Laminin enhances beta 2-adrenergic receptor stimulation of L-type Ca2+ current via cytosolic phospholipase A2 signalling in cat atrial myocytes

We previously reported that attachment of atrial myocytes to the extracellular matrix protein laminin (LMN), decreases adenylate cyclase (AC)-cAMP and increases beta 2-adrenergic receptor (AR) stimulation of L-type Ca2+ current (ICa,L). This study therefore sought to determine whether LMN enhances beta 2-AR signalling via a cAMP-independent mechanism, i.e. cytosolic phospholipase A2 (cPLA2) signalling. Studies were performed on acutely isolated atrial myocytes plated on uncoated coverslips (-LMN) or coverslips coated with LMN (+LMN). As previously reported, 0.1 mu m zinterol (zint- beta 2-AR) stimulation of ICa,L was larger in +LMN than -LMN myocytes. In +LMN myocytes, zint- beta 2-AR stimulation of ICa,L was inhibited by inhibition of cPLA2 by arachidonyltrifluoromethyl ketone (AACOCF3; 10 mu m), inhibition of Gi by pertussis toxin and chelation of intracellular Ca2+ by 10 mu m BAPTA-AM. In contrast to zinterol, stimulation of ICa,L by fenoterol (fen- beta 2-AR), a beta 2-AR agonist that acts exclusively via Gs signalling, was smaller in +LMN than -LMN myocytes. Arachidonic acid (AA; 5 mu m) stimulated ICa,L to a similar extent in -LMN and +LMN myocytes. Inhibition of cAMP-dependent protein kinase A (cAMP-PKA) by either 5 mu m H-89 or 1 mu m KT5720 in -LMN myocytes mimicked the effects of +LMN myocytes to enhance zint- beta 2-AR stimulation of ICa,L, which was blocked by 10 mu m AACOCF3. In contrast, H-89 inhibited fen- beta 2-AR stimulation of ICa,L, which was unchanged by AACOCF3. Inhibition of ERK1-2 by 1 mu m U0126 inhibited zint- beta 2-AR stimulation of ICa,L in +LMN myocytes and -LMN myocytes in which cAMP-PKA was inhibited by KT5720. In -LMN myocytes, cytochalasin D prevented inhibition of cAMP-PKA from enhancing zint- beta 2-AR stimulation of ICa,L. We conclude that LMN enhances zint- beta 2-AR stimulation of ICa,L via Gi-ERK1-2-cPLA2-AA signalling which is activated by concomitant inhibition of cAMP-PKA signalling and dependent on the actin cytoskeleton. These findings provide new insight into the cellular mechanisms by which the extracellular matrix can remodel beta 2-AR signalling in atrial muscle.