Src Family Tyrosine Kinase Signaling in Lens Epithelium is Linked to Calcium-activated Adenylate Cyclase

Abstract

Purpose : Previously we showed exposure of intact lenses to hyposmotic solution causes a Src Family Kinase (SFK)-dependent increase in Na,K-ATPase activity in the epithelium. Here we examined the mechanism that links osmotic stress to SFK activation.

Methods : Intact porcine lenses were exposed to hyposmotic solution (200 mOsm) for 2-10 min and then the epithelium was isolated and used for measurement of cAMP, Na,K-ATPase activity and SFK phosphorylation. SFK phosphorylation (activation) was studied by Western blot analysis, cAMP by RIA and Na,K-ATPase activity as ouabain-sensitive ATP hydrolysis in cell homogenates. Results (mean ± SE) were analyzed by t-test or 1-way ANOVA (p <0.05 considered significant).

Results : When lenses were exposed to hyposmotic solution (2-5 min), SFK phosphorylation in the epithelium was increased (1.45± 0.07 fold, p=0.0001, n=3). The SFK phosphorylation response was not altered in lenses exposed to hyposmotic solution in the presence of a soluble guanylate cyclase inhibitor ODQ (10 µM) that inhibits the Ca²⁺-NOS-cGMP-PKG pathway. In contrast, the hypotonicity-induced SFK phosphorylation was abolished by a cytoplasmic Ca²⁺ chelator, BAPTA-AM (10µM) (Con 1.0±0., hypo 1.59±0.17 vs BAPTA+hypo 0.66±0.15, p=0.0009, n=3), by a TRPV4 antagonist, HC 067047 (10 µM) (Con 1.0±0, hypo 1.47±0.10 vs HC+Hypo 1.12±0.08 p=0.0325, n=3), and by a selective protein kinase A inhibitor, H89 (10 µM) (Con 1.0±0, Hypo 1.45±0.15 vs H89+hypo 1.08±0.02, p=0.02, n=3). The data suggest hypotonicity-induced SFK activation may follow TRPV4-mediated Ca²⁺ entry and subsequent activation of a Ca-dependent AC. Consistent with this notion, hyposmotic solution caused a rapid increase of cAMP in the lens epithelium at 2 min (6.17±0.42 vs 8.48±0.6, p=0.014, n=8) and at 5 min 5.54 ±0.34 vs 12.50±2.20 (p=0.011, n=6) pmol cAMP/mg protein. Moreover, BAPTA-AM suppressed the hypotonicity-induced Na,K-ATPase activity increase (Control 48.32±3.05, hypo 88.6±6.64 vs BAPTA-treated 44.33±3.34, p=0.0011, n=4) nmoles ATP hydrolyzed/mg protein/30 min. Ca-dependent adenylate cyclase 3 (ADCY3) and 8 (ADCY8) are expressed in the lens epithelium.

Conclusions : The findings indicate SFK activation in the epithelium of lenses exposed to hyposmotic solution is linked to stimulation of a Ca-dependent adenylate cyclase, production of cAMP and activation of protein kinase A. The mechanism depends on Ca²⁺ entry via TRPV4 channels.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.