A cAMP-dependent mechanism of Src Family Kinase activation in the porcine lens

Abstract

Purpose: Na,K-ATPase activity in the lens epithelium is subject to control by Src family tyrosine kinases (SFKs). Previously we showed exposure of intact lenses to hyposmotic solution causes an SFK-dependent increase in Na,K-ATPase activity in the epithelium. Here we examined the mechanism of hyposmotic solution-induced SFK activation.

Methods: Intact porcine lenses were exposed to hyposmotic solution (200 mOsm) for 2-10 min. SFK phosphorylation (activation) in the epithelium was studied by Western blot and cAMP was measured by RIA. Results as mean ± SE are analyzed by t-test or 1-way ANOVA (p <0.05 considered significant).

Results: When lenses were exposed to hyposmotic solution, SFK phosphorylation in the epithelium was approx. double the control value (2.02 ± 0.03, p=0.001, n=3). The soluble guanylate cyclase inhibitor, ODQ (10 µM), did not alter the magnitude of hyposmotic solution-induced SFK phosphorylation (p=0.5, n=3) excluding the involvement of the Ca²⁺-NOS-cGMP-PKG pathway. However, the magnitude of the SFK response to hyposmotic solution was reduced by a TRPV4 antagonist, HC 067047 (10 µM) (p=0.001, n=3), a cytoplasmic calcium chelator, BAPTA-AM (p=0.001, n=3) and a selective inhibitor of protein kinase A, H89 (10 µM) (p=0.01, n=3). Similar responses were found in primary cultured lens epithelium exposed to hyposmotic solution. The data suggest SFK activation may follow activation of a calcium-dependent soluble adenylate cyclase in the lens epithelium. Consistent with this notion, hyposmotic solution caused a rapid transient increase of cAMP in the lens epithelium: 2 min = 8.5±0.6, 5 min = 12.5±2.2, 10 min = 8.3±1.0 pmol/mg protein, (p=0.001, n=5) compared to control (3.7±0.2). Moreover, the cytoplasmic calcium chelator BAPTA-AM suppressed the Na,K-ATPase activity increase in the epithelium of hyposmotic solution-treated lenses (54.2±6.7 vs 88.6±6.7 nmoles ATP hydrolyzed/mg protein/30 min, p=0.001, n=4).

Conclusions: Taken together the findings indicate SFK activation in the epithelium of lenses exposed to hyposmotic solution is linked to stimulation of a calcium-activated adenylate cyclase, production of cAMP and activation of protein kinase A.