Hyposmotically activated chloride channels in cultured rabbit non-pigmented ciliary epithelial cells.
1. We used whole-cell patch-clamp recording techniques and noise analysis of whole-cell current to investigate the properties of hyposmotic shock (HOS)-activated Cl- channels in SV40-transformed rabbit non-pigmented ciliary epithelial (NPCE) cells. 2. Under conditions designed to isolate Cl- currents, exposure of cells to hyposmotic external solution reversibly increased the whole-cell conductance. 3. The whole-cell current activated with a slow time course (> 15 min), exhibited outward rectification and was Cl- selective. 4. The disulphonic stilbene derivatives 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS, 0.5 mM), 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS, 0. 5 mM) and 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS, 0.5 mM) produced a voltage-sensitive block of HOS-activated Cl- current at depolarized potentials, whereas niflumic acid produced a voltage-independent block of the current. 5. Under Ca2+-free conditions, HOS stimulation still reversibly activated the Cl- current, but the amplitude of current was reduced and the time course of current activation was slower compared with control (P < 0. 05). 6. The non-specific kinase inhibitor H-7 (100 microM), upregulated HOS-activated Cl- current amplitude in all cells tested (P < 0.05). 7. Noise analysis of whole-cell Cl- current indicated that cell swelling activated a high density of small conductance Cl- channels (< 1 pS). 8. We conclude that HOS primarily activates a high density of volume-sensitive small conductance Cl- channels in rabbit NPCE cells, and that Ca2+ and phosphorylation are involved in channel regulation.
Shi
C
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Department of Pharmacology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7.
Ryan
J S
JS
French
A S
AS
Coca-Prados
M
M
Kelly
M E
ME
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000494:pharmacology; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D018118:Chloride Channels / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000378:metabolism*; D002924:Ciliary Body / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D009544:Niflumic Acid / Q000494:pharmacology; D009997:Osmotic Pressure; D018408:Patch-Clamp Techniques; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors; D011817:Rabbits; D015290:Second Messenger Systems; D013267:Stilbenes / Q000494:pharmacology
D018118:Chloride Channels; D004791:Enzyme Inhibitors; D047428:Protein Kinase Inhibitors; D013267:Stilbenes; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D009544:Niflumic Acid; C022306:4,4'-dinitro-2,2'-stilbenedisulfonic acid; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D002118:Calcium