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In order to clarify how PGE2 regulates gastric mucosal integrity, we examined the effects of PGE2 on ethanol-caused injury of isolated gastric chief cells, cultured gastric mucous cells from guinea pigs and Balb/c 3T3 fibroblasts. Pretreatment of these cells with PGE2 reduced ethanol-caused injury of the cells. Furthermore, pretreatment of gastric mucous cells with indomethacin enhanced ethanol-caused injury, suggesting that endogenous PGE2 may be involved in the cell protection. PGE2 stimulated an increase in diacylglycerol (DG) accumulation in chief cells and treatment of chief cells with synthetic DG reduced the injury of the cells. However, DG accumulation was not observed in gastric mucous cells treated with PGE2. Therefore PGE2 may protect the cells from injury through a variety of mechanisms. In addition, PGE2 enhanced the survival of the quiescent fibroblasts cultured in the absence of serum, while PGE2 had no survival enhancing effect on gastric mucous cells. These results suggest that the mechanism by which PGE2 preserves the cell viability may depend on not only cell types used but also how the cells are injured.

To understand the mechanism by which prostaglandins (PGs) preserve gastric mucosal integrity, we established a primary cultured monolayer of guinea pig gastric mucous cells, and by using this culture system, we studied whether endogenously released PGE2 could influence the proliferation of the mucous cells. By the histochemical and morphological analysis at 24h of the culture periods, the cells were recognized to contain PAS-positive mucous granules with only 3% of them being parietal cells. Although the cells which were simultaneously labeled with [3H] arachidonic acid in 0.5% serum-containing medium synthesized and released radiolabeled PGE2, PGI2 and PGA2, the release of PGE2 was more markedly observed and was partially dependent upon arachidonic acid added to the culture medium. By radioimmunoassay of the culture media, the mucous cells were found to release PGE2 in a time-dependent manner in response to 10% serum. Pretreatment of the cells with 10(-4)M indomethacin not only inhibited PGE2 release but also inhibited increase in cell number. However, the addition of PGE2 dose-dependently restored the indomethacin-induced inhibition of cell growth with the maximal increase almost to the control level at 10(-6)M PGE2. These results suggest that PGE2 endogenously released from the cells may exert a proliferative effect on gastric mucous cells.

The muscarinic receptor system involved in pepsinogen secretion from isolated guinea pig gastric chief cells was investigated by evaluating the effect of muscarinic receptor antagonists on carbamylcholine (CCh)-stimulated chief cell responses. CCh stimulated the hydrolysis of polyphosphoinositide in chief cells at the same concentrations as it stimulated pepsinogen secretion. Each of five different muscarinic receptor antagonists, atropine, pirenzepine, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), AF-DX116 and scopolamine, inhibited both pepsinogen secretion and inositol phosphate accumulation stimulated by graded concentrations of CCh. The pA2 values of the antagonists calculated from data on inositol phosphate accumulation and pepsinogen secretion (atropine = scopolamine = 4-DAMP greater than pirenzepine greater than AF-DX116) suggest that the muscarinic acetylcholine receptor in gastric chief cells is the M3 subtype. On the other hand, CCh did not affect the adenylate cyclase/cAMP signaling pathway in gastric chief cells. All pA2 values of the antagonists were also in agreement with the Ki values determined by [3H]NMS binding to chief cells. Furthermore, GTP gamma S reduced [3H]acetylcholine binding to chief cell membranes in a concentration-dependent manner. The present study, therefore, suggests that muscarinic M3 receptors, which may be coupled to a G protein, mediate pepsinogen secretion, probably by activation of the polyphosphoinositide second messenger system in guinea pig gastric chief cells.

To investigate the pathogenesis of hereditary pancreatitis we determined whether pancreatic stone protein (PSP) gene was structurally altered in two independent families diagnosed as hereditary pancreatitis. Because, it has been shown that a decrease in the activity of PSP which inhibits CaCO3 crystal formation in pancreatic juice is closely related to the development of chronic calcifying pancreatitis. Southern blot analysis revealed neither a rearrangement nor a gross deletion of PSP gene in genomic DNA of affected members of both families. Furthermore, six exons of PSP gene amplified by polymerase chain reaction from genomic DNA was directly sequenced, while no apparent base mutation was observed. The immunohistochemical study utilizing monoclonal antibody to PSP showed the presence of immunoreactive PSP in the section of pancreatic tissue obtained from a patient affected with hereditary pancreatitis. However, the level of immunoreactive PSP in the remaining acinar cells of the patient pancreas was not reduced when compared with that of normal pancreas. Results, therefore, indicate that genetic alteration of PSP gene may not be responsible for the pathogenesis of hereditary pancreatitis.

To better understand the mechanism underlying the inhibition induced by cholecystokinin (CCK) of phosphatidylcholine (PC) synthesis, the effects of CCK treatment on the activities of enzyme involved in PC synthesis via CDP-choline pathway were studied in isolated rat pancreatic acini. CCK treatment of acini reduced CTP: phosphocholine cytidylyltransferase activity in both cytosolic and particulate fraction. However, CCK treatment of acini did not alter the activities of choline kinase and phosphocholinetransferase in acini. When acini were labeled with [3H] myristic acid and chased, CCK8 (1 nM) reduced the synthesis of [3H] myristic acid labeled-PC to 27% of control after 60 min-chase period. This inhibition of PC synthesis induced by CCK was accompanied by a delayed disappearance of [3H] diacylglycerol (DAG), the radioactivity of which was 225% of control at 60 min. CCK also induced an increase in [3H] triacylglycerol and [3H] phosphatidic acid in acini. These results suggest that CCK inhibits PC synthesis by inducing the inhibition of CTP: phosphocholine cytidylyltransferase activity. The inhibition by CCK of PC synthesis may contribute to the sustained accumulation of DAG in pancreatic acinar cells.

The effects of cholecystokinin (CCK) and other pancreatic secretagogues on phosphatidylcholine (PC) synthesis were studied in isolated rat pancreatic acini. When acini were incubated with [3H] choline in the presence of 1 nM CCK-octapeptide (CCK8) for 60 min, the incorporations of [3H] choline to both water soluble choline metabolites and PC in acini were reduced by CCK8 to 74% and 41% of control, respectively. Pulse-chase study revealed that CCK reduced both the disappearance of phosphocholine and the synthesis of PC. Ca(2+)-mobilizing secretagogues such as carbamylcholine and Ca2+ ionophore A23187 also reduced PC synthesis to the same extent as CCK8. By contrast, neither cAMP-dependent secretagogues such as secretin and dibutyryl cAMP nor a phorbol ester had any effect on PC synthesis in acini. These results suggest that CCK inhibits PC synthesis by inducing both the reduction of choline uptake into acini and the inhibition of CTP: phosphocholine cytidylyltransferase activity. This hormonal regulation of PC synthesis via CDP-choline pathway appears to be mediated by Ca(2+)-dependent pathway but not by cAMP- or protein kinase C-dependent pathway.

The mechanism by which PGE2 directly protects individual gastric cells from ethanol-induced injury was studied by using isolated gastric chief cells from guinea pig. Ethanol dose-dependently caused chief cell injury which was estimated by the release of lactate dehydrogenase (LDH) from chief cells. Pretreatment of chief cells with PGE2 reduced the cell damage caused by ethanol in time- and dose-dependent manner. The pretreatment at 37 degrees C and pH 7.4 with PGE2 maximally reduced the cell damage. This protective effect was reduced when the pretreatment was performed at either acid or alkaline pH or at reduced temperature. PGE2 did not stimulate any increase in cytosolic free Ca2+ concentration and initial Ca2+ influx rate. On the other hand, PGE2 stimulated an increase of cAMP accumulation in chief cells. However, pretreatment of chief cells with secretion, VIP, dbcAMP or forskolin failed to reduce subsequent injury caused by ethanol. These results suggest that PGE2 may protect chief cells against ethanol-caused injury probably via PGE type receptors coupled to as yet unidentified signal in gastric chief cells.

To clarify the mechanism by which the administration of anti-tumor drugs, antibiotics or hypoglycemic agents causes gastric mucosal injury, the effects of these drugs on phosphatidylcholine synthesis in isolated guinea pig gastric glands were examined in vitro. Anti-tumor drugs such as tegafur, cyclophosphamide, and mitomycin C decreased [3H]choline incorporation into phosphatidylcholine. Furthermore, tegafur at 0.4 mg/ml decreased [3H]choline incorporation in the glands that had been pulsed with [3H]choline incorporation, suggesting that tegafur exerts its effect by inhibiting late step of phosphatidylcholine synthesis in the stomach. On the other hand, cefaclor and glibenclamide had no effect on [3H]choline incorporation. Geranylgeranylacetone, an anti-ulcer drug partially restored tegafur-induced reduction of [3H]choline incorporation into phosphatidylcholine. These results suggest that the anti-tumor drug-induced gastric mucosal injury may be due to drug-induced decrease in phosphatidylcholine synthesis, which the restoration of phosphatidylcholine synthesis by geranylgeranylacetone may explain its anti-ulcer action on drug-induced gastric mucosal lesions in vivo.

The muscarinic receptor system involved in pepsinogen secretion from isolated guinea pig gastric chief cells was investigated by assessing the effect of muscarinic receptor antagonists on carbamylcholine (CCh)-induced pepsinogen secretion. CCh stimulated pepsinogen secretion in a dose dependent manner with the maximal and the half-maximal stimulatory concentrations at 10(-4) and 3 x 10(-6) M, respectively. Each of five different muscarinic receptor antagonists such as atropine, pirenzepine, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), AF-DX116 and scopolamine reduced pepsinogen secretion stimulated by graded concentration of CCh, but did not alter the maximum secretion. The increase in concentration of each antagonist caused parallel rightward shift of the dose response curve to CCh. Schild analysis of the inhibition of CCh-induced pepsinogen secretion by the antagonists showed that pA2 values of atropine, scopolamine and 4-DAMP are 8.8, 9.2 and 9.0, respectively. On the other hand, pA2 values of pirenzepine and AF-DX116 are 6.5 and 5.9, respectively, suggesting that the muscarinic receptor mediating pepsinogen secretion from chief cells has a intermediate affinity for pirenzepine and a low affinity for AF-DX116. These results suggest that the muscarinic acetylcholine receptor mediating pepsinogen secretion from gastric chief cells is the M3 subtype.

Cholecystokinin (CCK) binding to its receptors on guinea pig gastric chief cell membranes was characterized with 125I-COOH terminal octapeptide of CCK (125I-CCK8). Specific binding of 125I-CCK8 to chief cell membranes was maximal at pH 6.0 and 30 degrees C after 180 min of incubation and reversible upon the addition of 10(-7) M unlabeled CCK8. CCK analogs such as CCK8, gastrin-I, and COOH-terminal tetrapeptide of CCK (CCK4) competitively inhibited the labeled CCK8 binding with the half maximal inhibitory concentration of 10(-10) M, 3 X 10(-7) M and 10(-6) M, respectively. Furthermore, guanine nucleotide analogs such as GTP gamma S and Gpp(NH)p also inhibited the labeled CCK8 binding to chief cell membranes. Scatchard analysis of the binding data at pH 6.0 revealed two orders of the binding sites and GTP gamma S decreased the binding by converting two binding sites of the receptors to only one site of lower affinity. These results suggest that there are specific receptors for CCK, which are coupled to a guanine nucleotide regulatory protein on guiea pig gastric chief cell membranes.

To better understand the mechanism of phosphatidylcholine synthesis in the stomach, [3H] choline incorporation into phosphatidylcholine in response to the drugs which have been commonly used for treating the patients with gastric ulcer was examined using isolated guinea pig gastric glands. Teprenone stimulated [3H] choline incorporation into phosphatidylcholine in gastric glands, up to 146 +/- 11% of control (n = 6, P less than 0.05). On the other hand famotidine, ranitidine and cimetidine significantly decreased the incorporation to 73 +/- 8%, 72 +/- 11%, 67 +/- 11% of control, respectively (n = 6, P less than 0.05, P less than 0.05, P less than 0.05). When the glands were pulsed with [3H] choline followed by incubation in the presence of teprenone or each H2RA for 180 min to see the effects of the agents on the limiting step of the phosphatidylcholine synthesis, teprenone also significantly stimulated [3H] choline incorporation into phosphatidylcholine, but each H2RA did not affect phosphatidylcholine biosynthesis any more. Teprenone stimulated CTP:phosphocholine cytidylyltransferase (CTF) from a basal value of 0.92 +/- 0.10 to 1.69 +/- 0.39 (nmol/min/mg protein) (n = 3, P less than 0.05). These results suggest that teprenone may stimulate phosphatidylcholine biosynthesis through the activation of CTF, a late-limiting enzyme of PC biosynthesis, and H2RA may affect phosphatidylcholine synthesis by inhibiting choline transport or choline kinase in gastric glands.

The effects of ethanol on pepsinogen release from isolated guinea pig gastric chief cells were investigated. Ethanol, at concentrations of 300 mM to 900 mM, dose-dependently stimulated increases in pepsinogen release, initial Ca influx rate and intracellular free Ca concentration ([Ca2+]i) without affecting chief cells' viability. The increases in all parameters were inhibited by La3+ or EGTA, but not by nifedipine or verapamil. COOH-terminal octapeptide of cholecystokinin (CCK8) also stimulated increases in pepsinogen release, initial Ca influx rate and [Ca2+]i. However, the increases were dependent on not only extracellular Ca2+ but also intracellular Ca2+ mobilization. These results suggest that ethanol stimulates Ca2+ influx via the mechanism different from that of CCK8 and thereby stimulates pepsinogen release from isolated guinea pig gastric chief cells.

In isolated guinea pig gastric chief cells, gastrin-I and cholecystokinin-hexapeptide (CCK-4) stimulated pepsinogen release. However, the efficacies of these two peptide were 51% of that observed with CCK-octapeptide (CCK8). CR1409 and L-364718, both of which are new CCK receptor antagonists in pancreatic acinar cells, also inhibited 10(-8) M CCK8-stimulated pepsinogen release with a half-maximal inhibitory concentration (IC50) observed at 3 x 10(-9) M, respectively. The dose response curve to CCK8 for pepsinogen release shifted to the right in the presence of CR1409 or L-364718. The IC50 of these two antagonists for the CCK8-stimulated increase in cytosolic Ca2+ concentration monitored by Fura-2 were equal to those for CCK8-stimulated pepsinogen release. By contrast, the IC50 of dibutyryl cyclic GMP, a well-known CCK receptor antagonist, for CCK8-stimulated pepsinogen release was less than that for CCK8-stimulated increase in cytosolic Ca2+ concentration. Results suggested that CCK receptors in gastric chief cells are unique and may be different from CCK receptors in other tissues previously reported.

To better understand the mechanism of phosphatidylcholine synthesis in the stomach, [3H] choline incorporation into phosphatidylcholine in response to agents which have been shown to induce phosphatidylcholine biosynthesis in other tissues was examined using isolated guinea pig gastric glands. Palmitate and 12-O-tetradecanoyl phorbol 13-acetate (TPA) which has been shown to activate protein kinase C directly, stimulated [3H] choline incorporation into phosphatidylcholine in gastric glands, by 189 +/- 12.9%, and 129 +/- 10.4% of control, respectively (n = 4, p less than 0.01, p less than 0.05). On the other hand, dibutyryl cyclic AMP had no effect on the incorporation. When the glands were pulsed with [3H] choline followed by incubation in the presence of palmitate and TPA for 180 min to see the effects of the agents on the limiting step of the phosphatidylcholine synthesis, phosphatidyl-[3H] choline was increased to 167 +/- 7.5% and 142 +/- 7.5% of control respectively (n = 4, p less than 0.01, p less than 0.05). In parallel to the increase in phosphatidylcholine synthesis, phosphoryl-[3H] choline in the glands incubated with palmitate and TPA was decreased as compared with control. These results suggest that palmitate or TPA may stimulate phosphatidylcholine biosynthesis through the activation of cytidylyltransferase in the stomach.