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Urocortin 2 stimulates nitric oxide production in ventricular myocytes via Akt- and PKA-mediated phosphorylation of eNOS at serine 1177.

ABSTRACT: Urocortin 2 (Ucn2) is a cardioactive peptide exhibiting beneficial effects in normal and failing heart. In cardiomyocytes, it elicits cAMP- and Ca(2+)-dependent positive inotropic and lusitropic effects. We tested the hypothesis that, in addition, Ucn2 activates cardiac nitric oxide (NO) signaling and elucidated the underlying signaling pathways and mechanisms. In isolated rabbit ventricular myocytes, Ucn2 caused concentration- and time-dependent increases in phosphorylation of Akt (Ser473, Thr308), endothelial NO synthase (eNOS) (Ser1177), and ERK1/2 (Thr202/Tyr204). ERK1/2 phosphorylation, but not Akt and eNOS phosphorylation, was suppressed by inhibition of MEK1/2. Increased Akt phosphorylation resulted in increased Akt kinase activity and was mediated by corticotropin-releasing factor 2 (CRF2) receptors (astressin-2B sensitive). Inhibition of phosphatidylinositol 3-kinase (PI3K) diminished both Akt as well as eNOS phosphorylation mediated by Ucn2. Inhibition of protein kinase A (PKA) reduced Ucn2-induced phosphorylation of eNOS but did not affect the increase in phosphorylation of Akt. Conversely, direct receptor-independent elevation of cAMP via forskolin increased phosphorylation of eNOS but not of Akt. Ucn2 increased intracellular NO concentration ([NO]i), [cGMP], [cAMP], and cell shortening. Inhibition of eNOS suppressed the increases in [NO]i and cell shortening. When both PI3K-Akt and cAMP-PKA signaling were inhibited, the Ucn2-induced increases in [NO]i and cell shortening were attenuated. Thus, in rabbit ventricular myocytes, Ucn2 causes activation of cAMP-PKA, PI3K-Akt, and MEK1/2-ERK1/2 signaling. The MEK1/2-ERK1/2 pathway is not required for stimulation of NO signaling in these cells. The other two pathways, cAMP-PKA and PI3K-Akt, converge on eNOS phosphorylation at Ser1177 and result in pronounced and sustained cellular NO production with subsequent stimulation of cGMP signaling.

SUBMITTER: Walther S

PROVIDER: S-EPMC4187402 | biostudies-literature | 2014 Sep

REPOSITORIES: biostudies-literature

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Urocortin 2 stimulates nitric oxide production in ventricular myocytes via Akt- and PKA-mediated phosphorylation of eNOS at serine 1177.

Walther Stefanie S Pluteanu Florentina F Renz Susanne S Nikonova Yulia Y Maxwell Joshua T JT Yang Li-Zhen LZ Schmidt Kurt K Edwards Joshua N JN Wakula Paulina P Groschner Klaus K Maier Lars S LS Spiess Joachim J Blatter Lothar A LA Pieske Burkert B Kockskämper Jens J

American journal of physiology. Heart and circulatory physiology 20140711 5

Urocortin 2 (Ucn2) is a cardioactive peptide exhibiting beneficial effects in normal and failing heart. In cardiomyocytes, it elicits cAMP- and Ca(2+)-dependent positive inotropic and lusitropic effects. We tested the hypothesis that, in addition, Ucn2 activates cardiac nitric oxide (NO) signaling and elucidated the underlying signaling pathways and mechanisms. In isolated rabbit ventricular myocytes, Ucn2 caused concentration- and time-dependent increases in phosphorylation of Akt (Ser473, Thr3 ...[more]

PMID: 25015964

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Project description:KEY POINTS:Small-conductance Ca2+ -activated K+ (SK) channels expressed in ventricular myocytes are dormant in health, yet become functional in cardiac disease. SK channels are voltage independent and their gating is controlled by intracellular [Ca2+ ] in a biphasic manner. Submicromolar [Ca2+ ] activates the channel via constitutively-bound calmodulin, whereas higher [Ca2+ ] exerts inhibitory effect during depolarization. Using a rat model of cardiac hypertrophy induced by thoracic aortic banding, we found that functional upregulation of SK2 channels in hypertrophic rat ventricular cardiomyocytes is driven by protein kinase A (PKA) phosphorylation. Using site-directed mutagenesis, we identified serine-465 as the site conferring PKA-dependent effects on SK2 channel function. PKA phosphorylation attenuates ISK rectification by reducing the Ca2+ /voltage-dependent inhibition of SK channels without changing their sensitivity to activating submicromolar [Ca2+ ]i . This mechanism underlies the functional recruitment of SK channels not only in cardiac disease, but also in normal physiology, contributing to repolarization under conditions of enhanced adrenergic drive. ABSTRACT:Small-conductance Ca2+ -activated K+ (SK) channels expressed in ventricular myocytes (VMs) are dormant in health, yet become functional in cardiac disease. We aimed to test the hypothesis that post-translational modification of SK channels under conditions accompanied by enhanced adrenergic drive plays a central role in disease-related activation of the channels. We investigated this phenomenon using a rat model of hypertrophy induced by thoracic aortic banding (TAB). Western blot analysis using anti-pan-serine/threonine antibodies demonstrated enhanced phosphorylation of immunoprecipitated SK2 channels in VMs from TAB rats vs. Shams, which was reversible by incubation of the VMs with PKA inhibitor H89 (1 ?mol L-1 ). Patch clamped VMs under basal conditions from TABs but not Shams exhibited outward current sensitive to the specific SK inhibitor apamin (100 nmol L-1 ), which was eliminated by inhibition of PKA (1 ?mol L-1 ). Beta-adrenergic stimulation (isoproterenol, 100 nmol L-1 ) evoked ISK in VMs from Shams, resulting in shortening of action potentials in VMs and ex vivo optically mapped Sham hearts. Using adenoviral gene transfer, wild-type and mutant SK2 channels were overexpressed in adult rat VMs, revealing serine-465 as the site that elicits PKA-dependent phosphorylation effects on SK2 channel function. Concurrent confocal Ca2+ imaging experiments established that PKA phosphorylation lessens rectification of ISK via reduction Ca2+ /voltage-dependent inhibition of the channels at high [Ca2+ ] without affecting their sensitivity to activation by Ca2+ in the submicromolar range. In conclusion, upregulation of SK channels in diseased VMs is mediated by hyperadrenergic drive in cardiac hypertrophy, with functional effects on the channel conferred by PKA-dependent phosphorylation at serine-465.

Project description:RATIONALE:Large-conductance calcium-activated potassium channels (BK) are composed of pore-forming BK? and auxiliary ?1 subunits in arterial smooth muscle cells (myocytes). Vasoconstrictors, including endothelin-1 (ET-1), inhibit myocyte BK channels, leading to contraction, but mechanisms involved are unclear. Recent evidence indicates that BK? is primarily plasma membrane localized, whereas the cellular location of ?1 can be rapidly altered by Rab11A-positive recycling endosomes. Whether vasoconstrictors regulate the multisubunit composition of surface BK channels to stimulate contraction is unclear. OBJECTIVE:Test the hypothesis that ET-1 inhibits BK channels by altering BK? and ?1 surface trafficking in myocytes, identify mechanisms involved, and determine functional significance in myocytes of small cerebral arteries. METHODS AND RESULTS:ET-1, through activation of PKC (protein kinase C), reduced surface ?1 abundance and the proximity of ?1 to surface BK? in myocytes. In contrast, ET-1 did not alter surface BK?, total ?1, or total BK? proteins. ET-1 stimulated Rab11A phosphorylation, which reduced Rab11A activity. Rab11A serine 177 was identified as a high-probability PKC phosphorylation site. Expression of a phosphorylation-incapable Rab11A construct (Rab11A S177A) blocked the ET-1-induced Rab11A phosphorylation, reduction in Rab11A activity, and decrease in surface ?1 protein. ET-1 inhibited single BK channels and transient BK currents in myocytes and stimulated vasoconstriction via a PKC-dependent mechanism that required Rab11A S177. In contrast, NO-induced Rab11A activation, surface trafficking of ?1 subunits, BK channel and transient BK current activation, and vasodilation did not involve Rab11A S177. CONCLUSIONS:ET-1 stimulates PKC-mediated phosphorylation of Rab11A at serine 177, which inhibits Rab11A and Rab11A-dependent surface trafficking of ?1 subunits. The decrease in surface ?1 subunits leads to a reduction in BK channel calcium-sensitivity, inhibition of transient BK currents, and vasoconstriction. We describe a unique mechanism by which a vasoconstrictor inhibits BK channels and identify Rab11A serine 177 as a modulator of arterial contractility.

Dataset Information

Urocortin 2 stimulates nitric oxide production in ventricular myocytes via Akt- and PKA-mediated phosphorylation of eNOS at serine 1177.

Publications

Urocortin 2 stimulates nitric oxide production in ventricular myocytes via Akt- and PKA-mediated phosphorylation of eNOS at serine 1177.

Similar Datasets

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