Purinergic agonists stimulate lens Na-K-ATPase-mediated transport via a Src tyrosine kinase-dependent pathway

Shigeo Tamiya, Mansim C. Okafor, Nicholas A. Delamere

Research output: Contribution to journalArticle

21 Scopus citations

Abstract

The Na-K-ATPase is vital for maintenance of lens transparency. Past studies using intact lens suggested the involvement of tyrosine kinases in short-term regulation of Na-K-ATPase. Furthermore, in vitro phosphorylation of a lens epithelial membrane preparation by Src family kinases (SFKs), a family of nonreceptor tyrosine kinases, resulted in modification of Na-K-ATPase activity. Here, the effect of purinergic agonists, ATP and UTP, on Na-K-ATPase function and SFK activation was examined in the rabbit lens. Na-K-ATPase function was examined using two different approaches, measurement of ouabain-sensitive potassium (86Rb) uptake by the intact lens, and Na-K-ATPase activity in lens epithelial homogenates. ATP and UTP caused a significant increase in ouabain-sensitive potassium (86Rb) uptake. Na-K-ATPase activity was increased in the epithelium of lenses pretreated with ATP. Lenses treated with ATP or UTP displayed activation of SFKs as evidenced by increased Western blot band density of active SFK (phosphorylated at the active loop Y416) and decreased band density of inactive SFKs (phosphorylated at the COOH terminal). A single PY416-Src immunoreactive band at ∼60 kDa was observed, suggesting not all Src family members are activated. Immunoprecipitation studies showed that band density of active Src, and to a lesser extent active Fyn, was significantly increased, while active Yes did not change. Preincubation of the lenses with SFK inhibitor PP2 abolished the ATP-induced increase in ouabain-sensitive potassium (86Rb) uptake. The results suggest selective activation of Src and/or Fyn is part of a signaling mechanism initiated by purinergic agonists that increases Na-K-ATPase-mediated transport in the organ-cultured lens.

Original languageEnglish (US)
Pages (from-to)C790-C796
JournalAmerican Journal of Physiology - Cell Physiology
Volume293
Issue number2
DOIs
StatePublished - Aug 1 2007

Keywords

  • Receptors
  • Src kinase

ASJC Scopus subject areas

  • Physiology
  • Cell Biology

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