Inhibition of cAMP-Dependent Protein Kinase (PKA) Activates β2-Adrenergic Receptor (β2-AR) Stimulation of Cytosolic Phospholipase A2 (cPLA2) in Atrial Myocytes

Abstract

We reported that attachment of atrial myocytes to laminin (LMN) decreases adenylate cyclase (AC)/cAMP and increases β2-AR stimulation of L-type Ca2+ current (ICa,L). This study determined whether LMN enhances β2-AR signaling via a cAMP-independent mechanism, i.e. cPLA2 signaling. Atrial myocytes were plated on uncoated cover-slips (-LMN) or cover-slips coated with LMN (+LMN) (>2 hrs). As previously reported, 0.1 μM zinterol (β2-AR agonist) stimulation of ICa,L was larger in +LMN than -LMN myocytes. In +LMN myocytes, zinterol stimulation of ICa,L was inhibited by 10 μM AACOCF3 (cPLA2 inhibitor), pertussis toxin or 10 μM BAPTA-AM (intracellular Ca2+ chelator). Stimulation of ICa,L by fenoterol (β2-AR/Gs agonist) was smaller in +LMN than -LMN myocytes. Arachidonic acid (AA; 5 μM) stimulated ICa,L in -LMN and +LMN myocytes similarly. Inhibition of PKA by either 5 μM H-89 or 1 μM KT5720 in -LMN myocytes mimicked the effects of +LMN myocytes to enhance zinterol stimulation of ICa,L, which was blocked by AACOCF3. In contrast, H-89 inhibited fenoterol stimulation of ICa,L, which was unchanged by AACOCF3. Inhibition of ERK1/2 by 1 μM U-0126 inhibited zinterol stimulation of ICa,L in +LMN myocytes and -LMN myocytes in which PKA was inhibited (KT5720). Western blots showed that inhibition of PKA (KT5720) in -LMN myocytes markedly increased zinterol phosphorylation of ERK1/2. We conclude that inhibition of AC/cAMP/PKA by cell attachment to LMN or PKA by pharmacological agents in -LMN myocytes switches β2-AR signaling from predominantly Gs/AC/cAMP/PKA to Gi/ERK1/2/cPLA2/AA. These findings may be relevant to the remodeling of β-AR signaling in diseased (fibrotic) and/or aging atria, both of which exhibit decreases in AC activity.