Inhibition of cAMP-Dependent PKA Activates ?2-Adrenergic Receptor Stimulation of Cytosolic Phospholipase A2 via Raf-1/MEK/ERK and IP3-Dependent Ca2+ Signaling in Atrial Myocytes.

ABSTRACT: We previously reported in atrial myocytes that inhibition of cAMP-dependent protein kinase (PKA) by laminin (LMN)-integrin signaling activates ?2-adrenergic receptor (?2-AR) stimulation of cytosolic phospholipase A2 (cPLA2). The present study sought to determine the signaling mechanisms by which inhibition of PKA activates ?2-AR stimulation of cPLA2. We therefore determined the effects of zinterol (0.1 ?M; zint-?2-AR) to stimulate ICa,L in atrial myocytes in the absence (+PKA) and presence (-PKA) of the PKA inhibitor (1 ?M) KT5720 and compared these results with atrial myocytes attached to laminin (+LMN). Inhibition of Raf-1 (10 ?M GW5074), phospholipase C (PLC; 0.5 ?M edelfosine), PKC (4 ?M chelerythrine) or IP3 receptor (IP3R) signaling (2 ?M 2-APB) significantly inhibited zint-?2-AR stimulation of ICa,L in-PKA but not +PKA myocytes. Western blots showed that zint-?2-AR stimulation increased ERK1/2 phosphorylation in-PKA compared to +PKA myocytes. Adenoviral (Adv) expression of dominant negative (dn) -PKC?, dn-Raf-1 or an IP3 affinity trap, each inhibited zint-?2-AR stimulation of ICa,L in + LMN myocytes compared to control +LMN myocytes infected with Adv-?gal. In +LMN myocytes, zint-?2-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-?gal. In-PKA myocytes depletion of intracellular Ca2+ stores by 5 ?M thapsigargin failed to inhibit zint-?2-AR stimulation of ICa,L via cPLA2. However, disruption of caveolae formation by 10 mM methyl-?-cyclodextrin inhibited zint-?2-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 ?2-AR stimulation to act via PKC?/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 ?-AR signaling in failing and/or aging heart, both of which exhibit decreases in adenylate cyclase activity.

SUBMITTER: Pabbidi MR 

PROVIDER: S-EPMC5158063 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature