Hideyuki Hiraishi

Role of Superoxide and hydroxyl radicals in rat gastric mucosal injury induced by ethanol

SummaryIt has been reported that oxygen-derived free radicals play an important role in the pathogenesis of mucosal injury in the small intestine as well as in the stomach. The aims of this study were to test whether ethanol-induced damage in the rat stomach was prevented by the administration of (1) Superoxide dismutase (SOD; a scavenger of Superoxide radicals), (2) allopurionol (ALP; an inhibitor of xanthine oxidase), (3) dimethyl sulfoxide (DMSO; a scavenger of hydroxyl radicals). SOD significantly decreased the ulcer index from 100±8.5% (control) to 39.6±8.2% (P<0.001). Ethanol-induced damage was reduced by the administration of ALP by 37.4% (P<0.01). DMSO also diminished the ulcer index from 100±8.5% (control) to 31.6±5.8% (P<0.01). Histochemical studies supported these results. A scanning EM study, however, revealed that surface epithelial cells were not protected by SOD against ethanol-induced damage. These results demonstrated that SOD, ALP and DMSO had the ability to protect gastric mucosa against ethanol-induced injury. Accordingly, oxygen-derived free radicals may be involved in the pathogenesis of ethanol-induced gastric mucosal damage. Surface epithelial cells, however, were not protected even by SOD against ethanol-induced injury.

Protective effect of tauroursodeoxycholate against chenodeoxycholate-induced damage to cultured rabbit gastric cells

Ursodeoxycholate (UDC) and tauroursodeoxycholate (TUDC) have been reported to be protective against liver injury induced by other bile salts. UDC also has been shown to be effective against refluxed bile-induced gastritis after gastric surgery. However the mechanism of the therapeutic effect of UDC on gastric mucosa has not been known. In the present study, cytoprotective actions of UDC and TUDC against chenodeoxycholate (CDC)-induced gastric injury were investigated using rabbit gastric cell cultures without systemic factors. Rabbit gastric mucosal cells were cultured after the isolation of rabbit gastric cells with collagenase and ethylenediaminetetraacetic acid. Cytotoxicity was quantified by measuring51Cr release from prelabeled cells and MTT assay. Prostaglandin (PG) E2 was assayed by radioimmunoassay. Concentrations of CDC>0.5mM or UDC>5mM caused cellular damage and increased51Cr release in a dose-dependent and time-dependent fashion, while TUDC up to 10 mM did not. TUDC, but not UDC, showed a significant decrease of CDC (1.5 mM)-induced51Cr release dose dependently. The protective effect of TUDC against CDC-induced damage was confirmed by MTT assay. On phase-contrast microscopy, disruption of monolayers induced by CDC (1.5 mM) was clearly protected by TUDC (10 mM). Free radical scavengers (500 units/ml of superoxide dismutase, 300 units/ml of catalase, and 100 mM of dimethyl sulfoxide) or a calcium blocker (10−7–10−5 M verapamil) did not show significant protection against CDC-induced damage. Deprivation of Ca2+ in the media did not affect CDC-induced damage. Thus free radicals of Ca2+ might not be involved in the cell toxicity of CDC. Although TUDC (10 mM) significantly increased PGE2 production by cultured cells, indomethacin (10−4 M) did not reduce protective effects of TUDC, as assessed by51Cr release and MTT assay. In conclusion TUDC is cytoprotective against CDC-induced damage to cultured rabbit gastric cells. Neither free radicals, Ca2+, nor endogenous PGs may play leading roles in the mechanism of this action.

A monolayer culture of gastric mucous cells from adult rabbits

SummaryA new method for the primary monolayer cultures of adult rabbit gastric mucous cells has been developed. Rabbit gastric mucosal cells were isolated with etylenediaminetetraacetic acid and collagenase. Cells were cultured in Coon’s modified Ham’s F-12 medium supplemented with 10% fetal bovine serum, 15mM HEPES buffer, antibiotics, and antimycotic. The cells reached confluency on days 3–4. Histochemically 92% of the cells contained PAS positive gramules (mucous cells), 3% of cells showed a strong reaction for succinic dehydrogenase activity (parietal cells), 2% of the cells showed positive granules by Bowie staining (chief cells), and G6PDH staining was positive in 5% of the cells (surface mucous cells). Fibroblasts were rarely seen until day 7 (<1%). Thus rabbit cultured gastric cells were considered to be mainly comosed of mucous neck cells. These cells produced prostaglandin (PG) E2 and PGI2. Quantitatively cultured cells synthesized 1.475±0.039 ng/mg protein/hour of PGE2 and 0.244±0.042 pg/mg protein/hour of PGI2. This relatively simple and convenient technique provides a useful model for the study of cellular functions of gastric mucosa.

Adaptive cytoprotection in cultured rat gastric mucus-producing cells role of mucus and prostaglandin synthesis

In cultured gastric mucosal cells, we investigated whether: (1) adaptive cytoprotection was associated with stimulation of endogenous prostaglandin synthesis; (2) prostaglandins given exogenously were cytoprotective against ethanol-induced gastric mucosal cell damage; and (3) a relationship existed between cytoprotection and mucus release. Cytolysis was quantified by measuring51Cr release from prelabeled cells. Mucus release was determined by measurement of [3H]glucosamine release. Concentrations of ethanol >12% caused cell damage and increased51Cr release dose dependently. Pretreatment with low concentrations of ethanol (0.5–1.5%) decreased ethanol-induced51Cr release, but also decreased prostaglandin E2 synthesis. Prostaglandin E2 and 16,16-dimethyl prostaglandin E2 given exogenously were cytoprotective against ethanol-induced gastric mucosal cell damage. Treatment with low concentrations of ethanol (1.5%) increased mucus release from cultured gastric mucosal cells. However, prostaglandin E2 and 16,16-dimethyl prostaglandin E2 did not affect mucus release. We conclude that in cultured gastric mucus-producing cells: (1) adaptive cytoprotection occurs without stimulation of endogenous prostaglandin synthesis but with increase in mucus release; and (2) exogenous prostaglandins are cytoprotective against ethanol-induced gastric mucosal cell damage without stimulating mucus releasein vitro. We postulate that adaptive cytoprotection in cultured gastric mucus-producing cells is not mediated by prostaglandin, but by mucus released in response to a mild irritant.

Growth regulation of rabbit gastric epithelial cells and protooncogene expression

We recently developed a primary culture system for gastric epithelial cells from adult rabbits that allows the investigation of growth regulation at the cellular level. In this study, we demonstrated that epidermal growth factor (EGF), insulin, and dibutyryl adenosine 3′,5′-cyclic monophosphate (dBcAMP) all stimulated cell proliferation. Insulin and dB-cAMP potentiated the stimulation of cell proliferation by EGF, while transforming growth factor-β1 (TGF-β1) inhibited it. Expression of c-fos and c-myc was induced in response to the stimulation by these growth regulators, but the degree of expression did not necessarily correlate with the effects of these agents on cell proliferation. In conclusion, EGF, insulin, and dBcAMP were positive growth regulators, while TGF-β1 was a negative regulator in gastric epithelial cells. These growth modulators may exert their effects by distinct pathways from a standpoint of the expression of c-fos and c-myc.

Effect of adenosine and adenosine analogs on [14C]aminopyrine accumulation by rabbit parietal cells.

Adenosine receptors that modulate adenylate cyclase activity have been identified recently in a number of tissues. Adenosine A2 receptor is stimulatory to adenylate cyclase, whereas adenosine A1 receptor is inhibitory to adenylate cyclase. We investigated the effect of adenosine and its analogs on [14C]aminopyrine accumulation by rabbit parietal cells. Rabbit gastric mucosal cells were isolated by enzyme digestion. Parietal cells were enriched by nonlinear percoll gradients. [14C]Aminopyrine accumulation was used as an indicator of acid secretion. The effect of 2-chloroadenosine on histamine-stimulated [14C]aminopyrine accumulation was studied. The effects ofN-ethylcarboxamideadenosine, 2-chloroadenosine, stable analogs of adenosine, and adenosine on [14C]aminopyrine accumulation were assessed. Cyclic AMP content of parietal cells was determined by radioimmunoassay. Histamine and carbachol, known secretagogues, stimulated [14C]aminopyrine accumulation. 2-Chloroadenosine did not suppress histaminestimulated [14C]aminopyrine accumulation. 2-Chloroadenosine,N-ethylcarboxamideadenosine, and adenosine dose dependently increased [14C]aminopyrine accumulation. The order of potency wasN-ethylcarboxamideadenosine > 2-chloroadenosine > adenosine. 8-Phenyltheophylline and theophylline, adenosine-receptor antagonists, or cimetidine did not have significant effects on the increase of AP uptake induced by 2-chloroadenosine. Coadministration of dipyridamole, an adenosine uptake inhibitor, augmented the effect of adenosine on [14C]aminopyrine accumulation. 2-Chloroadenosine,N-ethylcarboxamideadenosine, and adenosine each induced a significant increase in cellular cyclic AMP. We conclude that there may be adenosine A2 receptors on rabbit parietal cells which modulate gastric acid secretion.

Roles of Ca2+ and protein kinase C in regulation of prostaglandin E2 release by cultured rabbit gastric epithelial cells

Prostaglandin (PG) has been reported to be one of the important protective factors in the gastric mucosa. However the mechanism of the regulation of endogenous PG production has not been well studied. We investigated the possible roles of Ca2+, cAMP, and protein kinase C (PKC) in the regulation of PGE2 release from cultured rabbit gastric mucosal cells. PGE2 was measured by radioimmunoassay. A23187 (Ca2+ ionophore) at 2×10−6 M significantly increased PGE2 release. Deprivation of Ca2+ from the medium blocked the A23187-induced increase of PGE2. TMB-8 (a putative inhibitor of Ca2+ release from intracellular stores) did not have any significant effects on the increase of PGE2-induced by A23187. Thus, A23187 increased PGE2 through the influx of extracellular Ca2+. W7 or compound 48/80 (calmodulin inhibitors) did not alter the response of PGE2 caused by A23187. Exogenous administration of cAMP, forskolin (an activator of adenylate cyclase), or 2-chloroadenosine (a possible activator of adenylate cyclase through adenosine A2 receptor) had neither significant effects on PGE2 release nor an effect on A23187-induced increase of PGE2 release. 12-O-tetradecanoylphorbol 13-acetate (TPA, an activator of PKC) significantly stimulated PGE2 release in a dose-dependent fashion, whereas another phorbol ester with no biological activity did not. A23187 at 0.8×10−6 M, but not cAMP, potentiated the TPA-induced increase of PGE2. Mepacrine (a phospholipase A2 inhibitor) reduced the A23187-and TPA-induced increase of PGE2. These results suggest that Ca2+ and protein kinase C may play important roles in the regulation of PGE2 release by cultured rabbit gastric cells.

Characterization of a newly established cell line (JR-St) derived from human gastric signet ring cell cancer, producing tumor markers.

SummaryDespite the importance of in vitro study of gastric cancer, there are very few established cell lines derived from human gastric carcinoma. We have recently established a new cell line derived from human gastric cancer which has the ability to produce tumor markers. This cell line has been designated JR-St. This cell line was derived from the cerebrospinal fluid of a 37-yr-old female patient who had metastatic brain tumor of signet ring cell gastric adenocarcinoma. This cell line has been maintained for more than 24 months through 80 passages with stable growth. PAS staining showed intracellular mucin granules. Transmission and scanning electron microscopy revealed cells with numerous microvilli and fine projections as well as intracellular granules, indicating mucin. This cell line had the ability to produce high concentrations of tumor markers such as carcinoembryonic antigen (CEA) and carbohydrate antigen (CA) 19-9. Thus this cell line should provide a very useful tool for the investigation of gastric cancer such as analysis of tumor markers as well as effects of anti-cancer drugs or growth factors.

Antioxidant defenses of cultured colonic epithelial cells against reactive oxygen metabolites.

Reactive oxygen metabolites produce colonic epithelial cellular injury. The present study evaluated the protective role of cellular superoxide dismutase, catalase, and glutathione (GSH) redox cycle in cultured rabbit colonic cells. Cultured rabbit colonic epithelial cells were exposed to reactive oxygen metabolites generated by hypoxanthine (1 mM) and xanthine oxidase (1 mU/ml) for up to 5 h. Cytotoxicity was quantified by measuring 51Cr release from prelabeled cells. Pretreatment with diethyldithiocarbamate (inhibitor of superoxide dismutase) reduced activity of cellular superoxide dismutase and increased 51Cr release caused by hypoxanthine/xanthine oxidase from colonic cells. Pretreatment with diethyl maleate (covalently binds GSH as catalyzed by GSH transferase), or buthionine sulfoximine (inhibitor of gamma-glutamylcysteine synthetase) decreased cellular GSH and enhanced reactive oxygen metabolites induced injury. Pretreatment with bis(chloroethyl)-nitrosourea (inhibitor of GSH reductase) inhibited activity of GSH reductase and increased 51Cr release from colonic cells. Preincubation with aminotriazole (inhibitor of catalase) reduced cellular catalase, but did not affect cellular injury. Therefore, we concluded that both cellular superoxide dismutase and the GSH redox cycle appeared to play a role in detoxifying reactive oxygen metabolites and that cellular catalase may be less important in rabbit colonic epithelial cells.

Arachidonic acid stimulation of mucus production by rat gastric cultured cells

The effect of arachidonic acid (AA) on mucus production (synthesis and secretion) by rat gastric monolayer-cultured cells was investigated. For the study of mucus synthesis, the rate of incorporation of [3H]glucosamine into the cultured cells was measured. The rate of release of glycoprotein into the culture media from the cells, which were incubated in the medium containing [3H]glucosamine for 24 hr in advance, was determined for the study of mucus secretion. Prostaglandins in the medium were measured by radio-immunoassay. AA (10−4 M)significantly increased mucus synthesis and secretion by the cultured cells (P<0.01). PG (E2 and I2) synthesis by the cultured cells was significantly enhanced by AA (10−4 M) (P<0.05). An addition of indomethacin to the culture medium abolished this effect of AA. These results suggested that gastric mucus production was enhanced by AAin vitro and that this effect may be mediated by endogenous PG production.