Ca2+-sensing receptor cleavage by calpain partially accounts for altered vascular reactivity in mice fed a high-fat diet.

The Ca-sensing receptor (CaSR) is expressed in endothelial and smooth muscle cells, but its role in regulating vascular reactivity is unclear, as are the effects of disease on CaSR function and expression. We studied vascular reactivity in aortic segments from healthy and diabetic mice, combined with in vitro proteolysis studies and Western blot analyses of CaSR expression in tissue samples. In endothelium-intact aortic rings, extracellular Ca elicited a nitric oxide-dependent relaxation that was attenuated by the CaSR antagonist, NPS2390. The calcimimetic, calindol, induced the endothelium-independent relaxation of aortic segments that was also sensitive to NPS2390. The antagonist failed to affect responses to acetylcholine or U46619 but attenuated contractions to phenylephrine and potassium. In mice fed a Western-type diet, phenylephrine-induced contractions and calindol-induced relaxations were markedly attenuated, and CaSR expression was decreased. The latter phenomenon could be attributed to the activation of the Ca-dependent protease, µ-calpain, and the subsequent proteolytic cleavage of the CaSR. CaSR activation in smooth muscle cells modulates vascular responsiveness to Ca-elevating agonists. These effects are blunted during metabolic stress because of the limited proteolysis of the CaSR by calpain. The loss of the CaSR function may predispose to the macrovascular late complications associated with diabetes.

11,12-EET stimulates the association of BK channel α and ß(1) subunits in mitochondria to induce pulmonary vasoconstriction.

In the systemic circulation, 11,12-epoxyeicosatrienoic acid (11,12-EET) elicits nitric oxide (NO)- and prostacyclin-independent vascular relaxation, partially through the activation of large conductance Ca(2+)-activated potassium (BK) channels. However, in the lung 11,12-EET contributes to hypoxia-induced pulmonary vasoconstriction. Since pulmonary artery smooth muscle cells also express BK channels, we assessed the consequences of BKß(1) subunit deletion on pulmonary responsiveness to 11,12-EET as well as to acute hypoxia. In buffer-perfused mouse lungs, hypoxia increased pulmonary artery pressure and this was significantly enhanced in the presence of NO synthase (NOS) and cyclooxygenase (COX) inhibitors. Under these conditions the elevation of tissue EET levels using an inhibitor of the soluble epoxide hydrolase (sEH-I), further increased the hypoxic contraction. Direct administration of 11,12-EET also increased pulmonary artery pressure, and both the sEH-I and 11,12-EET effects were prevented by iberiotoxin and absent in BKß(1)(-/-) mice. In pulmonary artery smooth muscle cells treated with NOS and COX inhibitors and loaded with the potentiometric dye, di-8-ANEPPS, 11,12-EET induced depolarization while the BK channel opener NS1619 elicited hyperpolarization indicating there was no effect of the EET on classical plasma membrane BK channels. In pulmonary artery smooth muscle cells a subpopulation of BK channels is localized in mitochondria. In these cells, 11,12-EET elicited an iberiotoxin-sensitive loss of mitochondrial membrane potential (JC-1 fluorescence) leading to plasma membrane depolarization, an effect not observed in BKß(1)(-/-) cells. Mechanistically, stimulation with 11,12-EET time-dependently induced the association of the BK α and ß(1) subunits. Our data indicate that in the absence of NO and prostacyclin 11,12-EET contributes to pulmonary vasoconstriction by stimulating the association of the α and ß(1) subunits of mitochondrial BK channels. The 11,12-EET-induced activation of BK channels results in loss of the mitochondrial membrane potential and depolarization of the pulmonary artery smooth muscle cells.