The Cl- channel blocker niflumic acid releases Ca2+ from an intracellular store in rat pulmonary artery smooth muscle cells

1 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). 2 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 (t(1/2)) 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 t(1/2) to peak for the caffeine-induced Ca2+ transient, it was significantly increased by A-9-C. 3 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. 4 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. 5 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.