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 NG-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-nitroso-acetyl-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.

Effect of nitric oxide on integrity, blood flow and cyclic GMP levels in the rat gastric mucosa: modulation by sialoadenectomy.

1. The effects of the nitrosothiol, S-nitroso N-acetylpenicillamine (SNAP) which liberates nitric oxide (NO), on ethanol-mediated gastric damage, blood flow and cyclic GMP levels in siaoloadenectomized (SALX) rats have been investigated. 2. Intraluminal instillation of ethanol (5-50% w/v) dose-dependently induced haemorrhagic damage and decreased NO synthase activity in the gastric mucosa. Both the extent of mucosal damage and inhibition of NO synthase activity were exacerbated in SALX rats. 3. Epidermal growth factor administration (5 and 10 micrograms kg-1, s.c.) reduced mucosal damage but did not restore NO synthase activity in ethanol-treated SALX rats. 4. SNAP infusion (0.01-1.0 micrograms kg-1 min-1, i.v.) attenuated haemorrhagic damage in ethanol-treated rats. The reduction in mucosal damage was significantly greater in SALX rats. 5. SNAP administration also caused an increase in gastric mucosal blood flow and cyclic GMP levels in control rats and both responses were augmented in SALX animals. 6. These data suggest that SALX is associated with increases in mucosal susceptibility to ethanol-mediated damage and reduces mucosal NO synthase activity. Epidermal growth factor does not appear to influence mucosal NO synthase in ethanol-treated rats. Furthermore, SALX augments the responsiveness of the gastric mucosa to NO administration. Therefore, factors from the salivary glands influence gastric NO formation and mucosal responsiveness to a NO donor.