The effect of endothelin-1 on Src-family tyrosine kinases and Na,K-ATPase activity in porcine lens epithelium

Previous studies show Src family kinase (SFK) activation is involved in a response that stimulates Na,K-ATPase. Here, we tested whether SFK activation is involved in the Na,K-ATPase response to endothelin-1 (ET-1). Intact porcine lenses were exposed to 100nM ET-1 for 5-30min. Then, the epithelium was removed and used for Na,K-ATPase activity measurement and Western blot analysis of SFK activation. Na,K-ATPase activity was reduced by ~30% in lenses exposed to ET-1 for 15min. The response was abolished by the SFK inhibitor PP2 or the ET receptor antagonist, PD145065. Activation of a ~61kDa SFK was evident from an increase in Y416 phosphorylation, which reached a maximum at 15min ET-1 treatment, and a decrease in Y527 phosphorylation. PP2 prevented SFK activation. Since Fyn, Src, Hck, and Yes may contribute to the observed 61kDa band, these SFKs were isolated by immunoprecipitation and analyzed. Based on Y416 phosphorylation, ET-1 appeared to activate Fyn, while Src and Hck were inhibited and Yes was unaltered. ET-1 requires SFK activation to cause Na,K-ATPase inhibition. ET-1 elicits a different pattern of SFK activation from that reported earlier for purinergic agonists that stimulate Na,K-ATPase activity and activate Src. In the ET-1 response Src is inhibited and Fyn is activated. The findings suggest SFK phosphorylation is involved in a regulatory mechanism for Na,K-ATPase. Knowing this may help us understand drug actions on Na,K-ATPase. Faulty regulation of Na,K-ATPase in the lens could contribute to cataract formation since an abnormal sodium content is associated with lens opacification.