Inhibition Of Camp-Dependent Pka Activates Beta(2)-Adrenergic Receptor Stimulation Of Cytosolic Phospholipase A(2) Via Raf-1/Mek/Erk And Ip3-Dependent Ca2+ Signaling In Atrial Myocytes

We previously reported in atrial myocytes that inhibition of cAMP-dependent protein kinase (PKA) by laminin (LMN)-integrin signaling activates beta(2)-adrenergic receptor (beta(2)-AR) stimulation of cytosolic phospholipase A(2) (cPLA(2)). The present study sought to determine the signaling mechanisms by which inhibition of PKA activates beta(2)-AR stimulation of cPLA(2). We therefore determined the effects of zinterol (0.1 mu M; zint-beta(2)-AR) to stimulate I-Ca,I- L in atrial myocytes in the absence (+ PKA) and presence (-PKA) of the PKA inhibitor (1 mu M) KT5720 and compared these results with atrial myocytes attached to laminin (+ LMN). Inhibition of Raf-1 (10 mu M GW5074), phospholipase C (PLC; 0.5 mu M edelfosine), PKC (4 mu M chelerythrine) or IP3 receptor (IP3R) signaling (2 mu M 2-APB) significantly inhibited zint-beta(2)-AR stimulation of ICa, L in-PKA but not + PKA myocytes. Western blots showed that zint-beta(2)-AR stimulation increased ERK1/2 phosphorylation in-PKA compared to + PKA myocytes. Adenoviral (Adv) expression of dominant negative (dn)-PKC alpha, dn-Raf-1 or an IP3 affinity trap, each inhibited zint-beta(2)-AR stimulation of ICa, L in + LMN myocytes compared to control + LMN myocytes infected with Adv-beta gal. In + LMN myocytes, zint-beta(2)-AR stimulation of ICa, L was enhanced by adenoviral overexpression of wild-type cPLA(2) and inhibited by double dn-cPLA2 S505A/S515A mutant compared to control + LMN myocytes infected with Adv-beta gal. In-PKA myocytes depletion of intracellular Ca2+ stores by 5 mu M thapsigargin failed to inhibit zint-beta(2)-AR stimulation of ICa, L via cPLA(2). However, disruption of caveolae formation by 10 mM methyl-beta-cyclodextrin inhibited zint-beta(2)-AR stimulation of ICa, L -PKA myocytes significantly more than in + PKA myocytes. We conclude that inhibition of PKA removes inhibition of Raf-1 and thereby allows beta(2)-AR stimulation to act via PKCa/Raf-1/MEK/ERK1/2 and IP3-mediated Ca2+ signaling to stimulate cPLA(2) signaling within caveolae. These findings may be relevant to the remodeling of beta-AR signaling in failing and/or aging heart, both of which exhibit decreases in adenylate cyclase activity.