Reactive oxygen species and calcium homeostasis in cultured human intestinal smooth muscle cells

Abstract

Reactive oxygen species (ROS) significantly alter cell function. We examined the effects of hydrogen peroxide (H2O2) and xanthine/xanthine oxidase (X/XO) on isolated intestinal muscle cells. We assessed cell viability with the exclusion dye trypan blue and assayed the effects of H2O2 and X/XO on the intracellular redox state with the fluorescent probe 2',7'-dichlorofluorescein. Intracellular calcium concentration was measured in cells loaded with fura 2-acetoxymethyl ester, and we recorded whole membrane currents with conventional patch-clamp methods. Cells remained viable after a 5-min exposure to H2O2 and X/XO. H2O2 and X/XO led to a significant rise of the intracellular concentration of ROS. H2O2 (270 microM to 2.7 mM) as well as X/XO (0.25-16 mU; 0.5 mM xanthine) significantly increased intracellular calcium concentrations. Depletion of intracellular calcium with ryanodine or thapsigargin did not abolish the effect of ROS on the intracellular calcium concentration. In the absence of external calcium or in the presence of the calcium channel blocker nifedipine, H2O2 and X/XO still increased the intracellular calcium level. Thus calcium influx and calcium release from internal stores contributed to this rise in cytosolic calcium. Catalase and superoxide dismutase blunted or completely abolished the changes in calcium concentration elicited by H2O2 and X/XO. Exposure to ROS resulted in a rapid decline of the membrane resistance without significant changes in voltage-sensitive ion currents. We conclude that ROS disrupt the calcium homeostasis of cells at concentrations that do not lead to immediate cell death. The resulting elevation in cytosolic free calcium will activate a variety of biochemical reactions and may thus contribute to the cytotoxicity of reactive oxygen molecules.