Role of calcium in nitric oxide-mediated injury to rat gastric mucosal cells
Abstract
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. (Gastroenterology 1996 Jul;111(1):65-72)
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Role of calcium homeostasis in gastric mucosal injury and protection
2001, Life SciencesUsing a human gastric mucosal cell line, known as AGS cells, we determined the role that perturbations in intracellular Ca2+ concentration [Ca2+]i might play in cellular injury induced by various damaging agents. For deoxycholate (CD) and ethanol (EtOH) induced damage, a concentration related increase in [Ca2+]i was noted that preceded and closely paralleled the magnitude of injury. Thus, the higher the concentration of DC or EtOH, the more profound were the changes in [Ca2+]i and the resultant degree of cellular injury. Pretreatment with a low concentration of DC (50 μM; called a mild irritant) that was not damaging by itself attenuated injury induced by a damaging concentration (i.e. 250 μM) of DC, and appeared to elicit this protective action through mechanisms that resisted intracellular Ca2+ accumulation. Additional studies indicated that the mechanism of aspirin damage may be similar and that other protective agents such as prostaglandins and growth factors appear to mediate their protective properties through prevention of intracellular Ca2+ alterations. We propose that agents that prevent mucosal injury mediate this activity through a cellular response (involving active Ca2+ efflux) that subsequently provides a protective action by limiting the magnitude of intracellular Ca2+ accumulation.
Effect of nitric oxide on apoptotic activity in the rat gastrointestinal tract
2001, European Journal of PharmacologyThe effect of nitric oxide (NO) on apoptosis in the gastrointestinal mucosa was investigated. Experiments involved long-term exposure of rat gastric mucosal cells in vitro to exogenous NO delivered from the NO, donor S-nitroso-N-acetyl-penicillamine, and the effect of intravenous administration of lipopolysaccharide in vivo, in the presence and absence of the selective inhibitor of inducible NO synthase N-(3-(aminomethyl)benzyl) acetamidine (1400 W). S-nitroso-N-acetyl-penicillamine produced a dose-related inhibition of caspase 3-like activity and DNA fragmentation in isolated gastric mucosal cells. Caspase 3-like activity and DNA fragmentation in gastric, ileal and colonic mucosa were increased both 5 and 24 h after injection of lipopolysaccharide (3 mg/kg, i.v.) to rats in vivo. Administration of 1400 W (5 mg/kg, i.v.) immediately after lipopolysaccharide enhanced caspase 3-like activity and DNA fragmentation above that found with lipopolysaccharide alone. In conclusion, data obtained both in vitro and in vivo suggest that NO exerts an anti-apoptotic effect on rat gastrointestinal mucosal cells.
Induction of heat shock protein 72 by a nitric oxide donor in guinea-pig gastric mucosal cells
1998, European Journal of PharmacologyGastric mucosal cells may be exposed to exogenous nitric oxide (NO) from a variety of sources. The response of primary cultures of guinea-pig gastric mucosal cells to the NO donor S-nitroso-N-acetyl-penicillamine was therefore investigated. Exposure to S-nitroso-N-acetyl-penicillamine for 8 h caused a concentration-dependent induction of heat shock protein 72 (HSP 72). Induction was inhibited by the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, and by blockade of transcription with actinomycin D. Induction of HSP 72 by S-nitroso-N-acetyl-penicillamine was enhanced by diethyl maleate which decreased the intracellular reduced thiol content. By contrast, HSP 72 formation after heat shock was associated with an elevation of reduced thiol. Incubation with S-nitroso-N-acetyl-penicillamine for 18 h increased detachment of cells from the culture plate. The effect of S-nitroso-N-acetyl-penicillamine on detachment was exacerbated by the presence of actinomycin D. In conclusion, exogenous NO induces HSP 72 in guinea-pig gastric mucosal cells and this response may in part protect the cells from the deleterious effects of NO.
Intestinal epithelial hyperpermeability
1998, Gastroenterology Clinics of North AmericaThe lumen of the distal small intestine and colon represents an enormous reservoir of microbes and potentially toxic microbial products. Therefore, in addition to the absorption of water and nutrients, an important function of the gut is to serve as an effective barrier, limiting systemic contamination by intraluminal microbes or their products. Two approaches are commonly used in both clinical and experimental studies to assess the integrity of gut barrier function. One method estimates the degree of transmucosal movement of bacteria or yeast from the intestinal lumen to mesenteric lymph nodes (MLNs), other organs, or blood, a process referred to as microbial translocation. The other method quantitates the permeability of the gut to various water-soluble probes.
Translocation usually is quantitated by enumerating viable colony-forming units in MLNs, liver, spleen, lung, and blood. Simply culturing MLNs or other organs, however, substantially underestimates the extent of translocation because most microbes breaching the epithelial barrier are killed.53 Thus, the apparent increases in translocation, which have been observed under a variety of pathologic circumstances, probably reflect the combined effects of increased transmucosal penetration by microbes and decreased killing of penetrating organisms by the host's antimicrobial defense mechanisms.
To move from the lumen to the lamina propria, translocating microbes must breach the barrier imposed by the continuous epithelial lining of the intestine. Many species and strains of bacteria, such as Escherichia coli and Salmonella typhimurium, appear to translocate by moving through enterocytes, rather than by disrupting the junctional complexes between adjacent cells to traverse the epithelium via a paracellular route.3, 153 It is possible, however, that certain strains or species of bacteria pass through intercellular spaces.173 Moreover, if the epithelium is denuded or ulcerated, microbes can easily gain access to the lamina propria. After passing through the epithelial layer, bacteria can be ingested by macrophages and, if not killed, transported to regional lymph nodes. Microbes in the lamina propria also can directly enter lymphatics or capillaries.
Theoretically, there are two possible routes for passive permeation of the epithelium by hydrophilic molecules and ions. One is the transcellular pathway, and the other is the paracellular pathway. Because current in biologic systems is carried by the movement of small ions (e.g., Na+ and Cl−), one way to assess permeability is by measuring electrical resistance. It is noteworthy therefore that the electrical resistance of the cytosolic membranes of both the apical and the basolateral surfaces of enterocytes is about 106 to 109 ohm⋅cm2,61, 117 whereas the normal transepithelial resistance for the small intestinal epithelium is much lower (102 ohm⋅cm2).47, 87 Accordingly, it seems most likely that permeation of hydrophilic solutes occurs mainly via the paracellular pathway. More than 85% of the passive permeation by ions is thought to be paracellular.52
The paracellular pathway consists of a water-filled channel formed by adjacent enterocytes. The tight junction between adjoining cells acts as a limiting barrier, controlling permeation via the paracellular pathway and enabling the normal intestinal epithelium to exhibit a phenomenon called selective permeability.66 As a consequence, the gut readily absorbs small molecules and ions (e.g., water, Na+, Cl−, amino acids). The transepithelial passage of potentially toxic or proinflammatory hydrophilic molecules with a Stokes radius greater than about 11.5 Å, however, is precluded.87
The tight junction (or zonula occludens) is a narrow band, which consists of several strands wrapping around the circumference of the apical portion of epithelial cells. The number of the strands correlates with the tightness of the junction.31, 32 In the intestine, the tight junctions of the villus epithelium have a higher number of strands and are more restrictive than the tight junctions in the epithelium of the crypts, which have a lower number of strands. Several specialized proteins, including ZO-1 and ZO-2, have been identified as components of the tight junction and are thought to play an important functional role in the regulation of paracellular permeability.6 The tight junction also is closely linked to the cytoskeleton by a circumferential ring of actin and myosin, located just below the zonula occludens.86, 88 Mucosal permeability to hydrophilic solutes is regulated under physiologic conditions by intracellular signals, which are transmitted to the tight junction and translated into constriction or dilation of the apical pore by dynamic changes in the cytoskeleton. Under pathologic conditions, derangements in the regulation of epithelial permeability also are associated with changes in the actin-based cytoskeleton.
Key role of PLC-γ in EGF protection of epithelial barrier against iNOS upregulation and F-actin nitration and disassembly
2003, American Journal of Physiology - Cell PhysiologyIntestinal epithelial hyperpermeability: Update on the pathogenesis of gut mucosal barrier dysfunction in critical illness
2003, Current Opinion in Critical Care