The Cl~- channel blocker niflumic acid releases Ca~(2+) from an intracellular store in rat pulmonary artery smooth muscle cells

摘要

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 Ca~(2+) signalling in rat pulmonary artery smooth muscle cells was examined. Intracellular Ca~(2+) concentration ([Ca~(2+)]_i) was monitored with either fura-2 or fluo-4, and caffeine was used to activate the ryanodine receptor, thereby releasing Ca~(2+) from the sarcoplasmic reticulum (SR). 2. NFA and NPPB significantly increased basal [Ca~(2+)]_i and attenuated the caffeine-induced increase in [Ca~(2+)]_i. These Cl~- channel blockers also increased the half-time (t_(1/2)) to peak for the caffeine-induced [Ca~(2+)]_i transient, and slowed the removal of Ca~(2+) 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 Ca~(2+) in studies involving these Cl~- channel blockers. Both DIDS and A-9-C increased basal fluo-4 fluorescence, indicating an increase in intracellular Ca~(2+), and while DIDS had no significant effect on the t_(1/2) to peak for the caffeine-induced Ca~(2+) transient, it was significantly increased by A-9-C. 3. In the absence of extracellular Ca~(2+), NFA significantly increased basal [Ca~(2+)]_i, suggesting that the release of Ca~(2+) 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 [Ca~(2+)]_i induced by NFA. Additionally, incubating the cells with ryanodine also prevented the increase in basal [Ca~(2+)]_i induced by NFA. 5. These data show that Cl~- channel blockers have marked effects on Ca~(2+) signalling in pulmonary artery smooth muscle cells. Furthermore, examination of the NFA-induced increase in [Ca~(2+)]_i indicates that it is likely due to Ca~(2+) release from an intracellular store, most probably the SR.