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

Abstract

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 β₁-adrenergic receptor (β₁-AR) stimulation of L-type Ca²⁺ current (I_Ca,L). The present study sought to determine whether LMN-mediated down-regulation of β₁ signalling is due to down-regulation of adenylate cyclase and to gain insight into the signalling mechanisms responsible. β₁-AR stimulation was achieved by 0.01 μm isoproterenol (isoprenaline) plus 0.1 μm ICI 118551, a selective β₂-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, β₁-AR stimulation of I_Ca,L was significantly smaller in +LMN compared to −LMN atrial myocytes. In −LMN myocytes, 10 μm LY294002 (LY), a specific inhibitor of PI-(3)K, had no effect on β₁-AR stimulation of I_Ca,L. In +LMN myocytes, however, LY significantly increased β₁-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-β-galactosidase, cells infected with Adv-FRNK, Adv-Y397F-FAK or Adv-dnAkt each exhibited a significantly greater β₁-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 β₁-integrin receptors acts via FAK/PI-(3)K/Akt to inhibit adenylate cyclase activity and thereby down-regulates β₁-AR-mediated stimulation of I_Ca,L. These findings provide new insight into the cellular mechanisms by which the extracellular matrix can modulate atrial β-AR signalling.