In:
Biochemistry and Cell Biology, Canadian Science Publishing, Vol. 82, No. 6 ( 2004-12-01), p. 708-718
Abstract:
In mammalian nonpigmented ciliary epithelial (NPE) cells, hyposmotic stimulation leading to cell swelling activates an outwardly rectifying Cl conductance (I Cl,swell ), which, in turn, results in regulatory volume decrease. The aim of this study was to determine whether increased trafficking of intracellular ClC-3 Cl channels to the plasma membrane could contribute to the I Cl,swell following hyposmotic stimulation. Our results demonstrate that hyposmotic stimulation reversibly activates an outwardly rectifying Cl current that is inhibited by phorbol-12-dibutyrate and niflumic acid. Transfection with ClC-3 antisense, but not sense, oligonucleotides reduced ClC-3 expression as well as I Cl,swell . Intracellular dialysis with 2 different ClC-3 antibodies abolished activation of I Cl,swell . Immunofluorescence microscopy showed that hyposmotic stimulation increased ClC-3 immunoreactivity at the plasma membrane. To determine whether this increased expression of ClC-3 at the plasma membrane could be due to increased vesicular trafficking, we examined membrane dynamics with the fluorescent membrane dye FM1-43. Hyposmotic stimulation rapidly increased the rate of exocytosis, which, along with I Cl,swell , was inhibited by the phosphoinositide-3-kinase inhibitor wortmannin and the microtubule disrupting agent, nocodazole. These findings suggest that ClC-3 channels contribute to I Cl,swell following hyposmotic stimulation through increased trafficking of channels to the plasma membrane.Key words: ClC-3, NPE, cell swelling, membrane trafficking, ciliary body epithelium.
Type of Medium:
Online Resource
ISSN:
0829-8211
,
1208-6002
Language:
English
Publisher:
Canadian Science Publishing
Publication Date:
2004
SSG:
12
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