SHR arteries due to loss of the Ba(2+)-sensitive, inward-rectifier channel (K(IR)) component; the contribution of ouabain-sensitive, Na(+)/K(+)-ATPases was unaffected. Protein expression of both SK(Ca) and K(IR) channels was reduced in SHR arteries; the caveolin-1 monomer/dimer ratio was increased. CONCLUSIONS AND IMPLICATIONS: In SHRs, the distinct pathway that generates endothelium-dependent hyperpolarization in vascular myocyte by activation of IK(Ca) channels and Na(+)/K(+)-ATPases remains intact. The second pathway, initiated by endothelial SK(Ca) channel activation and amplified by K(IR) opening on both endothelial cells and myocytes is compromised in SHRs due to down-regulation of both SK(Ca) and K(IR) and to changes in caveolin-1 oligomers. These impairments in the SK(Ca)-K(IR) pathway shed new light on vascular control mechanisms and on the underlying vascular changes in hypertension.