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.     

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-1 (TGF-1) 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-1 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.     

Papaverine stimulation of prostaglandin E2 production by cultured rabbit gastric mucosa

Prostaglandins (PGs) are synthesised by gastric mucosa, and have been shown to inhibit gastric acid secretion and ulcer formation in man and experimental animals. Recently exogenous PGs, mainly of the E group, have been used for the treatment of peptic ulcer disease. We therefore searched for a drug that would stimulate endogenous gastric prostaglandin E₂ (PGE₂) synthesis. Rabbit gastric mucosa slices were cultured for 22 hours at 77°C. PGE₂, measured by radioimmunoassay, was found to be linearly secreted into the culture medium. PGE₂ accumulation in the medium during 22 hours of culture was 7·9±0·5 (SE) ng/mg tissue (N=20). Addition of papaverine (100 μ/ml), a cyclic nucleotide phosphodiesterase inhibitor, resulted in a significant increase (250% of control) in PGE₂ accumulation in the medium: 24·3±1·8 ng/mg tissue (N=25). Isobutylmethylxanthine (IBMX 100 μg/ml), another phosphodiesterase inhibitor, only slightly increased PGE₂ accumulation, while 8 bromo-cyclic AMP (1 mM) had no effect. Under these conditions IBMX increased by 20-fold mucosal cyclic AMP levels: 3·9±0·3 pmol/mg tissue (N=8) as compared with control levels: 0·2±0·03 pmol/mg tissue (N=8). Papaverine, however, did not alter mucosal cyclic AMP accumulation. These results indicate that papaverine stimulates PGE₂ production by cultured rabbit gastric mucosa and that this stimulation is not related to the inhibition of phosphodiesterase activity and accumulation of mucosal cyclic AMP. Papaverine induced stimulation of PGE₂ production should be further evaluated regarding its possible beneficial effects in protecting gastric mucosa and in reducing acid secretion in peptic ulcer patients.     

Cell cycle-dependent inhibition of DNA synthesis by prostaglandin I2 in cultured rabbit aortic smooth muscle cells

The role of prostaglandin I2 (PGI2) in the control of DNA synthesis during the cell cycle was investigated in cultured rabbit aortic smooth muscle cells (SMC). SMC at confluency in the G0 state reached the S phase about 16 h after stimulation with serum, as judged by measurement of [3H]thymidine incorporation into DNA (DNA synthesis). Cyclooxygenase inhibitors such as indomethacin and aspirin enhanced DNA synthesis, suggesting that endogenously synthesized prostaglandins inhibit DNA synthesis. Added PGE1 or PGE2 had little effect on DNA synthesis. PGI2 inhibited DNA synthesis only when added from 10 to 16 h after stimulation of SMC in the G0 state with serum. Addition of CS-570, a stable PGI2 analogue, inhibited DNA synthesis at any time after serum stimulation. The endogenous syntheses of PGI2 and DNA were negatively correlated. These results suggest that PGI2 inhibits DNA synthesis by acting on the progression stage of the G1 state.     

Effect of 16,16 dimethyl prostaglandin E2 on aspirin induced damage to rat gastric epithelial cells in tissue culture.

Prostaglandins (PGs) protect gastric mucosa against damage produced by acetylsalicylic acid (ASA). Whether this effect of prostaglandins is truly cytoprotective and whether cAMP plays an important role in this effect is uncertain. We studied the effect of: (1) 16,16 dimethyl prostaglandin E2 (dmPGE2), isobutylmethyl xanthine (IMX), and dibutyryl cAMP (DBcAMP) on ASA-induced damage to monolayer cultures of rat gastric mucosa composed primarily of mucus cells; (2) dmPGE2 on ASA absorption into the cultured cells. Cell damage was quantitated by 51Cr-release and trypan blue staining. Ten millimoles ASA significantly increased 51Cr-release (indicating cell damage) at pH 5.0, but not at pH 7.4. DmPGE2 significantly reduced ASA-induced increase of 51Cr-release. Isobutylmethyl xanthine did not change the rate of 51Cr-release caused by ASA plus dmPGE2. Dibutyryl cAMP did not significantly alter 51Cr-release caused by ASA. A dose response study of ASA damage showed close correlation between 51Cr-release and trypan blue staining (r = 0.93). Dimethyl prostaglandin E2 did not affect 14C-ASA incorporation by the cells at either pH 7.4 or pH 5.0. We conclude that: (1) dmPGE2 exerts a cytoprotective effect on cultured rat gastric cells; (2) cAMP does not play an important role in such cytoprotection; (3) this protection is not because of interference with ASA absorption by prostaglandin.     

Migration of primary cultured rabbit gastric epithelial cells requires intact protein kinase C and Ca2+/calmodulin activity

Superficial gastric mucosal injury is rapidly repaired by epithelial cell migration. This study aims to characterize the intracellular signal transduction pathways underlying the repair process. Primary monolayer cultures of rabbit gastric epithelial cells were wounded. The measured spontaneous cell migration speed at the edge of the wound was 457 ± 89 m/24 hr. Epidermal growth factor stimulated and genistein (receptor tyrosine protein kinase inhibitor) inhibited cell migration significantly. Down-regulation of protein Kinase C (PKC) with long-term phorbol 12-myristate 13-acsetate or inhibition with calphostin-C significantly inhibited cell migration. Blocking of Ca²⁺ channels with verapamil and endogenous Ca²⁺ release with TMB-8 or inhibition of the Ca²⁺/calmodulin complex with calmidazolium likewise significantly inhibited migration speed and also abolished the rise of [Ca²⁺]_i, which was measured in migrating cells. Modulation of the cAMP-PKA pathway or prostaglandin synthesis had no influence on cell migration. Gastric epithelial cell migration implies activation of receptor tyrosine kinase. It is associated with increased [Ca²⁺]_i and requires an intact Ca²⁺/calmodulin complex. Intact PKC activity also is needed.     

正常胃黏膜上皮细胞原代培养方法

胃黏膜上皮细胞是进行胃生理功能、病变机制、药物治疗等研究的重要工具，已被广泛应用于科学实验。原代培养细胞能反映原始组织的特点，与体内姊妹细胞的生理状态相似，是进行相关研究的理想材料。但胃黏膜上皮细胞对内环境要求较高，分离纯化后并不象肝细胞等易于存活和生长，不少实验因这一环节而不能进行下去，不得不采用从肿瘤组织衍生的各种细胞株，此类细胞株与正常胃黏膜上皮细胞在生长特性、形态结构等方面存在较大差异，不能代表正常胃黏膜上皮。在我国，几乎所有的相关研究都是采用各种细胞株，因此本文较详细全面地介绍了文献报道中较成功的正常胃黏膜上皮细胞原代培养方法，以期对相关研究有所帮助。     

Ethanol induces volume changes and gap junction closure via intracellular Ca2+ signalling pathway in cultured rabbit gastric epithelial cells

Background: Ethanol is a well‐established ‘barrier breaker’ in gastric mucosa, but its effects at cellular level remain to be detailed. Methods: Gastric epithelial cells were isolated from rabbits and cultured to monolayers. Intracellular calcium was measured spectrofluorometrically with fura‐2. The patency of gap junctions was assessed by photobleaching a small area of 5‐carboxyfluorescein loaded monolayer and measuring recovery of fluorescence. For cell volume measurements the change in fluorescence intensity was followed in calcein‐loaded monolayers with a confocal microscope. Results: Intracellular calcium concentration was increased from 65?±?9 to 140?±?17?nM; recovery of fluorescence signal after photobleaching was diminished from 53%?±?11% to 9%?±?3%; and cell volume was decreased significantly after 10?min exposure to 5% (vol/vol) ethanol. This volume decrease was prevented with serosal application of the potassium channel blocker, quinine, or by blocking the intracellular calcium signalling pathway with the intracellular calcium‐chelating agent BAPTA. This suggests that luminal ethanol opens the basolateral calcium‐dependent potassium selective channels via calcium signalling pathway, with resultant shrinkage of the cell. Conclusion: Intracellular calcium concentration is increased, gap junctions are closed and cell volume is decreased after exposure to 5% ethanol. Since gap junctions are known to be calcium gated, it is likely that their closure is secondary to the elevated cytosolic calcium in ethanol injured cells. This may have a protective function by limiting intercellular spread of impending cell injury. The opening of the basolateral potassium channel probably underlies the ethanol‐induced cell shrinkage and might contribute to the ethanol‐provoked epithelial damage.     

Ethanol induces volume changes and gap junction closure via intracellular Ca2+ signalling pathway in cultured rabbit gastric epithelial cells

BACKGROUND: Ethanol is a well-established 'barrier breaker' in gastric mucosa, but its effects at cellular level remain to be detailed. METHODS: Gastric epithelial cells were isolated from rabbits and cultured to monolayers. Intracellular calcium was measured spectrofluorometrically with fura-2. The patency of gap junctions was assessed by photobleaching a small area of 5-carboxyfluorescein loaded monolayer and measuring recovery of fluorescence. For cell volume measurements the change in fluorescence intensity was followed in calcein-loaded monolayers with a confocal microscope. RESULTS: Intracellular calcium concentration was increased from 65 +/- 9 to 140 +/- 17 nM; recovery of fluorescence signal after photobleaching was diminished from 53% +/- 11% to 9% +/- 3%; and cell volume was decreased significantly after 10 min exposure to 5% (vol/vol) ethanol. This volume decrease was prevented with serosal application of the potassium channel blocker, quinine, or by blocking the intracellular calcium signalling pathway with the intracellular calcium-chelating agent BAPTA. This suggests that luminal ethanol opens the basolateral calcium-dependent potassium selective channels via calcium signalling pathway, with resultant shrinkage of the cell. CONCLUSION: Intracellular calcium concentration is increased, gap junctions are closed and cell volume is decreased after exposure to 5% ethanol. Since gap junctions are known to be calcium gated, it is likely that their closure is secondary to the elevated cytosolic calcium in ethanol injured cells. This may have a protective function by limiting intercellular spread of impending cell injury. The opening of the basolateral potassium channel probably underlies the ethanol-induced cell shrinkage and might contribute to the ethanol-provoked epithelial damage.     

Effect of calcium channel blocker diltiazem on cytoprotection and prostaglandin and sulfhydryl production by rat gastric epithelial cells

The calcium channel blockers verapamil and diltiazem protect gastric mucosa against exogenous injury in vivo. Whether this protection is mediated by systemic factors, such as blood flow, is due to inhibition of gastric acid secretion, or is associated with stimulation of endogenous protective agents such as prostaglandins or sulfhydryls, is unknown. We have evaluated whether diltiazem protects rat gastric epithelial cells in tissue culture (a model which excludes the influence of systemic factors) against damage induced by sodium taurocholate, indomethacin, or ethanol. Further we have assessed the effect of diltiazem on prostaglandin and sulfhydryl production. 51Chromium release assay and phase contrast microscopy have been used to assess cell damage. Sodium taurocholate, indomethacin, and ethanol-damaged cultured cells in a dose-dependent manner. Pretreatment with diltiazem did not prevent the drug-induced damage. Diltiazem did not increase PGE2 and 6-keto PGF1a production by cultured cells nor did it affect the cellular level of endogenous sulfhydryls. In conclusion, the calcium channel blocker diltiazem is not directly protective to rat gastric mucosal cells in vitro. Diltiazem does not stimulate prostaglandin production by gastric cells nor does it increase the cellular level of protective sulfhydryls.     

Modulation of prostaglandin E2 synthesis in rabbit synoviocytes

Prostaglandin E2 (PGE2) production by rabbit synoviocytes was markedly stimulated by hydroxyapatite only after a 60-minute delay. Release of 3H-arachidonic acid (C20:4) and 3H-PGE2 from cells with phospholipids that were prelabeled with 3H-C20:4 did not occur in parallel. At 240 minutes, phospholipase activity in sonicated cell suspensions had increased only 38%, while cyclooxygenase activity had doubled (109%). This doubling, as well as the production of synoviocyte PGE2, was prevented by inhibiting the synthesis of protein. Cyclooxygenase is an inducible enzyme, and as such, it is a rate-controlling step in PGE2 production.     

Effects of myenteric denervation on gastric epithelial cells and gastric emptying

The influence of myenteric denervation on gastric emptying and epithelial cell population was studied by chemical lesion of myenteric neurons of rat stomach with benzalkonium chloride. Three groups were evaluated: denervated, denervated with pyloroplasty, and control. After three months, the animals were submitted to functional (gastric emptying of liquids) and morphological (neuronal counting; mucosal area, height, and volume; mucous, chief, and parietal cell population; parietal cell area; and gastrin-G and somatostatin-D cell population) studies. After benzalkonium chloride treatment, there was significant delay in gastric emptying of liquids and an increase in mucosal area of all gastric portions, in antral mucosal volume, in parietal cell area, and gastrin-G cell and somatostatin-D cell populations. These changes did not occur when denervation was combined with pyloroplasty, indicating that after denervation, gastric distension and stasis are the major stimuli for parietal cell hypertrophy and G and D cell hyperplasia     

Studies on the prostaglandin E-2-9-ketoreductase mediated production of prostaglandin F-2 alpha and its metabolism in cultured rabbit luteal cells

Luteal cells were isolated from pseudopregnant rabbits on days 3, 6, 9, 12 and 15 post ovulation. Prostaglandin concentration and the activities of the enzymes prostaglandin E-2-9-ketoreductase and prostaglandin-15-hydroxydehydrogenase were determined. Luteal cells from day 7 and 12 of pseudopregnancy were maintained in culture for 24 h and then exposed to a mixture of [1-14C]PGE2 (70 mumol/l) in the presence or absence of estradiol-17 beta. After a 24 h incubation period, the culture medium was adjusted to pH 3.5, immediately extracted and analysed for PG. Cultured luteal cells were able to convert exogenously applied PGE2 to PGF2 alpha and to metabolize both PGs. Primary PGs as well as their metabolites 15-keto PGE2, 15-keto PGF2 alpha, 13,14-dihydro-15-keto PGE2, and 13,14 dihydro-15-keto PGF2 alpha were detected in the culture medium and in the cells. The addition of estradiol-17 beta together with PGE2 caused a significant reduction in the PGE2-9-ketoreductase mediated PGF2 alpha synthesis, whereas the metabolism of PGE2 remained unchanged. This inhibitory effect of estradiol-17 beta was dose-dependent on day 12 of pseudopregnancy. The results demonstrate that isolated rabbit luteal cells are able to synthesize and metabolize the luteolytic factor PGF2 alpha. Whether the inhibitory effect of estradiol-17 beta may have any physiological relevance has to be examined in further studies.     

Effects of epidermal growth factor and insulin on migration and proliferation of primary cultured rabbit gastric epithelial cells

We assessed the influence of epidermal growth factor (EGF) and insulin on gastric epithelial restoration in vitro. Rabbit gastric epithelial cells were cultured and formed a complete monolayer cell sheet in 2 days. We created a wound (1.8±0.05 mm²) by denuding an area of cells, and EGF (0.1–30 ng/ml) and/or insulin (1nM–1μM) was added. The restoration process, which included cell migration and proliferation, was monitored by measuring the cell-free area every 12 h for 2 days. Proliferating cells were detected by sequential staining with bromodeoxyuridine (BrdU). Control cells showed complete repair in 36–48h and restoration was accelerated dose-dependently by EGF or insulin. EGF plus insulin further accelerated restoration, which was then completed in 12–24h. EGF and/or insulin increased the number of BrdU- positive cells. The results indicated that EGF and insulin additively accelerated gastric epithelial wound repair by stimulating both the migration and the proliferation of gastric epithelial cells (particularly the former).     

胃癌患者外周血单个核细胞产生前列腺素E2的研究

目的：探讨前列腺素Ｅ２（ＰＧＥ２）在胃癌中的应用价值。方法：用放免法测定３０例胃癌、３０例良性胃病患者和２３例健康人外周血单个核细胞产生ＰＧＥ２水平，同时还检测了ＴＮＦα和ＩＬ－２受体（ＩＬ－２Ｒ）表达水平，并使用消炎痛体外干预。结果：胃癌患者ＰＧＥ２和ＴＮＦα水平显著高于正常对照及良性胃病组（Ｐ＜００５），而胃癌患者ＩＬ－２Ｒ表达水平显著降低（Ｐ＜００１）；消炎痛体外干预可显著提高胃癌患者ＴＮＦα和ＩＬ－２Ｒ表达水平（Ｐ＜００１）。结论：研究ＰＧＥ２产生情况，对反映胃癌患者免疫功能状态及改善免疫治疗效果可能有一定意义     

HTRA1 gene expression in gastric epithelial cells

Objective:To explore HtrA1 gene expression aud its regulation in human gastric cancers.Methods:The HtrA1 mRNA levels were examined by QPCR analysis and coufirmed its expression with Northern blot analysis.The HtrA1 protein levels in all six gastric epithelial cell lines were investigated by Western blot analysis.Gene copy number was accessed and then sequenced the coding region from each mRNA in all six cell lines.The HtrA1 promoter region DNA methylation status was detected by using bisulfite sequeucing analysis.Effect of decitabine and TSA on HTRA1 expression in gastric cancer cell line was determined by RTPCR.Results:HIC analysis indicated that HtrA1 was highly expressed in normal epithelium,but dramatically down-regulated in gastric carcinoma tissues and variably expressed in tumor-adjacent tissues.HtrA1 gene expression was dramatically decreased in gastric carcinoma cells compared to nontumorigenic counterparts.The HtrA1 gene loss in any of the 4 breast cancer cell lines was not detected.Total 14 CpGs in this region were all methylated in gastric cancer cells,whereas two normal cells.GES-1 and HFI-145,were having several unmethylated cytosines in this region.HtrA1 showed as~Mr 44,000,Expression of HtrA1 protein was not observed in any of the four gastric caucer cell lines.BGC-823.MKN-45.SGC-7901and MKN-28.HtrA1 expression was observed in the HF1-145and GES-1 cell lines.Conclusions:The epigenetic silencing for HtrA1gene expression could provide a possible strategy for re-activating Htrt1 gene expression in gastric cancer cells.thus facilitating further investigation of HtrA1's role in chemotherapy.     

A monolayer culture of human gastric epithelial cells

Our aim was to develop a fibroblast-free monolayer culture of human gastric mucosal cells, using the specimens obtained by routine endoscopic biopsy. Human gastric mucosa obtained from normal volunteers by endoscopic biopsy was dissociated from collagenase and hyaluronidase. Dissociated cells were cultured in supplemented Coon's modified Ham's F-12 medium. Within 24 hr of inoculation, the cells were attached to the culture dishes. This was followed by cellular outgrowth. On phase-contrast microscopy, all cells had epithelial characteristics and fibroblasts were not observed. Ninety percent of cells contained periodic acid Schiff reaction-positive mucous granules after diastase digestion consistent with mucous epithelial cells. Two percent of the cells gave a strong reaction for succinic dehydrogenase activity (parietal cells). Immunohistochemical staining for pepsinogen in cultured cells was negative. On EM, microvilli-like projections, junctional complexes, Golgi apparatus, and mucous granules were apparent in the majority of cells. Mitotic figures were observed by day 3 with Giemsa staining. Autoradiographically, these cells were able to incorporate [3H]TdR into the nuclei. Cells were capable of synthesizing DNA, and this function was inhibited by cycloheximide. Cells could be cultured for up to two weeks without fibroblast contamination. A method of primary monolayer culture of human gastric mucosa obtained by a routine endoscopic biopsy has been successfully developed.