Nitric oxide donors increase cytosolic ionized calcium in cultured human intestinal epithelial cells

We used fluorescence spectrophotometry, digital-imaging fluorescence microscopy, and the fluorescent dye, Fura-2, to measure the effects of several different nitric oxide (NO .) donors on intracellular levels of ionized calcium ([Ca2+](i)) in cultured Caco-2(BBe) cells, a human enterocytic cell line. Incubation of Caco-2(BBe) cells with sodium nitroprusside (SNP) or isosorbide dinitrate for 2 h significantly increased [Ca2+](i). The effects of both agents were concentration dependent. The lowest concentrations of SNP or isosorbide dinitrate capable of increasing [Ca2+](i) were .625 mM and 10 mM, respectively. Chelation of extracellular Ca2+ with 2 mM EGTA abrogated the increase in [Ca2+](i) induced by SNP. In contrast, blockade of voltage-gated Ca2+ channels with 2 mM NiCl2 or 200 mu M verapamil failed to affect SNP-induced alterations in [Ca2+](i). Using NBD-phallicidin to stain polymerized (F)-actin, we found that incubation of Caco-2(BBe) cells with SNP results in actin polymerization, particularly in the periphery of cells. We conclude that NO increases [Ca2+](i) and promotes actin polymerization in a cultured enterocytic cell line.