Inhibition of cAMP-Dependent PKA Activates [beta].sub.2-Adrenergic Receptor Stimulation of Cytosolic Phospholipase A.sub.2 via Raf-1/MEK/ERK and IP.sub.3-Dependent Ca.sup.2+ 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].sub.2 -adrenergic receptor ([beta].sub.2 -AR) stimulation of cytosolic phospholipase A.sub.2 (cPLA.sub.2). The present study sought to determine the signaling mechanisms by which inhibition of PKA activates [beta].sub.2 -AR stimulation of cPLA.sub.2 . We therefore determined the effects of zinterol (0.1 [mu]M; zint-[beta].sub.2 -AR) to stimulate I.sub.Ca,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 IP.sub.3 receptor (IP.sub.3 R) signaling (2 [mu]M 2-APB) significantly inhibited zint-[beta].sub.2 -AR stimulation of I.sub.Ca,L in-PKA but not +PKA myocytes. Western blots showed that zint-[beta].sub.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 IP.sub.3 affinity trap, each inhibited zint-[beta].sub.2 -AR stimulation of I.sub.Ca,L in + LMN myocytes compared to control +LMN myocytes infected with Adv-[beta]gal. In +LMN myocytes, zint-[beta].sub.2 -AR stimulation of I.sub.Ca,L was enhanced by adenoviral overexpression of wild-type cPLA.sub.2 and inhibited by double dn-cPLA.sub.2 .sup.S505A/S515A mutant compared to control +LMN myocytes infected with Adv-[beta]gal. In-PKA myocytes depletion of intracellular Ca.sup.2+ stores by 5 [mu]M thapsigargin failed to inhibit zint-[beta].sub.2 -AR stimulation of I.sub.Ca,L via cPLA.sub.2 . However, disruption of caveolae formation by 10 mM methyl-[beta]-cyclodextrin inhibited zint-[beta].sub.2 -AR stimulation of I.sub.Ca,L in-PKA myocytes significantly more than in +PKA myocytes. We conclude that inhibition of PKA removes inhibition of Raf-1 and thereby allows [beta].sub.2 -AR stimulation to act via PKC[alpha]/Raf-1/MEK/ERK1/2 and IP.sub.3 -mediated Ca.sup.2+ signaling to stimulate cPLA.sub.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.