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M.M. Civan, K. Peterson–Yantorno, C.W. Do; Activation of PKC Modulates Cl– Release by Non–Pigmented Ciliary Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3321.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To examine the putative role of ClC–3, a PKC–inhibitable Cl– channel, in mediating the Cl– release in native bovine non–pigmented ciliary epithelial (NPE) cells. Methods: Native bovine NPE cells were freshly isolated by enzymatic treatment. Cell volume was determined by monitoring quenching of total calcein fluorescence after validating its application to NPE cells. Whole–cell currents were recorded by patch–clamp measurements. The effects of PKC modulators on both cell volume and whole–cell currents were investigated. Results: Following hypotonic cell swelling of native bovine NPE cells, the rate of the regulatory volume decrease (RVD) was monitored. The RVD reflects NPE–cell release of KCl and, secondarily, water. Pretreatment with the Cl––channel blocker 5–nitro–2–(phenylpropylamino)–benzoate (NPPB, 100 µM) inhibited the rate of the RVD. Like NPPB, activation of PKC by pretreatment with phorbol 12,13–dibutyrate (PDBu, 100 nM) inhibited the RVD, whereas inhibition of PKC by pretreatment with 300 nM staurosporine enhanced the RVD. In parallel with the volumetric measurements, extracellular hypotonicity stimulated swelling–activated Cl– currents (ICl,swell). The stimulation was sufficiently delayed and inhibited by pretreatment with 100 nM PDBu. However, the inhibition was not sustained; no effects on ICl,swell were observed under steady–state conditions. Conclusions: Consistent with our recent findings, ClC–3 may be functionally important in mediating Cl– release in NPE cells. Blocking ClC–3 by activating PKC significantly inhibited the swelling–triggered Cl– efflux by the NPE cells, thereby reducing the rate of volume recovery. The unsustained effect of PDBu on ICl,swell might suggest the possible up–regulation of other swelling–activated Cl– channels that mediate ICl,swell.
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