Abstract:
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)EEIPI), 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.
journal_name
Invest Ophthalmol Vis Scijournal_title
Investigative ophthalmology & visual scienceauthors
Shi C,Barnes S,Coca-Prados M,Kelly MEkeywords:
subject
Has Abstractpub_date
2002-05-01 00:00:00pages
1525-32issue
5eissn
0146-0404issn
1552-5783journal_volume
43pub_type
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journal_title:Investigative ophthalmology & visual science
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journal_title:Investigative ophthalmology & visual science
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journal_title:Investigative ophthalmology & visual science
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