Src family tyrosine kinase activation is required for endothelin‐1 to inhibit Na,K‐ATPase in porcine lens epithelium

Abstract

Abstract Purpose Earlier studies point to the involvement of Src family tyrosine kinases (SFKs) in the stimulation of of Na,K‐ATPase activity by purinergic receptor agonists ATP and UTP. Src itself was activated (Tamiya, et al. 2007, Am. J. Physiol. 293: C790‐6). Here, we examined the role of SFKs in the response to endothelin‐1 (ET‐1), an ET receptor agonist that causes Na,K‐ATPase inhibition. Methods Porcine lenses were incubated 30 min in Krebs’ solution with ET‐1 or other test agents. The epithelium was removed, homogenized and analyzed by western blot or Na,K‐ATPase activity assay. Results Exposure of the intact lens to ET‐1 caused a reduction in Na,K‐ATPase activity. The Na,K‐ATPase response was not observed when lenses were pretreated with 10 uM PP2, a selective inhibitor of SFKs. ET‐1 caused SFK activation evident from an increase in Y416 phosphorylation and decrease in Y527 phosphorylation of a ~61kDa SFK. The SFK inhibitor PP2 abolished the SFK phosphorylation response. Since SFKs Fyn, Src, Hck and Yes may contribute to the observed 61kDa band, these SFKs were isolated by immunoprecipitation and analyzed separately. Based on Y416 phosphorylation, ET‐1 appeared to activate Fyn, while Src and Hck were inhibited. Yes activity was unaltered. Conclusion ET‐1, which causes Na,K‐ATPase inhibition, elicits a different pattern of SFK activation from that reported earlier for purinergic agonists which stimulate Na,K‐ATPase activity. Previously, Na,K‐ATPase stimulation was observed when Src was activated. The ET‐1 response points to Na,K‐ATPase inhibition when Fyn kinase is activated.