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The effects of intracellular [Cl.sup.-] concentration ([[[Cl.sup.-]].sup.i]) on acetylcholine (ACh)-stimulated exocytosis were studied in guinea pig antral mucous cells by video microscopy. ACh activated [Ca.sup.2+]-regulated exocytosis (an initial phase followed by a sustained phase). Bumetanide (20 [micro]M) or a [Cl.sup.-]-free (N[O.sup.-.sub.3]) solution enhanced it; in contrast, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, a [Cl.sub.-] channel blocker) decreased it and eliminated the enhancement induced by bumetanide or N[O.sup-.sub.3] solution. ACh and [Ca.sup.2+] dose-response studies demonstrated that N[O.sup-.sub.3] solution does not shift their dose-response curves, and ATP depletion studies by dinitrophenol or anoxia demonstrated that exposure of N[O.sup-.sub.3] solution prior to ATP depletion induced an enhanced initial phase followed by a sustained phase, whereas exposure of N[O.sup-.sub.3] solution after ATP depletion induced only a sustained phase. Intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sup.i]) measurements showed that bumetanide and N[O.sup-.sub.3] solution enhanced the ACh-stimulated [[[Ca.sup.2+]].sup.i] increase. Measurements of [[[Cl.sub.-]].sub.i] revealed that ACh decreases [[[C1.sup.-]].sub.i] and that bumetanide and N[O.sup-.sub.3] solution decreased [[[C1.sup.-]].sub.i] and enhanced the ACh-evoked [[[Cl.sub.-]].sub.i] decrease; in contrast, NPPB increased [[[C1.sub.-]].sub.i] and inhibited the [[[Cl.sub.-]].sub.i] decrease induced by ACh, bumetanide, or N[O.sup-.sub.3] solution. These suggest that [[[Cl.sub.-]].sub.i] modulates [[[Ca.sup.2+]].sub.i] increase and ATP-dependent priming. In conclusion, a decrease in [[[C1.sub.-]].sub.i] accelerates ATP-dependent priming and [[[Ca.sup.2+]].sub.i] increase, which enhance [Ca.sup.2+]-regulated exocytosis in ACh-stimulated antral mucous cells. gastric antrum; mucin exocytosis; acetylcholine; intracellular [Cl.sub.-] concentration

In guinea pig antral mucous cells, ACh stimulates the [Ca.sup.2+]-regulated exocytosis, which has a characteristics feature: an initial transient phase followed by a sustained phase. The effects of cGMP on ACh-stimulated exocytosis were studied in guinea pig antral mucous cells using video microscopy. cGMP enhanced the frequency of ACh-stimulated exocytotic events, whereas cGMP alone did not induce any exocytotic events under the ACh-unstimulated condition, cGMP did not stimulate either [Ca.sup.2+] mobilization or cAMP accumulation. The [Ca.sup.2+] dose-response studies demonstrated that cGMP shifted the dose-response curve upward with no shift to the lower concentration. This indicates that cGMP increased maximal responsiveness of the [Ca.sup.2+]-regulated exocytosis, but not the [Ca.sup.2+] sensitivity. Moreover, under a condition of ATP depletion by dinitrophenol (DNP) or anoxia ([N.sub.2] bubbling), ACh evoked only a sustained phase in exocytotic events with no initial transient phase. However, ACh evoked an initial transient phase followed by a sustained phase with addition of cGMP before ATP depletion, whereas only a sustained phase was evoked in a case of cGMP addition after ATP depletion. Thus cGMP-induced enhancement in ACh-stimulated exocytotic events requires ATP, suggesting that cGMP modulates ATP-dependent priming of [Ca.sup.2+]-regulated exocytosis in antral mucous cells. In conclusion, cGMP increases the number of primed granules via acceleration of the ATP-dependent priming, which enhances the [Ca.sup.2+]-regulated exocytosis stimulated by ACh. gastric mucin secretion; guanosine 3'-cyclic monophosphate; exocytosis; acetylcholine; intracellular calcium concentration

The effects of indomethacin (IDM) and aspirin (ASA) on ACh (10 [micro]M) -stimulated exocytotic events were studied in guinea pig antral mucous cells by using video optical microscopy. IDM or ASA, which inhibits cyclooxygenase (COX), decreased the frequency of ACh-stimulated exocytotic events by 30% or 60%, respectively. The extent of inhibition induced by ASA (60%) decreased by 30% when IDM or arachidonic acid (AA, the substrate of COX) was added. IDM, unlike ASA, appears to induce the accumulation of AA, which enhances the frequency of ACh-stimulated exocytotic events in ASA-treated cells. ONO-8713 (100 [micro]M; an inhibitor of the EP1-EP4 prostaglandin receptors) and N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, HCl (H-89, 20 [micro]M; an inhibitor of PKA) also decreased the frequency of ACh-stimulated exocytotic events by 60%. However, the supplementation of PGE2 (1 [micro]M) prevented the 1DM-induced decrease in the frequency of ACh-stimulated exocytotic events. SC-560 (an inhibitor of COX-I) decreased the frequency of ACh-stimulated exocytotic events by 30%, but NS-398 (an inhibitor of COX-2) did not. Moreover, IDM decreased the frequency of exocytotic events stimulated by ionomycin, suggesting that COX-1 activity is stimulated by an increase in intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]). ACh and ionomycin increased PG[E.sub.2] release in antral mucosal cells. In conclusion, in ACh-stimulated antral mucous cells, an increase in [[[Ca.sup.2+]].sub.i] activates [Ca.sup.2+]-regulated exocytotic events and PG[E.sub.2] release mediated by COX-I. The released PG[E.sub.2] induces the accumulation of cAMP, which enhances the [Ca.sup.2+]-regulated exocytosis. The autocrine mechanism mediated by PG[E.sub.2] maintains the high-level mucin release from antral mucous cells during ACh stimulation. gastric mucin secretion; cyclic adenosine 5'-monophosphate; cyclooxygenase-1; indomethacin; aspirin

The effects of intracellular Cl- concentration ([Cl-]i) on acetylcholine (ACh)-stimulated exocytosis were studied in guinea pig antral mucous cells by video microscopy. ACh activated Ca2+-regulated exocytosis (an initial phase followed by a sustained phase). Bumetanide (20 μM) or a Cl- -free (NO3-) solution enhanced it; in contrast, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, a Cl- channel blocker) decreased it and eliminated the enhancement induced by bumetanide or NO3- solution. ACh and Ca2+ dose-response studies demonstrated that NO3- solution does not shift their dose-response curves, and ATP depletion studies by dinitrophenol or anoxia demonstrated that exposure of NO3- solution prior to ATP depletion induced an enhanced initial phase followed by a sustained phase, whereas exposure of NO3- solution after ATP depletion induced only a sustained phase. Intra-cellular Ca2+ concentration ([Ca2+]i) measurements showed that bumetanide and NO3- solution enhanced the ACh-stimulated [Ca2+]i increase. Measurements of [Cl-]i revealed that ACh decreases [Cl-]i and that bumetanide and NO3- solution decreased [Cl-]i and enhanced the ACh-evoked [Cl-]i decrease; in contrast, NPPB increased [Cl-]i and inhibited the [Cl-]i decrease induced by ACh, bumetanide, or NO3- solution. These suggest that [Cl-]i modulates [Ca2+]i increase and ATP-dependent priming. In conclusion, a decrease in [Cl-]i accelerates ATP-dependent priming and [Ca2+]i increase, which enhance Ca2+-regulated exocytosis in ACh-stimulated antral mucous cells. [ABSTRACT FROM AUTHOR]

Prostaglandin E2 (PGE2), which is generated by two isoforms of cyclo-oxygenase (COX1 and COX2), is a key mediator in gastric mucosal defense. In the present study, antral mucosa of guinea-pigs was incubated with various agonists or antagonists in a medium, the PGE2 concentration of which was measured using a PGE2 EIA kit. Prostaglandin E2 was released from the antral mucosa spontaneously (basal PGE2 release) and acetylcholine (ACh, 10 μm) enhanced the PGE2 release (ACh-stimulated PGE2 release) was mediated via intracellular Ca2+ concentration ([Ca2+]i). Arachidonic acid enhanced both forms of PGE2 release, and a phospholipase A2 inhibitor (amylcinnamoyl anthranilic acid) and COX inhibitors (acetylsalicylic acid and indomethacin) decreased them. 5-(4-Chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethylpyrazol (SC560, 100 nm, a COX1-selective inhibitor) inhibited ACh-stimulated PGE2 release without any decrease in basal PGE2 release. N-(2-Cyclohexyloxy-4-nitrophenyl) methanesulphonamide (NS398, 20 μm, a COX2-selective inhibitor) decreased basal PGE2 release without any reduction of ACh-stimulated PGE2 release. However, ionomycin (a Ca2+ ionophore) increased PGE2 release from antral mucosa in the presence of SC560 or NS398, suggesting that COX1 and COX2 are regulated by [Ca2+]i. These findings indicate that COX1-containing cells have ACh receptors but COX2-containing cells do not. Moreover, in isolated antral epithelial cells, SC560 decreased basal and ACh-stimulated PGE2 release, but NS398 did not. In conclusion, in antral mucosa, basal PGE2 release is mainly maintained by COX2 of non-epithelial cells, and ACh-stimulated PGE2 release is maintained by COX1 of epithelial cells. [ABSTRACT FROM AUTHOR]

Ca2+-regulated exocytosis is enhanced by an autocrine mechanism via the PGE2–cAMP pathway in antral mucous cells of guinea-pigs. The inhibition of the PGE2–cAMP pathway by H-89 (an inhibitor of protein kinase A, PKA) or aspirin (ASA, an inhibitor of cyclo-oxygenase, COX) decreased the frequency of ACh-stimulated exocytotic events by 60%. Indomethacin (IDM, an inhibitor of COX), however, decreased the frequency of ACh-stimulated exocytotic events only by 30%. Moreover, IDM increased the frequency of ACh-stimulated exocytotic events by 50% in H-89-treated or ASA-treated cells. IDM inhibits the synthesis of Prostaglandin (PGG/H) and (15R)-15-hydroxy-5,8,11 cis-13-trans-eicosatetraenoic acid (15R-HPETE), while ASA inhibits only the synthesis of PGG/H. Thus, IDM may accumulate arachidonic acid (AA). AACOCF3 or N-( p-amylcinnamoyl) anthranilic acid (ACA; both inhibitors of phospholipase A2, PLA2), which inhibits AA synthesis, decreased the frequency of ACh-stimulated exocytotic events by 60%. IDM, however, did not increase the frequency in AACOCF3-treated cells. AA increased the frequency of ACh-stimulated exocytotic events in AACOCF3- or ASA-treated cells, similar to IDM in ASA- and H-89-treated cells. Moreover, in the presence of AA, IDM did not increase the frequency of ACh-stimulated exocytotic events in ASA-treated cells. The PGE2 release from antral mucosa indicates that inhibition of PLA2 by ACA inhibits the AA accumulation in unstimulated and ACh-stimulated antral mucosa. The dose–response study of AA and IDM demonstrated that the concentration of intracellular AA accumulated by IDM is less than 100 nm. In conclusion, IDM modulates the ACh-stimulated exocytosis via AA accumulation in antral mucous cells. [ABSTRACT FROM AUTHOR]

Terbutaline (10 µM) induced a triphasic volume change in alveolar type II (AT-II) cells: an initial shrinkage (initial phase) followed by cell swelling (second phase) and a gradual shrinkage (third phase). The present study demonstrated that the initial and the third phases are evoked by the activation of K+ and Cl- channels and the second phase is evoked by the activation of Na+ and Cl- channels. Ouabain blocked the third phase, although it did not block the initial and second phases. This suggests that the third phase is triggered by the Na+-K+ pump. Tetraethylammonium (TEA, a K+terbutaline-stimulated third phase, although quinidine, another K+ channel blocker, increased the volume of AT-II cells. The TEA-induced cell shrinkage was inhibited by ouabain, suggesting that TEA increases Na+-K+ pump activity. Ba2+, 2,3-diaminopyridine and a high [K+] ]o] (30 mM) similarly decreased the volume of AT-II cells. These findings suggest that depolarization induced by TEA increases Na+-K+ pump activity, which increases [K+]i. This [K+]i increase, in turn, hyperpolarizes membrane potential. Valinomycin (a K+ ionophore), which induces hyperpolarization, decreased the volume of AT-II cells and enhanced the third phase in these cells. In conclusion, in terbutaline-stimulated AT-II cells, an increase in Na+-K+ pump activity hyperpolarizes the membrane potential and triggers the third phase by switching net ion transport from NaCl entry to KCl release. [ABSTRACT FROM AUTHOR]

Background: Elderly people frequently have symptoms of fullness and appetite loss due to impaired gastric motor activity. These symptoms may cause malnutrition, immunosuppression and other complications.Objective: The effects of aging and daily activity on gastric motility in the elderly were investigated by electrogastrography and the (13)C-acetate breath test.Methods: We enrolled seven active elderly subjects (active elderly group), seven elderly subjects staying at a geriatric facility who had reduced mental and physical capacities (inactive elderly group) and seven healthy young volunteers (young group). Electrogastrography was recorded before and after ingestion of a (13)C-acetate-mixed liquid meal. Expired air was sampled every 10 min after the meal to measure the (13)CO(2) concentration.Results: The ratio of the incidence of the 3-cpm wave (gastric intrinsic frequency) during the postprandial period compared to the fasting state was reduced in both elderly groups compared to young subjects, and the reduction was greater in the inactive elderly than in the active elderly group. The ratio of the amplitude of the peak frequency during the postprandial period to that in the fasting state (power ratio) was also lower in the elderly groups. The time of peak (13)CO(2) expiration was delayed in the active elderly and more so in the inactive elderly group.Conclusions: Postprandial peristalsis and gastric contractile force are reduced in the elderly, and gastric emptying is delayed indicating a reduction in gastric motor activity. [ABSTRACT FROM AUTHOR]

Focuses on a study on the cAMP modulation of calcium&sup2+;-regulated exocytosis in ACh-stimulated antral mucous cells of guinea pig. Materials and methods; Results; Discussion.

OBJECTIVES:We investigated which neutrophil-derived proteins in whole gut lavage fluid (WGLF) most accurately reflect disease activity in inflammatory bowel disease.METHODS:WGLF was obtained from patients undergoing whole gut lavage as a bowel preparation for colonoscopy. Twenty-seven patients with ulcerative colitis (UC), 23 patients with Crohn’s disease (CD), and 35 control subjects were examined. The concentrations of lactoferrin, polymorphonuclear neutrophil elastase (PMN-E), myeloperoxidase, and lysozyme in WGLF were measured by ELISA. For the assessment of stability, WGLF samples were stored at 37°C for various periods.RESULTS:In UC, the concentrations of lactoferrin, myeloperoxidase, and lysozyme in WGLF had good correlations with colonoscopic grading. Zero, 12, five, and 10 of 28 samples from active UC patients showed normal concentrations of lactoferrin, PMN-E, myeloperoxidase, and lysozyme, respectively. In CD, the concentrations of lactoferrin and myeloperoxidase had good correlations with the Crohn’s disease activity index. Thirteen and seven of 36 samples from inactive CD patients (Crohn’s disease activity index ≤ 150) showed high concentrations of lactoferrin and myeloperoxidase, respectively. Most of them (11/13, 6/7) were found to have ulceration by colonoscopy or small bowel x-ray. The ratio of the lactoferrin concentration in the WGLF supernatant to that in total WGLF was highest among these proteins in all disease groups and control subjects. Lactoferrin and myeloperoxidase showed good stability in WGLF, whereas PMN-E and lysozyme did not.CONCLUSION:Lactoferrin is the most suitable of these proteins for use as a neutrophil-derived WGLF marker of intestinal inflammation. [Copyright &y& Elsevier]

Abstract Background and Aim: It has been suggested that dietary fat exacerbates intestinal inflammation. We investigated the effect of fatty acids on interleukin (IL)-8 production in a human intestinal epithelial cell line (Caco-2). Methods: The cells were cultured as monolayers on microporous membranes in culture inserts. Oleic acid (OA), capric acid (CA), docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) were applied to the apical compartment of Caco-2 cell monolayers. The concentration of IL-8 in the basolateral medium was measured by using enzyme-linked immunosorbent assay, and the expression of IL-8 mRNA was measured by using competitive reverse transcription–polymerase chain reaction. Protein kinase C inhibitors (GF109203X and calphostin C) and H-7 (a protein kinase inhibitor) were used to study the mechanisms by which IL-8 production is stimulated. Results: Both OA and CA enhanced IL-8 production (approximately fivefold), whereas DHA and EPA did not. Both OA and CA also enhanced IL-1-induced IL-8 production. The onset of OA-induced IL-8 production was delayed compared with that of CA-induced IL-8 production. Both OA and CA enhanced IL-8 mRNA expression (approximately fivefold) after 6 and 3 h, respectively. The protein kinase inhibitor (H-7) reduced both OA- and CA-induced IL-8 production by 88.0 and 85.9%, respectively. The protein kinase C inhibitors (GF109203X and calphostin C) reduced OA-induced IL-8 production by 29.3 and 54.5%, respectively, but showed no effect on CA-induced IL-8 production. Conclusions: These findings suggest that not only OA but also CA stimulates IL-8 production in intestinal epithelial cells, and the mechanisms of action differ between OA and CA. [ABSTRACT FROM AUTHOR]

In order to investigate the mucosal injury mechanism in UC, we made dextran sulfate sodium (DSS)-induced colitis in rat and examined pathological findings, MPO activity, PGE[sub 2] level, and local mRNA expression and secretion of IL-1β, TNF-α, GRO/CINC-1 and IL-10 in DSS colitis mucosa. Moreover, we estimated the correlation between the severity of mucosal damage and changes of these local inflammatory mediators’ values. Neutrophil infiltration was marked and MPO activity was locally increased in proportion to the severity of mucosal damage. The mRNA expression and secretion of IL-1β, GRO/CINC-1 and IL-10 were increased. Especially, the secretions of IL-1β and GRO/CINC-1 were increased in proportion to the severity of mucosal damage. However, those of TNF-α were not increased in the colitis mucosa. An abnormal macrophage function and the presence of macrophage subtypes producing different cytokines would be predicted from our TNF-α data. The lesion was less severe in the colonic mucosa with higher levels of endogenous PGE[sub 2] , while it was more severe in the colonic mucosa with lower levels of endogenous PGE[sub 2] , implicating this compound as an inhibitory factor against the development of inflammation in the affected mucosa. Our results suggest that PGE[sub 2] might have therapeutic applicability to UC.Copyright © 2001 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: To clarify the similarities and dissimilarities in the macroscopic classification criteria for early colorectal carcinoma (CRC) between Japan and China. Methods: Six early CRC cases were included in this study. Eleven Japanese and Chinese endoscopists were asked to review the colonoscopic pictures of these cases, including before and after indigocarmine spraying. After viewing the pictures, all the endoscopists individually made their classificatory diagnoses of these cases and indicated the findings on which they based each diagnosis. Results: Some lesions diagnosed by Japanese endoscopists as IIa or IIa + IIc, might be classified as Is or Isp by Chinese endoscopists. For superficial lesions consisting of elevation with central depression, IIa + depression, IIa + IIc or IIc + IIa were classified according to the ratio of elevated area/depressed area. However, international as well as interobserver differences still existed in the classification of such lesions. In addition, most Chinese endoscopists overlooked the slightly depressed part on the top of a protruded lesion. Conclusion: Discrepancies on macroscopic classification for early CRC do exist between Japanese and Chinese endoscopists, which were found not only in terminology, but also in recognition of some lesions. In order to develop a universal macroscopic classification, there is a great need for international communication and cooperation. [ABSTRACT FROM AUTHOR]

To investigate the development and progression of colorectal carcinoma, submucosal invasive carcinoma (SMC) with residual intramucosal neoplasm was studied histopathologically. Intramucosal neoplasm was confirmed by immunohistochemical staining against anti-α-smooth muscle actin antibody. Submucosal invasive carcinoma was classified into polypoid growth-type carcinoma (PGC) and non-polypoid growth-type carcinoma (NPGC), depending on the presence of intramucosal tumor proliferation. Tumors were > 15 mm in size in 78.2% of the PGC lesions studied, but the degree of submucosal invasion was minimal (invasion of the upper 500 μm of the submucosal layer) in 52.9% of the PGC lesions. Conversely, 64.4% of NPGC lesions were &figfua; 15 mm in size and the degree of submucosal invasion was moderate or severe (involving the middle and deeper layer of the submucosa) for 72.9% of NPGC. In other words, lesions of NPGC were significantly smaller in size but showed deeper infiltration than PGC lesions. Furthermore, PGC was derived from intramucosal polypoid carcinoma (including carcinoma with adenoma) and was morphologically identical to polyp cancer as reported previously. However, NPGC was derived from the flat and/or depressed type of intramucosal carcinoma classified not as polyp type, but as the superficial type. Typical NPGC was, therefore, also of the superficial type. In addition, approximately 25% of PGC lesions were identified as having an adenoma–carcinoma sequence. There was no coexistence with adenoma in the NPGC lesions, suggesting de novo development. When the degree of histologic atypia in the two types of intramucosal carcinoma was compared, 74.7% of PGC lesions showed low-grade carcinoma, regardless of tumor size, while 62.7% of NPGC lesions showed high-grade carcinoma in the intramucosal lesion. Approximately 25% of carcinomas with low-grade atypia were positive for p53 (as were the high-grade lesions), but it was not expressed in the adenoma.... [ABSTRACT FROM AUTHOR]