Laminin enhances beta(2)-adrenergic receptor stimulation of L-type Ca(2+) current via cytosolic phospholipase A(2) 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 Ca(2+) current (I(Ca,L)). This study therefore sought to determine whether LMN enhances beta(2)-AR signalling via a cAMP-independent mechanism, i.e. cytosolic phospholipase A(2) (cPLA(2)) 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 microm zinterol (zint-beta(2)-AR) stimulation of I(Ca,L) was larger in +LMN than LMN myocytes. In +LMN myocytes, zint-beta(2)-AR stimulation of I(Ca,L) was inhibited by inhibition of cPLA(2) by arachidonyltrifluoromethyl ketone (AACOCF(3); 10 microm), inhibition of G(i) by pertussis toxin and chelation of intracellular Ca(2+) by 10 microm BAPTA-AM. In contrast to zinterol, stimulation of I(Ca,L) by fenoterol (fen-beta(2)-AR), a beta(2)-AR agonist that acts exclusively via G(s) signalling, was smaller in +LMN than LMN myocytes. Arachidonic acid (AA; 5 microm) stimulated I(Ca,L) to a similar extent in LMN and +LMN myocytes. Inhibition of cAMP-dependent protein kinase A (cAMP/PKA) by either 5 mum H89 or 1 microm KT5720 in LMN myocytes mimicked the effects of +LMN myocytes to enhance zint-beta(2)-AR stimulation of I(Ca,L), which was blocked by 10 microm AACOCF(3). In contrast, H89 inhibited fen-beta(2)-AR stimulation of I(Ca,L), which was unchanged by AACOCF(3). Inhibition of ERK1/2 by 1 microm U0126 inhibited zint-beta(2)-AR stimulation of I(Ca,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 I(Ca,L). We conclude that LMN enhances zint-beta(2)-AR stimulation of I(Ca,L) via G(i)/ERK1/2/cPLA(2)/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.