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S. Tamiya, M. C. Okafor, N. A. Delamere; Stimulation of Lens Na,K-ATPase-Mediated Transport by Purinergic Receptor Agonists via a Src Tyrosine Kinase-Dependent Pathway. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2823.
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© ARVO (1962-2015); The Authors (2016-present)
Previous studies from our group have demonstrated that lens Na,K-ATPase activity can be modulated by Na,K-ATPase α subunit phosphorylation using Src family of tyrosine kinases in vitro. In the present study experiments were conducted to test whether Src family kinases (SFKs) are involved in the regulation of Na,K-ATPase function by purinergic receptor agonists in the organ-cultured rabbit lens.
Na,K-ATPase function was examined using two distinct approaches, measurement of ouabain-sensitive potassium (86Rb) uptake by the intact lens and measurement of Na,K-ATPase activity in lens epithelial homogenates. For Western blot analysis, lens epithelium was lysed and subjected to SDS-PAGE, transferred to nitrocellulose membrane and probed using an antibody against active Src family kinases (phosphorylated at the active loop tyrosine), Src, Yes, Fyn and ß-actin.
ATP and UTP significantly increased ouabain-sensitive potassium uptake. The response was blocked by suramin, a purinergic receptor antagonist. Na,K-ATPase activity was also significantly increased in epithelia obtained from lenses previously exposed to ATP. The abundance of active SFKs was significantly increased in lens epithelium of ATP- or UTP-treated lenses as judged by an increase in the band density of SFKs phosphorylated at the active loop tyrosine. Immunoprecipitation studies revealed that the band density was significantly increased for active Src, and to a lesser extent active Fyn, while active Yes did not change. ATP failed to increase the ouabain-sensitive potassium uptake in the presence of Src family kinase inhibitor PP2.
The results show that purinergic receptor agonists stimulates Na,K-ATPase-mediated transport and activates Src family kinases in the intact rabbit lens. Selective activation of Src and possibly Fyn is involved in the observed increase in Na,K-ATPase-mediated transport.
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