Abstract
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.
Highlights
We previously reported that attachment of atrial myocytes to the extracellular matrix protein laminin (LMN) acts via β1 integrin receptors to decrease β1-adrenergic receptor (AR) and increase β2-AR stimulation of L-type Ca2+ current (ICa,L) [1]
This scenario is consistent with the fact that cytosolic phospholipase A2 (cPLA2) activation requires both elevation of submicromolar intracellular [Ca2+] and phosphorylation by various kinases. These findings may have important implications with respect to the aging and/or failing heart, both of which exhibit decreases in adenylate cyclase activity. In both animal models [37] and in the human right atrium [38], increasing age is associated with a decrease in β1-AR function that results from a decrease in adenylate cyclase activity
The present studies suggest that feline atrial cardiomyocytes exhibit a similar regulation as that seen in gastric muscle
Summary
We previously reported that attachment of atrial myocytes to the extracellular matrix protein laminin (LMN) acts via β1 integrin receptors to decrease β1-adrenergic receptor (AR) and increase β2-AR stimulation of L-type Ca2+ current (ICa,L) [1]. Cell attachment to LMN acts via inhibition of adenylate cyclase/PKA to both inhibit β1-AR signaling and enhance β2-AR signaling through activation of cPLA2. In embryonic chick ventricular myocytes [4] and rat ventricular myocytes [5] β2-AR stimulation activates cPLA2/AA signaling These authors proposed that activation of β2-AR/cPLA2 signaling may compensate for depressed cAMP signaling [4]. In both of these studies by Pavoine et al (1999) and Ait-Mamar et al, (2005) cardiomyocytes were cultured on LMN, supporting our findings that cell attachment to LMN may be responsible for inhibition of PKA and activation of β2-AR/cPLA2 signaling. The mechanism by which PKA inhibition activates β2-AR/cPLA2 signaling is not clear
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