NITRIC OXIDE DONORS INCREASE CYTOSOLIC IONIZED CALCIUM IN CULTURED HUMAN INTESTINAL EPITHELIAL CELLS

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Abstract
We used fluorescence spectre-photometry, 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-2BBe cells, a human enterocytic cell line. Incubation of Caco-2BBe cells with sodium hitroprusside (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 NiCI2 or 200 ±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-2BBe 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.

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