Inhibition of cAMP-Dependent PKA Activates β2-Adrenergic Receptor Stimulation of Cytosolic Phospholipase A2 via Raf-1-MEK-ERK and IP3-Dependent Ca2 Signaling in Atrial MyocytesReportar como inadecuado




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We previously reported in atrial myocytes that inhibition of cAMP-dependent protein kinase PKA by laminin LMN-integrin signaling activates β2-adrenergic receptor β2-AR stimulation of cytosolic phospholipase A2 cPLA2. The present study sought to determine the signaling mechanisms by which inhibition of PKA activates β2-AR stimulation of cPLA2. We therefore determined the effects of zinterol 0.1 μM; zint-β2-AR to stimulate ICa,L in atrial myocytes in the absence +PKA and presence -PKA of the PKA inhibitor 1 μM KT5720 and compared these results with atrial myocytes attached to laminin +LMN. Inhibition of Raf-1 10 μM GW5074, phospholipase C PLC; 0.5 μM edelfosine, PKC 4 μM chelerythrine or IP3 receptor IP3R signaling 2 μM 2-APB significantly inhibited zint-β2-AR stimulation of ICa,L in–PKA but not +PKA myocytes. Western blots showed that zint-β2-AR stimulation increased ERK1-2 phosphorylation in–PKA compared to +PKA myocytes. Adenoviral Adv expression of dominant negative dn -PKCα, dn-Raf-1 or an IP3 affinity trap, each inhibited zint-β2-AR stimulation of ICa,L in + LMN myocytes compared to control +LMN myocytes infected with Adv-βgal. In +LMN myocytes, zint-β2-AR stimulation of ICa,L was enhanced by adenoviral overexpression of wild-type cPLA2 and inhibited by double dn-cPLA2S505A-S515A mutant compared to control +LMN myocytes infected with Adv-βgal. In–PKA myocytes depletion of intracellular Ca2+ stores by 5 μM thapsigargin failed to inhibit zint-β2-AR stimulation of ICa,L via cPLA2. However, disruption of caveolae formation by 10 mM methyl-β-cyclodextrin inhibited zint-β2-AR stimulation of ICa,L in–PKA myocytes significantly more than in +PKA myocytes. We conclude that inhibition of PKA removes inhibition of Raf-1 and thereby allows β2-AR stimulation to act via PKCα-Raf-1-MEK-ERK1-2 and IP3-mediated Ca2+ signaling to stimulate cPLA2 signaling within caveolae. These findings may be relevant to the remodeling of β-AR signaling in failing and-or aging heart, both of which exhibit decreases in adenylate cyclase activity.



Autor: M. R. Pabbidi , X. Ji, J. T. Maxwell, G. A. Mignery, A. M. Samarel, S. L. Lipsius

Fuente: http://plos.srce.hr/



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