Ca2+-mediated damage to rabbit gastric mucosal cells: modulation by nitric oxide

Perturbations in cellular Ca²⁺ homeostasis can lead to oxidative stress whereas nitric oxide has been shown to inactive oxygen radicals. Therefore the effects of inhibition of nitric oxide (NO) synthase activity on Ca²⁺-mediated disruption to rabbit dispersed gastric mucosal cells have been examined. Addition of the Ca²⁺ ionophore A23187 (3–25 μM) to the incubation medium induced a concentration-dependent increase in cell damage is assessed by trypan blue dye uptake and decreased cellular metabolic activity as estimated by alamar blue absorbance. These responses were exacerbated by inhibition of NO synthase activity with $\text{N}{}^{\mathrm{<mtext>G</mtext>}}\text{-}\text{monomethyl-}\text{L}\text{-arginine}$ (300 μM). The deleterious effects of ionophore A23187 and $\text{N}{}^{\mathrm{<mtext>G</mtext>}}\text{-}\text{monomethyl-}\text{L}\text{-arginine}$ were ameliorated by addition of the NO donor S-nitroso-acetyl-penicillamine to the cell suspension. An increase in cellular Ca²⁺ in response to ionophore A23187 (12.5 μM) resulted in enhaced 2′7′-dichlorofluorescein fluorescence suggesting an elevation in oxidative stress. Ca²⁺-mediated cell injury was abolished by the oxygen radical scavengers, catalase and 2′,2′-dipyridyl. However, the cytotoxic effect of combined treatment with A23187 and $\text{N}{}^{\mathrm{<mtext>G</mtext>}}\text{-}\text{monomethyl-}\text{L}\text{-arginine}$ was not reduced by administration of oxygen radical scavengers. $\text{N}{}^{\mathrm{<mtext>G</mtext>}}\text{-}\text{monomethyl-}\text{L}\text{-arginine}$ treatment exacerbated the increase in cytosolic Ca²⁺ in response to ionophore A23187 as assessed by indo-1 fluorescence. Furthermore this increase in cytosolic Ca²⁺ was reduced by addition of S-nitroso-acetyl-penicillamine to the incubation medium. These data suggest that NO synthase inhibition in gastric mucosal cells exacerbates the damaging actions of the Ca²⁺ ionophore A23187. The increase in cell damage in response to the NO synthase inhibitor $\text{N}{}^{\mathrm{<mtext>G</mtext>}}\text{-}\text{monomethyl-}\text{L}\text{-arginine}$ does not appear to be mediated by an increase in oxidative stress and may be associated in part with changes in cellular Ca²⁺ flux.