Inhibition of carrageenin-induced paw edema by a superoxide dismutase derivative that circulates bound to albumin.

Although the possible involvement of superoxide radical and its metabolite(s) in the pathogenesis of various types of edema have been suggested, direct evidence supporting this concept is lacking. Since intravenously administered Cu2+Zn2(+)-type superoxide dismutase (SOD) rapidly disappeared from the circulation with a half-life of 4 min, the enzyme could not be used to test whether superoxide radicals played a critical role in the modulation of vascular permeability. We previously synthesized a SOD derivative (SM-SOD) by linking poly(styrene co-maleic acid butyl ester) (SM) to the enzyme (Ogino, T., Inoue, M., Ando, Y., Awai, M., Maeda, H. and Morino Y. (1988) Int. J. Pept. Protein Res. 32, 1583-1588); SM-SOD circulates bound to albumin with a half-life of 6 h. To test whether superoxide radicals play an important role in the regulation of vascular permeability, the effect of SM-SOD on experimental paw edema was studied in the rat. Subcutaneous injections of carrageenin to the paw rapidly induced local edema by increasing vascular permeability. Intravenous administration of SM-SOD markedly inhibited the carrageenin-induced increase in vascular permeability and suppressed the development of paw edema. In contrast, the same dose of SOD showed no such inhibitory effect. These results suggest that superoxide radical and/or its metabolite(s) might play a critical role in the pathogenesis of carrageenin-induced vasogenic edema.     

Inhibition of ischemia and reflow-induced liver injury by an SOD derivative that circulates bound to albumin

Ischemia followed by reflow often results in tissue injury. Although reactive oxygens seem to play an important role in the pathogenesis of postischemic reflow-induced tissue injury, the mechanism and an efficient way to inhibit oxidative injury are not known. We studied the mechanism by which hepatic transport function was inhibited by a transient occlusion followed by reflow of the portal vein and hepatic artery by using a superoxide dismutase (SOD) derivative (SM-SOD) which circulates bound to albumin with a half-life of 6 h. Occlusion of the hepatic vessels for 20 min followed by reflow for 60 min significantly inhibited transhepatic transport of cholephilic ligands, such as bromosulfophthalein (BSP) and taurocholic acid. Intravenous administration of SM-SOD markedly inhibited the reflow-induced decrease in transhepatic transport of these ligands. Thiobarbituric acid - reactive metabolites (TBAR) in the liver and plasma remained unchanged during occlusion and reflow, while TBAR in the bile increased significantly. Intravenous injection of SM-SOD inhibited the reflow-induced increase in biliary TBAR. Xanthine oxidase activity in plasma also increased during occlusion and reflow by an SM-SOD-inhibitable mechanism. Polymorphonuclear leukocyte-dependent chemiluminescence of the peripheral blood remained unchanged during occlusion, but increased markedly with time after reflow. SM-SOD also inhibited the increase in chemiluminescence almost completely. These and other results suggested that the superoxide radical and/or its metabolite(s) might play an important role in the pathogenesis of the reflow-induced liver injury and that SM-SOD might be useful for studying the mechanism for tissue injury caused by oxygen toxicity.     

Synthesis of a superoxide dismutase derivative that circulates bound to albumin and accumulates in tissues whose pH is decreased

Protection of tissues from oxidative stress is one of the major prerequisites for aerobic life. Since intravenously injected Cu2+/Zn2+-type superoxide dismutase (SOD) disappears from the circulation with a short half-life of 5 min, its clinical use as a scavenger for superoxide radical is limited. We synthesized a human erythrocyte type SOD derivative (SM-SOD) by linking 2 mol of hydrophobic organic anion, alpha-4-[( 6-(N-maleimido)hexanoyloxymethyl]cumyl]half-butyl-esterified poly(styrene-co-maleic acid) (SM), to the cysteinyl residues of the dimeric enzyme without decreasing enzymic activity. SM-SOD, but not SOD, bound to an albumin-Sepharose column; the bound SM-SOD was eluted by a buffer solution containing 0.5% sodium dodecyl sulfate or 10 mM warfarin, suggesting that SM-SOD reversibly binds to the warfarin site on albumin. Due to the amphipathic nature of the SMI moiety, SM-SOD bound also to cell membranes particularly when the pH was decreased. In vivo analysis in the rat revealed that intravenously injected SM-SOD circulated bound to albumin with a half-life of 6 h. Postischemic reperfusion arrhythmias were almost completely prevented by a single dose of SM-SOD, but not SOD. Thus, the prolonged half-life of SM-SOD in the circulation and its preferential accumulation in an injured site with decreased pH appeared to be responsible for preventing myocardial injury. These results suggest that superoxide radical and/or its metabolite(s) would play an important role in the pathogenesis of postischemic reperfusion arrhythmias and that SM-SOD may be useful for decreasing tissue injury in ischemic heart disease.     

Inhibition of posthemorrhagic transfusion-induced gastric injury by a long-acting superoxide dismutase derivative

Although oxygen-free radicals have been postulated to play an important role in the pathogenesis of gastric mucosal injury induced by posthemorrhagic blood transfusion, direct evidence supporting this hypothesis is lacking. Superoxide dismutase (SOD) has been shown to inhibit oxygen toxicity in vitro in various types of cell injury. However, in some cases, oxidative tissue injury cannot be decreased efficiency predominantly due to its rapid elimination by renal glomerular filtration. To overcome such frustrating situations, we have synthesized a SOD derivative that circulates bound to albumin with a half-life of 6 hr. When blood was withdrawn from the rat (22 ml/kg) for 30 min followed by transfusion of the extracted blood, marked gastric mucosal lesions occurred within 30 min after transfusion. Intravenously injected SOD derivative markedly decreased gastric mucosal injury. Kinetic analysis using 125I-labeled albumin revealed that the vascular permeability of the stomach increased significantly after transfusion by a SOD derivative inhibitable mechanism. Thus, superoxide radical and/or its metabolite(s) play a critical role in the pathogenesis of posthemorrhagic transfusion-induced gastric injury.     

Inhibition of fibroblast chemotaxis by superoxide dismutase

Superoxide radicals are known to be important mediators in chronic inflammatory and fibrotic processes, in which accumulation of fibroblasts is thought to play a major role in the pathogenetic events. The enzyme superoxide dismutase removes these radicals by a catalytic reaction. Chemotactic response of human fibroblasts and fibrosarcoma-derived cells (HT-1080) to fibroblast conditioned medium, fibronectin and platelet-derived growth factor was inhibited in a dose-dependent manner in the presence of superoxide dismutase, while random migration, cell proliferation, cell viability and synthesis of collagen and non-collagenous proteins was not altered. In contrast, phorbol myristate acetate, an inducer of superoxide generation, stimulated the chemotactic movement of fibroblasts to the attractants. Evidence for the formation of superoxide is provided by the reduction of tetrazolium salt by activated fibroblasts which could be inhibited by superoxide dismutase. Thus, it is concluded that superoxide in small amounts is involved in the mechanism of fibroblast chemotaxis. Superoxide dismutase may, therefore, reduce fibroblast migration into sites of injury or inflammation.     

Inhibition of cytotoxicity by intracellular superoxide dismutase supplementation

The role of intracellular oxyradicals in H2O2 and neutrophil-induced cytotoxicity is suggested by previous studies showing protection by inhibitors such as deferroxamine, dimethylthiourea, and dimethyl sulfoxide. In the current studies, the role of intracellular O2- is specifically examined by evaluating the effects of intracellular superoxide dismutase (SOD) supplementation on cytotoxicity of rat pulmonary artery endothelial cells induced by H2O2 and activated neutrophils. To minimize in vitro manipulation, supplementation was accomplished by incubating endothelial cells in the presence of SOD (1-20 mg/mL). Increases up to greater than 17-fold the baseline SOD activity were achievable using this approach, with uptake being maximal after 6 h of incubation. This increase was resistant to trypsin digestion, suggesting the intracellular location of SOD. Compared to controls, SOD-supplemented cells showed significantly increased resistance to killing by H2O2 and activated neutrophils. Inactive SOD failed to provide protection. The degree of protection was dependent on the dose of cytotoxic agent and the extent of SOD supplementation. The results provide new evidence that intracellular O2- participates in the killing process induced by these two stimuli. The intracellular source of O2- remains to be determined, although previous studies suggest xanthine oxidase as a likely candidate.     

Inhibition by superoxide dismutase of methemoglobin formation from oxyhemoglobin.

The formation of methemoglobin from oxyhemoglobin in a solution containing photoreduced riboflavin and oxygen was inhibited by superoxide dismutase. The rate of the reaction was pH-dependent in the range of 6.8 to 7.8, increasing as the pH was reduced. Inhibition by superoxide dismutase was enhanced as the EDTA concentration increased and was dependent on enzymatic activity. Under conditions in which superoxide dismutase inhibition was incomplete, catalase inhibited the reaction but mannitol had no effect. The data support the mediation of methemoglobin formation by superoxide. The hypothesis is offered that superoxide anion reduced the heme-bound oxygen in oxygemoglobin by one electron, permitting the subsequent dissociation of ferrihemoglobin and peroxide. The ability of superoxide dismutase to inhibit the formation of methemoglobin may represent one of its functions in the mature erythrocyte.     

Inhibition of yeast ribosomal stalk phosphorylation by Cu-Zn superoxide dismutase

Reversible phosphorylation of acidic ribosomal proteins of Saccharomyces cerevisiae is an important mechanism, regulating the number of active ribosomes. The key role in regulation of this process is played by specific, second messenger-independent protein kinases. A new protein-inhibitor regulating activity of PK60S kinase has been purified from yeast extracts and characterised. Peptide mass fingerprinting (PMF) and amino-acid sequence analysis by Post Source Decay (PSD) have identified the inhibitor as a Cu-Zn superoxide dismutase (SOD). Inhibition by SOD is competitive with respect to protein substrates-P proteins and 80S ribosome-with K(i) values of 3.7 microM for P2A protein and 0.6 microM for 80S ribosomes. A close correlation was found between the state of phosphorylation of P proteins in diauxic shift and logarithmic growth yeast cells and activity of SOD. The possible mechanism of regulation of PK60S activity, and participation of SOD protein in regulation of 80S-ribosome activity in stress conditions, is discussed.     

Clozapine,a dopamine DA4 receptor antagonist,reduces gastric acid secretion and stress-induced gastric mucosal injury

Dopamine and its agonists modulate a variety of gastrointestinal functions. In light of the increasing attention directed toward novel dopamine receptors and compounds that are active at these sites, we examined the effects of a dopamine D₄ antagonist and putative antipsychotic, clozapine, in a model of conscious basal gastric acid secretion and in a model of stress-induced gastric mucosal injury. At a dose of 10.0 mg/kg i.p., clozapine significantly inhibited basal gastric acid secretion by 84% relative to vehicle. Lower doses (2.5 and 5.0 mg/kg) were inactive. Doses of 2.5, 5.0 and 7.5 mg/kg i.p. all significantly reduced restraint stress-induced gastric mucosal injury in rats. The highest dose inhibited gastric lesions by 70% relative to vehicle. We conclude that dopamine D₄ receptors, present in high concentrations in mesolimbic brain regions, modulate gastric function and pathology in addition to mesolimbic D₁ receptors, whose role in gastrointestinal function is already established.     

Inhibition of surfactant function by copper-zinc superoxide dismutase (CuZn-SOD)

Haddad, Imad Y., Bedford Nieves-Cruz, and Sadis Matalon.Inhibition of surfactant function by copper-zinc superoxide dismutase (CuZn-SOD). J. Appl.Physiol. 83(5): 1545-1550, 1997.The efficacy ofantioxidant enzymes to limit oxidant lung injury by instillation withsurfactant mixtures in preterm infants with hyaline membrane disease isunder investigation. However, there is concern that instillation ofproteins in the alveolar space may inactivate pulmonary surfactant. Westudied the effects of bovine copper-zinc superoxide dismutase(CuZn-SOD) on the biophysical properties of two distinct surfactantpreparations. Incubation of calf lung surfactant extract (CLSE, 1 mgphospholipid/ml) and Exosurf (0.1 mg phospholipid/ml) with CuZn-SOD(1-10 mg/ml) prevented the fall of surface tension at minimalbubble radius (T_min) to lowvalues with dynamic compression in a pulsating bubble surfactometer. CuZn-SOD also enhanced the sensitivity to inactivation by albumin, normal human serum, and after treatment with peroxynitrite. The inhibitory effects of CuZn-SOD on CLSE, but not Exosurf, were abolishedat high lipid concentrations (3 mg/ml) and after the addition of humansurfactant protein A (by weight). We conclude that CuZn-SOD mayinterfere with the surface activity of surfactant mixtures, leading todecreased effectiveness of surfactant replacement therapy.

     

Activation of p38 MAPK by reactive oxygen species is essential in a rat model of stress-induced gastric mucosal injury

Stress ulceration is a common complication in critically ill patients and can result in significant upper gastrointestinal bleeding associated with a high morbidity and mortality. At present, little is known of the molecular mechanisms underlying the incidence of this type of gastric damage. In the present study, we investigated the temporal activation of the redox-sensitive p38 signaling transduction cascade and its roles in a well-defined experimental model of cold immobilization stress-induced gastric ulceration. Exposure of Sprague-Dawley rats to 6 h of cold immobilization stress led to a rapid activation of p38 in the gastric mucosa at as early as 15 min after stress, and this activation was maximal after 1.5 h of stress and still persisted until the end of stress. Selectively blocking p38 by pretreatment with SB 239063, a potent and selective p38 inhibitor, suppressed the stress-promoted TNF-alpha, IL-1beta, and CINC-1 production and then prevented the subsequent neutrophil infiltration, gastric mucosal epithelial necrosis and apoptosis, and the ulcerative lesions formation. Prior administration of the free radical scavengers, tempol and N-acetyl-L-cysteine, abolished the stress induction of p38 activation and the resulting mucosal inflammation and gastric injury. These results demonstrate that reactive oxygen species-mediated p38 activation plays an essential role in the pathogenesis of stress-induced gastric inflammatory damage in the rat model of cold immobilization stress. Our findings suggested that inhibition of p38 activation might be a potential strategy for the prophylaxis and treatment of stress ulceration.     

Loss of parietal cell superoxide dismutase leads to gastric oxidative stress and increased injury susceptibility in mice

Mitochondrial superoxide dismutase (SOD2) prevents accumulation of the superoxide that arises as a consequence of oxidative phosphorylation. However, SOD2 is a target of oxidative/nitrosative inactivation, and reduced SOD2 activity has been demonstrated to contribute to portal hypertensive gastropathy. We investigated the consequences of gastric parietal cell-specific SOD2 deficiency on mitochondrial function and gastric injury susceptibility. Mice expressing Cre recombinase under control of the parietal cell Atpase4b gene promoter were crossed with mice harboring loxP sequences flanking the sod2 gene (SOD2 floxed mice). Cre-positive mice and Cre-negative littermates (controls) were used in studies of SOD2 expression, parietal cell function (ATP synthesis, acid secretion, and mitochondrial enzymatic activity), increased oxidative/nitrosative stress, and gastric susceptibility to acute injury. Parietal cell SOD2 deficiency was accompanied by a 20% (P < 0.05) reduction in total gastric SOD activity and a 93% (P < 0.001) reduction in gastric SOD2 activity. In SOD2-deficient mice, mitochondrial aconitase and ATP synthase activities were impaired by 36% (P < 0.0001) and 44% (P < 0.005), respectively. Gastric tissue ATP content was reduced by 34% (P < 0.002). Basal acid secretion and peak secretagogue (histamine)-induced acid secretion were reduced by 43% (P < 0.0001) and 40% (P < 0.0005), respectively. There was a fourfold (P < 0.02) increase in gastric mucosal apoptosis and 41% (P < 0.001) greater alcohol-induced gastric damage in the parietal cell SOD2-deficient mice. Our findings indicate that loss of parietal cell SOD2 leads to mitochondrial dysfunction, resulting in perturbed energy metabolism, impaired parietal cell function, and increased gastric mucosal oxidative stress. These alterations render the gastric mucosa significantly more susceptible to acute injury.     

Interrelationships between the gastric cytoprotective effects of vitamin A and beta-carotene and the gastric mucosal superoxide dismutase activity in rats

Gastric mucosal damage was produced in rats by the intragastric administration of 96% ethanol or 0.6 M HCl, according to the method of Robert et al. Vitamin A or beta-carotene, in doses of 10 mg/kg, given intragastrically 30 min before the administration of the necrotizing agents. The animals were killed 1 hr after the administration of the necrotizing agents. The following experimental parameters were studied, without and with application of vitamin A and beta-carotene; number of gastric lesions (ulcers); severity of gastric mucosal lesions (ulcers); gastric mucosal superoxide dismutase (SOD) activity. It was found that; vitamin A and beta-carotene, in doses of 10 mg/kg, are able to prevent significantly both the number and severity of gastric mucosal lesions (ulcers) produced by the application of 96% ethanol or 0.6 M HCl; the significant increase of ethanol-induced gastric mucosal SOD activity can be inhibited by the application of vitamin A and beta-carotene; vitamin A and beta-carotene are capable of preventing the development of gastric mucosal lesions (ulcers) produced by the intragastric administration of 0.6 M HCl, while these agents fail to compensate for the HCl-induced decrease of gastric mucosal SOD activity. It has been suggested that; vitamin A and beta-carotene are gastric cytoprotective agents; the ulcer preventive effects of vitamin A and beta-carotene are partly dependent on their scavanger behaviour.     

Identification of a peptide sequence in albumin that potentiates superoxide production by microglia

Microglial activation has recently been recognized as a cause of damage in various neurodegenerative diseases. A possible mechanism underlying this damage is the activation of microglia by serum factors leaked through a disruption of the blood-brain barrier, which in turn trigger microglial cell proliferation and the release of various substances toxic to neurons, such as superoxide (O(2)(-)). We recently reported that serum albumin enhanced O(2)(-) production in cultured rat microglia stimulated by phorbol ester. In the present report, we identify the active site of this enhancement within the albumin molecule. We purified an active subfragment from trypsin-treated bovine serum albumin that was composed of 12-mer and 33-mer peptides connected by a disulfide bond. The chemically synthesized 12-mer peptide showed activity within a concentration range ( approximately 10(-7) M:) equivalent to that of albumin. The activities of a series of synthesized peptides conclusively indicated that the minimum active sequence was Leu-His-Thr-Leu. The present study may shed light on the mechanism of neuronal cell damage in various neurodegenerative diseases.     

The interrelationships between the development of ethanol-, HCl, NaOH and NaCl-induced gastric mucosal damage and the gastric mucosal superoxide dismutase activity in the rats

Gastric mucosal damage was produced by intragastric administration of 96% ethanol, 0.6 M HCl, 0.2 M NaOH or 25% NaCl. The animals were killed 1 hr later, when the number and severity of gastric lesions (ulcers) was recorded. At the time of the sacrifice of the animals gastric mucosal superoxide dismutase (SOD) activity was measured. It was found that (1) the gastric mucosal damage could be induced by the administration of any of the necrotizing agents in all animals, (2) superoxide dismutase (SOD) activity increased significantly in the damaged gastric mucosa following 96% ethanol, while its activity decreased significantly during the development of gastric mucosal damage produced by the intragastric administration of 0.6 M HCl, 0.2 M NaOH or 25% NaCl. It has been concluded that: (1) the enzyme systems necessary to generate the superoxide free radical anions can be stimulated by ethanol, and they can be inhibited by the application of 0.6 M HCl, 0.2 M NaOH and 25% NaCl: (2) the observed stimulation or inhibition of the enzyme systems to generate the superoxide free radical anions may be of pathological significance in the development of gastric mucosal damage produced by the intragastric administration of 96% ethanol, 0.6 M HCl, 0.2 M. NaOH or 25% NaCl.     

Inhibition of 2,3-dimethyl-1,4-naphthohydroquinone auto-oxidation by copper and by superoxide dismutase.

2,3-Dimethyl-1,4-naphthohydroquinone undergoes auto-oxidation to the corresponding quinone at pH 7.4, with stoichiometric consumption of oxygen and formation of hydrogen peroxide. In an unpurified buffer, the rate of oxidation was low, but it increased nearly 9-fold when trace metals were removed from the buffer by treatment with Chelex resin. A similar increase in rate was achieved by addition of DTPA or bathophenanthroline sulfonate to unpurified buffer, whereas EDTA and desferal were less effective. Addition of copper to purified buffer led to inhibition of oxidation, with a 50% decrease in rate being observed at a metal concentration of 7.1 nM, and it is likely that the low auto-oxidation rate recorded in unpurified buffer was due to copper contamination of the latter. The auto-oxidation of 2,3-dimethyl-1,4-naphthohydroquinone was exceptionally sensitive to inhibition by superoxide dismutase, with a concentration of only 4.5 ng/ml being sufficient for a 50% decrease in rate, and the inhibitory effect of copper may be due to the ability of this metal to catalyse the dismutation of superoxide. Previous studies have shown that the rates of auto-oxidation of 1,4-naphthohydroquinone and 2-methyl-1,4-naphthohydroquinone are influenced by copper contamination of buffer and the present study shows that this is also true for a di-substituted naphthohydroquinone. For accurate assessment of rates of naphthohydroquinone auto-oxidation, it is important that purified buffers or appropriate chelating agents, are employed.     

Inhibition of vancomycin-induced nephrotoxicity by targeting superoxide dismutase to renal proximal tubule cells in the rat

Vancomycin, a glycopeptide antibiotic, has a broad spectrum against methicillin-resistant Staphylococcus aureus (MRSA). Because vancomycin induces renal dysfunction, the dose and the duration of its administration are limited. The mechanism of vancomycin-induced renal dysfunction is not known. We recently synthesized a hexamethylenediamine-conjugated cationic superoxide dismutase (AH-SOD) which rapidly accumulates in renal proximal tubule cells and inhibits oxidative injury of the kidney. The present work reports the protective effects of AH-SOD against vancomycin-induced renal dysfunction. Male Wistar rats (200-210 g) were intraperitoneally administered with either 200 or 400 mg/kg of vancomycin twice a day for 7 days. Either 5 mg/kg/day AH-SOD or saline was subcutaneously injected 5 min before every vancomycin injection. Biochemical analysis revealed that plasma levels of blood urea nitrogen and creatinine increased significantly in vancomycin-treated group by an AH-SOD-inhibitable mechanism. Histological examination revealed that vancomycin also elicited a marked destruction of glomeruli and necrosis of proximal tubule by an AH-SOD inhibitable mechanism. These results suggest that oxidative stress underlies the pathogenesis of vancomycin-induced nephrotoxicity and that targeting SOD and/or related antioxidants to renal proximal tubule cells might permit the administration of higher doses of vancomycin sufficient for eradication of MRSA without causing renal injury.     

Modulation of oxidant lung injury by using liposome-entrapped superoxide dismutase and catalase

Increased cellular generation of partially reduced species of oxygen mediates the toxicity of hyperoxia to cultured endothelial cells and rats exposed to 95-100% oxygen. Liposomal entrapment and intracellular delivery of superoxide dismutase (SOD) to cultured porcine aortic endothelial cells increased the specific activity of cellular SOD up to 15-fold. The liposome-mediated augmentation of SOD activity persisted in cell monolayers and rendered these cells resistant to oxygen-induced injury in a cell SOD activity-dependent manner. Addition of free SOD to culture medium had no effect on cell SOD activity or resistance to oxygen toxicity. SOD and catalase-containing liposomes injected i.v. into rats increased lung-associated enzyme specific activities two- to fourfold. Liposome entrapment of both SOD and catalase significantly increased the circulating half-lives of these enzymes and was critical for prevention of in vivo oxygen toxicity. Free SOD and catalase injected i.v. in the absence or presence of control liposomes did not increase corresponding lung enzyme activities or survival time in 100% oxygen. These studies show that O2- and H2O2 are important mediators of oxygen toxicity and that intracellular delivery of oxygen protective enzymes can reduce tissue injury owing to overproduction of partially reduced oxygen species.     

Ammonia aggravates stress-induced gastric mucosal oxidative injury through the cancellation of cytoprotective heat shock protein 70

The relationship between Helicobacter pylori colonization and the formation of stress-induced gastric mucosal injury remains unknown. Since ammonia (NH(3)) is known as one of the injurious factors in H. pylori-colonized gastric mucosa, the present study is designed to investigate the level of stress-induced gastric mucosal oxidative injury with or without intragastric NH(3) overloading. To apply emotional stress, the communication box paradigm was used in the mouse model. Mice (C57BL/6, male) were pretreated with distilled water (responder-H(2)O) or 0.01% NH(3) (responder-NH(3)) through a gastric tube once a day for a week. Emotional stress was then applied to the responder mice for 3 h per day for 3 d by watching and hearing the behavior of the sender mice subjected to electric shocks to the feet (2 mA, 10 s, 50 s interval). After the communication box protocol, the tissue MPO activity, the contents of TBA-reactive substances (TBARS), and the level of gastric mucosal HSP70 were examined. Responder-NH(3) mice developed more severe gastric lesions than the responder-H(2)O subjects. MPO activity and TBARS contents were enhanced significantly in the responder-NH(3) group compared with the responder-H(2)O subjects. Although the contents of HSP70 in the gastric mucosa increased in the responder-H(2)O group compared with the control-H(2)O animals, they were significantly attenuated in the responder-NH(3) mice. Excess intragastric NH(3) was able to enhance the formation of emotional stress-induced gastric mucosal lesions. This injury may be associated with the enhanced production of oxygen free radicals from accumulated neutrophils under the NH(3)-mediated cancellation of gastric mucosal cytoprotective HSP70.