The Cl(-) channel blocker niflumic acid releases Ca(2+) from an intracellular store in rat pulmonary artery smooth muscle cells.
The effect of the Cl- channel blockers niflumic acid (NFA), 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), and anthracene-9-carboxylic acid (A-9-C), on Ca2+ signalling in rat pulmonary artery smooth muscle cells was examined. Intracellular Ca2+ concentration ([Ca2+]i) was monitored with either fura-2 or fluo-4, and caffeine was used to activate the ryanodine receptor, thereby releasing Ca2+ from the sarcoplasmic reticulum (SR). NFA and NPPB significantly increased basal [Ca2+]i and attenuated the caffeine-induced increase in [Ca2+]i. These Cl- channel blockers also increased the half-time (t1/2) to peak for the caffeine-induced [Ca2+]i transient, and slowed the removal of Ca2+ from the cytosol following application of caffeine. Since DIDS and A-9-C were found to adversely affect fura-2 fluorescence, fluo-4 was used to monitor intracellular Ca2+ in studies involving these Cl- channel blockers. Both DIDS and A-9-C increased basal fluo-4 fluorescence, indicating an increase in intracellular Ca2+, and while DIDS had no significant effect on the t1/2 to peak for the caffeine-induced Ca2+ transient, it was significantly increased by A-9-C. In the absence of extracellular Ca2+, NFA significantly increased basal [Ca2+]i, suggesting that the release of Ca2+ from an intracellular store was responsible for the observed effect. Depleting the SR with the combination of caffeine and cyclopiazonic acid prevented the increase in basal [Ca2+]i induced by NFA. Additionally, incubating the cells with ryanodine also prevented the increase in basal [Ca2+]i induced by NFA. These data show that Cl- channel blockers have marked effects on Ca2+ signalling in pulmonary artery smooth muscle cells. Furthermore, examination of the NFA-induced increase in [Ca2+]i indicates that it is likely due to Ca2+ release from an intracellular store, most probably the SR.
Cruickshank
Stuart F
SF
Department of Physiology and Pharmacology, University of Strathclyde, 27 Taylor Street, Glasgow G4 ONR.
Baxter
Lynne M
LM
Drummond
Robert M
RM
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000818:Animals; D000873:Anthracenes / Q000494:pharmacology; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects; D002478:Cells, Cultured; D018118:Chloride Channels / Q000037:antagonists & inhibitors* / Q000502:physiology; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism*; D009544:Niflumic Acid / Q000494:pharmacology*; D009579:Nitrobenzoates / Q000494:pharmacology; D011651:Pulmonary Artery / Q000166:cytology / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012433:Ryanodine / Q000494:pharmacology; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism
D000873:Anthracenes; D018118:Chloride Channels; D009579:Nitrobenzoates; D012433:Ryanodine; C058176:5-nitro-2-(3-phenylpropylamino)benzoic acid; D002110:Caffeine; D009544:Niflumic Acid; C010030:9-anthroic acid; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D002118:Calcium