Laminin acts via focal adhesion kinase/phosphatidylinositol‐3′ kinase/protein kinase B to down‐regulate β₁‐adrenergic receptor signalling in cat atrial myocytes

We previously reported that short-term (2 h) plating of cat atrial myocytes on the extracellular matrix protein, laminin (LMN) decreases adenylate cyclase activity and beta(1)-adrenergic receptor (beta(1)-AR) stimulation of L-type Ca(2+) current (I(Ca,L)). The present study sought to determine whether LMN-mediated down-regulation of beta(1) signalling is due to down-regulation of adenylate cyclase and to gain insight into the signalling mechanisms responsible. beta(1)-AR stimulation was achieved by 0.01 microm isoproterenol (isoprenaline) plus 0.1 microm ICI 118551, a selective beta(2)-AR antagonist. Atrial myocytes were plated for at least 2 h on uncoated cover-slips (-LMN) or cover-slips coated with LMN (+LMN). As previously reported, beta(1)-AR stimulation of I(Ca,L) was significantly smaller in +LMN compared to -LMN atrial myocytes. In -LMN myocytes, 10 microm LY294002 (LY), a specific inhibitor of PI-(3)K, had no effect on beta(1)-AR stimulation of I(Ca,L). In +LMN myocytes, however, LY significantly increased beta(1)-AR stimulation of I(Ca,L). Western blots revealed that compared with -LMN myocytes, +LMN myocytes showed a significant increase in Akt phosphorylation at Ser-473, which was prevented by LY. In another approach, +LMN myocytes were infected (multiplicity of infection (MOI), 100; 24 h) with replication-defective adenoviruses (Adv) expressing dominant-negative inhibitors of focal adhesion kinase (FAK) (Adv-FRNK or Adv-Y397F-FAK) or Akt (Adv-dnAkt). Compared with control cells infected with Adv-beta-galactosidase, cells infected with Adv-FRNK, Adv-Y397F-FAK or Adv-dnAkt each exhibited a significantly greater beta(1)-AR stimulation of I(Ca,L). In -LMN myocytes LY had no effect on forskolin (FSK)-stimulated I(Ca,L). However, in +LMN myocytes LY significantly increased FSK-stimulated I(Ca,L). Similar results were obtained in +LMN atrial myocytes infected with Adv-FRNK. We conclude that LMN binding to beta(1)-integrin receptors acts via FAK/PI-(3)K/Akt to inhibit adenylate cyclase activity and thereby down-regulates beta(1)-AR-mediated stimulation of I(Ca,L). These findings provide new insight into the cellular mechanisms by which the extracellular matrix can modulate atrial beta-AR signalling.