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

• Published in: British journal of pharmacology 2009-02, Vol.140 (8), p.1442-1450
• Main Authors: Cruickshank, Stuart F, Baxter, Lynne M, Drummond, Robert M
• Format: Article
• Language: English
• ISSN: 0007-1188; 1476-5381
• DOI: 10.1038/sj.bjp.0705571

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). 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. 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. 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. 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. British Journal of Pharmacology (2003) 140 , 1442–1450. doi: 10.1038/sj.bjp.0705571