Role of calcium in nitric oxide-mediated injury to rat gastric mucosal cells

Background & Aims: Perturbations in Ca2+ homeostasis as well as high levels of nitric oxide have been associated with gastric cellular injury. The purpose of this study was to examine whether high levels of endogenous or exogenous NO damage gastric cells by altering intracellular Ca2+. Methods: Epithelial cells were isolated from the rat stomach, and cell integrity was estimated by trypan blue exclusion and alamar blue dye absorption. Cytosolic intracellular Ca2+ concentrations ([Ca2+](i)) were determined by indo-1 dye fluorescence. NO synthase activity was assessed radioenzymatically. Results: induction of Ca2+-independent NO synthase in response to endotoxin challenge resulted in decreased viability and an increase in [Ca2+](i) in gastric mucosal cells. These responses were ameliorated by pretreatment with N-G-nitro-L-arginine methyl ester or dexamethasone. Treatment of cells with the NO donor S-nitrosoacetyl-penicillamine also decreased cell integrity and increased [Ca2+](i). The actions of S-nitrosoacetyl-penicillamine could be reduced by decreasing intracellular or extracellular Ca2+, by chelating Ca2+ with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid or 1,2,-bis(2-aminophenoxy)ethane-N,N,N, N'-tetraacetic acid acetoxymethyl ester, by Ca2+ channel antagonism (nifedipine), or by displacing surface-bound Ca2+ (lanthanum). Furthermore, cell damage was reduced by inhibiting protein kinase C activity with either H-7 or staurosporine. Conclusions: Excessive levels of NO from either endogenous or exogenous sources results in a reduction in gastric cellular viability. This response seems to be related causally to an increase in [Ca2+](i) and protein kinase C activation.