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The augmentation of BK channel activity by nitric oxide signaling in rat cerebral arteries involves co-localized regulatory elements.

ABSTRACT: Large conductance, Ca²⁺-activated K⁺ (BK) channels control cerebrovascular tone; however, the regulatory processes influencing these channels remain poorly understood. Here, we investigate the cellular mechanisms underlying the enhancement of BK current in rat cerebral arteries by nitric oxide (NO) signaling. In isolated cerebral myocytes, BK current magnitude was reversibly increased by sodium nitroprusside (SNP, 100??M) and sensitive to the BK channel inhibitor, penitrem-A (100?nM). Fostriecin (30?nM), a protein phosphatase type 2A (PP2A) inhibitor, significantly prolonged the SNP-induced augmentation of BK current and a similar effect was produced by sildenafil (30?nM), a phosphodiesterase 5 (PDE5) inhibitor. Using proximity ligation assay (PLA)-based co-immunostaining, BK channels were observed to co-localize with PP2A, PDE5, and cGMP-dependent protein kinase (cGKI) (spatial restriction?

SUBMITTER: Kyle BD

PROVIDER: S-EPMC5718322 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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The augmentation of BK channel activity by nitric oxide signaling in rat cerebral arteries involves co-localized regulatory elements.

Kyle Barry D BD Mishra Ramesh C RC Braun Andrew P AP

Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism 20170203 12

Large conductance, Ca<sup>2+</sup>-activated K<sup>+</sup> (BK) channels control cerebrovascular tone; however, the regulatory processes influencing these channels remain poorly understood. Here, we investigate the cellular mechanisms underlying the enhancement of BK current in rat cerebral arteries by nitric oxide (NO) signaling. In isolated cerebral myocytes, BK current magnitude was reversibly increased by sodium nitroprusside (SNP, 100 μM) and sensitive to the BK channel inhibitor, penitrem- ...[more]

PMID: 28155571

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Project description:Nitric oxide (NO) relaxes vascular smooth muscle cells (SMCs) and dilates blood vessels by increasing intracellular levels of cyclic guanosine monophosphate (cGMP), which stimulates the activity of cGMP-dependent protein kinase (PKG). However, the vasodilator mechanisms downstream of PKG remain incompletely understood. Here, we found that transient receptor potential melastatin 4 (TRPM4) cation channels, which are activated by Ca2+ released from the sarcoplasmic reticulum (SR) through inositol triphosphate receptors (IP3Rs) under native conditions, are essential for SMC membrane depolarization and vasoconstriction. We hypothesized that signaling via the NO/cGMP/PKG pathway causes vasodilation by inhibiting TRPM4. We found that TRPM4 currents activated by stretching the plasma membrane or directly activating IP3Rs were suppressed by exogenous NO or a membrane-permeable cGMP analog, the latter of which also impaired IP3R-mediated release of Ca2+ from the SR. The effects of NO on TRPM4 activity were blocked by inhibition of soluble guanylyl cyclase or PKG. Notably, upon phosphorylation by PKG, IRAG (IP3R-associated PKG substrate) inhibited IP3R-mediated Ca2+ release, and knockdown of IRAG expression diminished NO-mediated inhibition of TRPM4 activity and vasodilation. Using superresolution microscopy, we found that IRAG, PKG, and IP3Rs form a nanoscale signaling complex on the SR of SMCs. We conclude that NO/cGMP/PKG signaling through IRAG inhibits IP3R-dependent activation of TRPM4 channels in SMCs to dilate arteries.Significance statementNitric oxide is a gaseous vasodilator produced by endothelial cells that is essential for cardiovascular function. Although NO-mediated signaling pathways have been intensively studied, the mechanisms by which they relax SMCs to dilate blood vessels remain incompletely understood. In this study, we show that NO causes vasodilation by inhibiting the activity of Ca2+-dependent TRPM4 cation channels. Probing further, we found that NO does not act directly on TRPM4 but instead initiates a signaling cascade that inhibits its activation by blocking the release of Ca2+ from the SR. Thus, our findings reveal the essential molecular pathways of NO-induced vasodilation-a fundamental unresolved concept in cardiovascular physiology.

Project description:Activation of the apelin/APJ receptor signaling system causes endothelium-dependent and nitric oxide (NO)-dependent relaxation in several peripheral arteries. The effects of apelin in cerebral arteries are unknown; however, apelin inhibits voltage-dependent increases in large-conductance, calcium-activated K channel (BKCa) currents in cerebral artery smooth muscle cells. Because NO-induced relaxation of cerebral arteries is mediated, in part, by activation of BKCa channels, the goals of this study were to determine the net effect of apelin in cerebral arteries, as well as test the hypothesis that the actions of apelin in cerebral arteries are secondary to stimulation of APJ receptors. Immunoblot and quantitative reverse transcription polymerase chain reaction analyses detected APJ receptors in cerebral arteries of male Sprague-Dawley rats, and immunofluorescence studies using confocal microscopy confirmed APJ receptor localization in smooth muscle cells. In myograph studies, apelin itself had no direct vasomotor effect but inhibited relaxations to the NO-donor, diethylamine NONOate, and to the endothelium-dependent vasodilator, bradykinin. These effects of apelin were mimicked by the selective BKCa-channel blocker, iberiotoxin, and suppressed by the APJ receptor antagonist, F13A. Apelin also inhibited relaxations evoked by the BKCa-channel openers, NS1619 and BMS 191011, but had no effect on relaxation to levcromakalim, a selective KATP-channel opener. Apelin had no effect on diethylamine NONOate-induced or bradykinin-induced increases in cyclic guanosine monophosphate levels. Patch clamp recordings demonstrated that apelin and iberiotoxin each suppressed the increase in BKCa currents induced by DEA and NS1619 in freshly isolated cerebral artery smooth muscle cells. The results demonstrate that apelin inhibits NO-induced relaxation of cerebral arteries through a mechanism involving activation of APJ receptors and inhibition of BKCa channels in cerebral arterial smooth muscle cells.

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The augmentation of BK channel activity by nitric oxide signaling in rat cerebral arteries involves co-localized regulatory elements.

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The augmentation of BK channel activity by nitric oxide signaling in rat cerebral arteries involves co-localized regulatory elements.

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