Protein tyrosine kinase and protein phosphatase signaling pathways regulate volume-sensitive chloride currents in a nonpigmented ciliary epithelial cell line

PURPOSE. To investigate whether signaling pathways that incorporate protein tyrosine kinases and phosphatases regulate PKC-sensitive, volume-sensitive Cl^- currents (I_Cl.vol) in cultured rabbit nonpigmented ciliary epithelial cells. METHODS. Activation of I_Cl.vol in response to hyposmotic stimulation was recorded with whole-cell patch-clamp techniques in the presence of pharmacologic agents that activate or block kinases and phosphatases. RESULTS. I_Cl.vol in rabbit nonpigmented ciliary epithelial cells was identified as a PKC-sensitive, volume-sensitive Cl^- current, because current was downregulated during cell swelling by phorbol-12-dibutyrate, a PKC activator, and the PKC inhibitors, calphostin and chelerythrine, enhanced the current. Activation of c-Src tyrosine kinases, with an Src activator peptide (EPQ-(pY)EEIPD, increased I_Cl.vol after hyposmotic stimulation, whereas the protein tyrosine kinase inhibitor, genistein, but not its inactive analogue daidzein, inhibited the current. The phosphatidylinositol-3-kinase (PI3K) inhibitor, wortmannin, inhibited I_Cl.vol. Wortmannin did not further inhibit I_Cl.vol in cells pretreated with the protein tyrosine kinase inhibitor, genistein, but blocked enhancement of I_Cl.vol by PKC inhibitors. The serine-threonine protein phosphatase (PP) inhibitor, okadaic acid, blocked activation of I_Cl.vol whereas insulin, which activates PI3K and PP-1, enhanced the current. The insulin-enhanced current was also blocked by okadaic acid. I_Cl.vol was not activated under isosmotic conditions by the simultaneous inhibition of PKC with calphostin and activation of PP-1 by insulin. CONCLUSIONS. These data show that PKC-sensitive Cl^- currents activated in response to cell swelling in nonpigmented ciliary epithelial cells are modulated by protein tyrosine kinase, PI3K, and PP signaling pathways. Activation of PP and PKC may involve the upstream intermediaries Src tyrosine kinase and PI3K.