Laminin Acts Via Focal Adhesion Kinase/Phosphatidylinositol-3' Kinase/Protein Kinase B To Down-Regulate Beta(1)-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 Ca2+ current (I-Ca,I-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 mu m isoproterenol (isoprenaline) plus 0.1 mu m 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,I-L was significantly smaller in +LMN compared to -LMN atrial myocytes. In -LMN myocytes, 10 mu m LY294002 (LY), a specific inhibitor of PI-(3)K, had no effect on beta(1)-AR stimulation of I-Ca,I-L. In +LMN myocytes, however, LY significantly increased beta(1)-AR stimulation of I-Ca,I-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,I-L. In -LMN myocytes LY had no effect on forskolin (FSK)-stimulated I-Ca,I-L. However, in +LMN myocytes LY significantly increased FSK-stimulated I-Ca,I-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,I-L. These findings provide new insight into the cellular mechanisms by which the extracellular matrix can modulate atrial beta-AR signalling.