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Adaptive gastric cytoprotection is mediated by prostaglandin EP1 receptors: A study using rats and knockout mice
723 Role of ECL-Cell Histamine in Acute Gastric Mucosal Damage Induced by ischemiaReperfusion. Masayuki Kitano, Dept of Gastroenteroland Hepatol, Kinki Univ, Oeaka-SayamaJapan; Yosuke Kishimoto, Dept of Clin Pharmacol, Tottori Univ, Yonago Japan; Roll Hakanson, Dept of Pharmacol, Univ of Lund, Lund Sweden; Yoko Haenuki, Satoshi Onoda, Dept of Clin Pharmacol, Tottori Univ, Yonago Japan; Masatoshi Kudo, Dept of Gastroenteroland Hepatol, Kinki Univ, Osaka-SayamaJapan; Junichi Hasegawa,Oept of Clin Pharmacol, Kinki Univ, Yonago Japan BACKGROUND:ECL cells are histamine-forming endocrine ceils in the oxyntic mucosa. There are no reports to monitor mobilization of ECL-cell histamine in any models of gastric ulcer. Temporal clamping of the celiac artery (ischemia-reperfusion) is accompanied by gastric mucosal damage. Using microdialysis probes implanted in the gastric submucosa, we have studied the role of ECL-cell histamine in acute gastric mucosal damageinduced by ischemiareperfusion. METHOD:Wistar rats and Ws/Ws rats were used. Ws/Ws rats with a muta~on of c-kit receptors are completely deficient in mast cells. Both types of rats were fasted for 18 hours prior to the experiments,but were allowed free accessto water. Under pentobarbital anesthesia, a flexible microdialysis probe was inserted into the submecosal layer of the acid producing part of the stomach. The inlet tube was connectedto a microinfusion pump and the outlet was allowed to drain into polystyrenevials. The microdialysis probes were perfused with degassedsaline (1.2 p.I/min). Samplesfor histamine measurementwere collected every 10 min. After collecting basal fractions, the celiac artery was occludedfor 30 rain (ischemla) followed by removal of the clamp (repertusion). The total area of erosions was measured 60 min after removal of the clamp. Some rats were pretreatedwith ~-fluoromethylhistidine (aFMH), histamine H1 antagonist or H2antagonist. ~-FMH is known to depletethe ECL cells of histamine without affecting mast-cell histamine. Ws/Ws rats were compared with wild type rats. Histamine was measured by ELISA. RESULTS: Immediately after the ischemia, the microdialysatehistamine startedto rise in Wister rats. After 30 rain of ischemla,the micrndialysate histamine increased50-fold. The histamine H1 and H2 receptor antagonists dose-dependently reduced the total area of erosions induced by ischemia-reperfusion without affecting the increase of microdialysate histamine. Ischemia also raised the micrndlalysate histamine 7g-fold in Ws/Ws rats. There was no significant difference betweenWs/Ws rats and wild type rats with respect to the total area of erosions. In both types of rats, ~-FMH prevented the rise in microdialysatehistamineand protectedthe gastric mucosafrom damage.CONCLUSION: The histamine mobilized during ischemia seems to derive from ECL cells rather than mast cells and plays a pivotal role in the pathogenesisof gastric mucocal damage induced by ischemia-reperfusion.
724 Adaptive Gastric Cytoprofeetion Is Mediated by Prostaglandle EP1 Receptors: A Study Using Rats and Knockout Mice Koji Takeuchi, Shinichi Kato, Hideo Araki, Yusaku Komoike, Masamori Takesda, Kyoto Pharm Univ, Kyoto Japan Endogenous prostaglandins(PG) play a central role in adaptivecytoprotection induced in the stomach by mild irritants, yet the EP-receptorsubtype involved in this phenomenon remains unexplored. In the present study, we used taurocholate (TC) as a mild irritant in both rats and EP-receptorknockout mice, and examinedwhich EP receptor subtype is responsiblefor the adaptivegastric cytoprotection. Methods: Male SD rats and C57BL/6 mice, both wild-type and knockout one lacking EP1 or EP3-receptors,were used after 18 h fasting. Gastric lesions were induced by PO administration of HCI/ethanol (60% ethanol in 150 mM HCI), and the animals were killed 1 h later. PGE2(O.3mg/kg) or TC (5-20 mM) was administered PO 30 min before HCI/ethanol. Results: HCI/ethanol-inducedgastric lesions were dose-dependently prevented by TC, and the effect at 20 mM was equivalentto that induced by PGE2at0.03 mg/ kg. The protective effect of TC was significantly attenuated by indomethacin as well as ONOAE-829 the EP1 antagonist, but not affected by either NS-398 the selective COX-2 inhibitor or chemical ablation of capsaicin-sensitivesensory neurons, Likewise, the protective action of PGE2againstHCI/ethanol-induceddamage was also antagonized by ONO-AE-829 but not chemical deafferentation.TC significantly increasedthe mucocal PGE2contentsin the stomach, with or without chemical deafferentation, and this effect was blocked in the presence of indomethacin but not NS-398 or ONO-AE-829. In addition, TC also increased the mucosal PGE2contentssimilarly in both wild-type and knockout mice lacking EP1- or EP3-receptors, yet the protective action of TC against HCVethanolwas observed in both wild-type and EP3 receptor knockout mice, but not in mice lacking EPl-receptors. On the other hand,the adaptive cytoprotection induced by TC was similarly observed in iP-receptor knockout mice, whereas the capsaicinprotection was totally attenuatedin the animals lacking IP receptors.Conclusion: The presentfindings confirmed a critical role for endogenousPGE2produced mainly by COX1 in adaptive gastric cytoprotection and suggestedthat this action is mediated by activation of EPl-receptors but not associatedwith capsaicin-sensifiveafferent neurons. This study also suggests the involvement of IP-receptor in gastric protection induced by capsaicin.
725 The Role Of Apoptosis And Cytokines In Gastric Preconditioning Tomasz Brzozowski, Dept of Physiology, Univ Sch of Medicine, Cracow Poland; Peter C. Konturek, Dept of Medicine, Univ of Erlangen-Nuremberg,Eitangen Germany; Zbigniew Sliwowski, Malgorzata Mitis-Musiol, Robert Pajdo, Slawomir Kwiecien, Dept of Physiology, Univ Sch of Medicine, Cracow Poland; Eckhart G. Hahn, Dept of Medicine, Univ of Erlangen-Nuremberg, Erlangen Germany; Stanislaw J. Konturek, Dept of Physiology, Univ Sch of Medicine, Cracow Poland Background:Gastricpreconditioning(GPC)refersto an increasedresistanceof gastric mucosa which is subjected to repeated brief episodes of ischaemiathat limit the mucosal damage caused by subsequentmore prolonged ischeamic insult. Aim: We havedemonstratedrecently that COX-derivedprostaglandinsand nitric oxide are involved in the mechanismof GPC but the role in this phenomenonof geneticallyprogrammedcell death (apoptosis)andproinflammatory
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cytokines remains unknown. Methods: GPCwas induced in rats by short ischaemia(occlusion of celiac artery twice for 5 rain) applied 30 rain before subsequent exposure to 0.5 h of ischaemia induced by clamping of celiac artery followed by 3-12 h of repertusion (I/R). Some rats were pretreated 30 min before short ischaemia with ICE-1 (10 mg/kg i.p.), a caspase-1 inhibitor or pentoxifilline (PTX 50 mg/kg i.p.), an inhibitor of the production and release of TNF~. The area of gastric lesions was determined by planimetry, gastric blood flow (GBF) was measured by H~-gasclearancetechnique and blood was collected for plasma IL-1/3 and TNF~xlevels. Gastric mucosal sampleswere taken for determinationof IL-1/3 and TNFc~mRNA and Bax and Bcl-2 expressionassessedby transferase-mediateddUPT-biotin nick end-labeling (TUNEL) staining method, RT-PCR and Western blot. Results: Exposure to I/R produced numerous gastric erosions with a maximum at 12 h after reperfusion, being accompaniedby significant fall in the GSF and the rise in plasma IL-t/~ and TNF~ levels. Short ischaemia (5 min occlusion x 2) reduced I/R lesions and restored, in part, the GBF and plasma IL-lp and TNF~zlevels with the extent similar to that achieved in rats pretreated with ICE-1 or PTX. Expression of IL-lp and TNFa mRNA rose significantly up to 12 h after I/R and this was significandy inhibited by ICE-1 and PTX. Bax and Bcl-2 mRNA were undetectablein the intact gastric mucosa but after the end of I/R at 3h and 12h, a significant imbalance between Bax and Bcl-2 (Bax/Bcl-2 ratio>l) was observed. Expressionof Bax mRNA was detected in first 6 h while Bcl-2 mRNA was significantly decreasedup to 12 h after I/R and these effects were significantly attenuatedby GPC.Conclusions:1) I/R-inducedlesions involve an overexpression of inflammatory cytokines,the impairment in the GBFand enhancementin apoptosistriggered from the shift from cell death effector Bax to the cell death repressor Bcl-2 and 2) GPC renders the gastric mucosa more tolerant to prolonged ischaemic insult due to attenuation of apoptosis and suppression of cytokine expression and release.
726 Protein Kimice C Isozymes In TNPa-lnduced Cytotoxicity to Rat Intestinal Epithelial Ceils Oing Chang, Barry L. Tepperman, Univ of Western Ontario, London Canada Background:Tumour necrosisfactor (TNF,~)can inducecytotoxicity and apoptosis in a number of cell types and has been implicated in the regulation of many inflammatory processes. It has been suggested that protein kinase C (PKC) is one of the intracellular mediators of the actions of TNF<=.In the presentstudy, the role of PKCisoforms in TNF,~--mediatedcytotoxicity and apoptosisin rat intestinalepithelialcells was investigated.Methods:The intestinal epithelial cell line, IEC-18 were used in these studies. Cells were incubated with either TNFe or the PKC activator, phorbol myristate acetate(PMA)in the presenceor absenceof the transcription inhibitor, actinomycin D (AMD). Cytoxicity was assessedby a formazan-basedspectrophotometric assay.Apoptosis was determined by fluorescent microscopic examinationfor nuclear condensation and fragmentation as well as determination of DNA fragmentation by ELISA. Results: The extent of cell damage in responseto TNF<~treatment was enhancedwhen AMD was added to the incubation medium suggesting new protein synthesis plays a role in the cytotoxic actions of this cytokine. TNFe also induced the translocation of the PKC-~,8 and • isoforms from cytosol to the membrane fraction of the intestinal cells. Furthermore, the cytotoxic and apoptotic effects of TNF were reduced selectively by pretreating cells with the PKC-e translocation inhibitor, PKC-eV1-2. In contrast, while cells incubated with PMA also displayedan increase in cell injury, the extent of cytotoxicity and apoptosis was not enhanced by AMD. Furthermore. PMA-inducedcell damaoewas reduced by rottlerin, a PKC-~inhibitor. Caspase-3, an enzyme implicated in the mediation of apoptosis, was activated in cells in response to either TNF,,or PMA challenge to the cells and their effects on caspeseactivity were reduced by selective inhibition of PKC-• and 5 respectively. Furthermore, inhibition of caspase-3 activity reduced apoptosis in these cells. Conclusions: TNFa--and PMA-induced apoptosisand cytotoxicity in intestinalepithelial cells may be mediatedby differential regulation of selective PKC isoforms and these processes may occur through a caspase-dependent pathway.
Role of Herpes Simplex Virus Type I (HSV-1) in the Pathogenesis of Peptic Ulcer Disease Klisthenis X. Tsamakides, Saint Sawas Anticancer Hosp, Athens Greece; Evi Panotopoulou, G PapanikolaouResearch Unit, Athens Greece; Dimitrios A. Dimitrouldpoulos, Saint Sawas Anticancer Hosp, Athens Greece; Maria Christopouldu, G PapanikolaouResearch Unit, Athens Greece; Dimitrios Xinopoulos, Saint Sawas Anticancer Hosp, Athens Greece; E Goula, G PapanikolaouResearch Unit, Athens Greece; Marina Kontou, EmmanouU Arcbavlis, Saint Sawas Anticancer Hosp, Athens Greece; Stavros Kottaridis, G Papanikolaou Research Unit, Athens Greece; Emmanuel Paraskevas,Saint Sawas Anticancer Hosp, Athens Greece Aim of the study was the identification of the herpes simplex virus type 1 (HSV-t) from biopsy specimens of patients with active peptic ulcer as well as the investigation of the hypothesisthat HSV-1 may be associatedwith peptic ulcer disease.PATIENTSAND METHODS: 90 patients, 34 with prepyloric and 56 with duodenal ulcer as well as 50 patients with no evidenceof peptic ulcer consideredas a control group, were examined. Biopsies were taken from the crater and the rim of the ulcer, 3 cm away from the ulcer, as well as from endoscopicallyhealthy mucosa area of the patients and control group. Malignancyin patients with prepyloric ulcer was excluded histologically. The methods used for the identification of HSV-1 were polymerasechain reaction (PCR), in situ hybridization analysis, direct isolation and HSV-1 indirect immunofluorescence. Moreover, the detection of the HP was achievedby CLO-testat samplestaken from the antrum of the stomach. We also proceededto the statistical analysis of the results of the experimentaldata, as well as those epidemiologicalparameters, which may be associated with the pathogenesis of peptic ulcer disease. RESULTS: Using PCR method for the presenceof HSV-1, positive results were found in 28 out of 90 patients with peptic ulcer (31%) and specifically in 17 out of 56 patients with duodenal ulcer (30.4%) and in 11 out of 34 patients with prepyloric ulcer (32.4%). Using the isolation method HSV1 was isolated and identified in 8 out of 90 patients with peptic ulcer (8.9%) and more specifically in 7 out of 56 patients with duodenal ulcer (12.5%) and 1 out of 34 patients with prepyloric ulcer (2.9%). Positive samples for HSV-1 were obtained only from the crater or
724 Adaptive Gastric Cytoprofeetion Is Mediated by Prostaglandle EP1 Receptors: A Study Using Rats and Knockout Mice Koji Takeuchi, Shinichi Kato, Hideo Araki, Yusaku Komoike, Masamori Takesda, Kyoto Pharm Univ, Kyoto Japan Endogenous prostaglandins(PG) play a central role in adaptivecytoprotection induced in the stomach by mild irritants, yet the EP-receptorsubtype involved in this phenomenon remains unexplored. In the present study, we used taurocholate (TC) as a mild irritant in both rats and EP-receptorknockout mice, and examinedwhich EP receptor subtype is responsiblefor the adaptivegastric cytoprotection. Methods: Male SD rats and C57BL/6 mice, both wild-type and knockout one lacking EP1 or EP3-receptors,were used after 18 h fasting. Gastric lesions were induced by PO administration of HCI/ethanol (60% ethanol in 150 mM HCI), and the animals were killed 1 h later. PGE2(O.3mg/kg) or TC (5-20 mM) was administered PO 30 min before HCI/ethanol. Results: HCI/ethanol-inducedgastric lesions were dose-dependently prevented by TC, and the effect at 20 mM was equivalentto that induced by PGE2at0.03 mg/ kg. The protective effect of TC was significantly attenuated by indomethacin as well as ONOAE-829 the EP1 antagonist, but not affected by either NS-398 the selective COX-2 inhibitor or chemical ablation of capsaicin-sensitivesensory neurons, Likewise, the protective action of PGE2againstHCI/ethanol-induceddamage was also antagonized by ONO-AE-829 but not chemical deafferentation.TC significantly increasedthe mucocal PGE2contentsin the stomach, with or without chemical deafferentation, and this effect was blocked in the presence of indomethacin but not NS-398 or ONO-AE-829. In addition, TC also increased the mucosal PGE2contentssimilarly in both wild-type and knockout mice lacking EP1- or EP3-receptors, yet the protective action of TC against HCVethanolwas observed in both wild-type and EP3 receptor knockout mice, but not in mice lacking EPl-receptors. On the other hand,the adaptive cytoprotection induced by TC was similarly observed in iP-receptor knockout mice, whereas the capsaicinprotection was totally attenuatedin the animals lacking IP receptors.Conclusion: The presentfindings confirmed a critical role for endogenousPGE2produced mainly by COX1 in adaptive gastric cytoprotection and suggestedthat this action is mediated by activation of EPl-receptors but not associatedwith capsaicin-sensifiveafferent neurons. This study also suggests the involvement of IP-receptor in gastric protection induced by capsaicin.
725 The Role Of Apoptosis And Cytokines In Gastric Preconditioning Tomasz Brzozowski, Dept of Physiology, Univ Sch of Medicine, Cracow Poland; Peter C. Konturek, Dept of Medicine, Univ of Erlangen-Nuremberg,Eitangen Germany; Zbigniew Sliwowski, Malgorzata Mitis-Musiol, Robert Pajdo, Slawomir Kwiecien, Dept of Physiology, Univ Sch of Medicine, Cracow Poland; Eckhart G. Hahn, Dept of Medicine, Univ of Erlangen-Nuremberg, Erlangen Germany; Stanislaw J. Konturek, Dept of Physiology, Univ Sch of Medicine, Cracow Poland Background:Gastricpreconditioning(GPC)refersto an increasedresistanceof gastric mucosa which is subjected to repeated brief episodes of ischaemiathat limit the mucosal damage caused by subsequentmore prolonged ischeamic insult. Aim: We havedemonstratedrecently that COX-derivedprostaglandinsand nitric oxide are involved in the mechanismof GPC but the role in this phenomenonof geneticallyprogrammedcell death (apoptosis)andproinflammatory
A-136
cytokines remains unknown. Methods: GPCwas induced in rats by short ischaemia(occlusion of celiac artery twice for 5 rain) applied 30 rain before subsequent exposure to 0.5 h of ischaemia induced by clamping of celiac artery followed by 3-12 h of repertusion (I/R). Some rats were pretreated 30 min before short ischaemia with ICE-1 (10 mg/kg i.p.), a caspase-1 inhibitor or pentoxifilline (PTX 50 mg/kg i.p.), an inhibitor of the production and release of TNF~. The area of gastric lesions was determined by planimetry, gastric blood flow (GBF) was measured by H~-gasclearancetechnique and blood was collected for plasma IL-1/3 and TNF~xlevels. Gastric mucosal sampleswere taken for determinationof IL-1/3 and TNFc~mRNA and Bax and Bcl-2 expressionassessedby transferase-mediateddUPT-biotin nick end-labeling (TUNEL) staining method, RT-PCR and Western blot. Results: Exposure to I/R produced numerous gastric erosions with a maximum at 12 h after reperfusion, being accompaniedby significant fall in the GSF and the rise in plasma IL-t/~ and TNF~ levels. Short ischaemia (5 min occlusion x 2) reduced I/R lesions and restored, in part, the GBF and plasma IL-lp and TNF~zlevels with the extent similar to that achieved in rats pretreated with ICE-1 or PTX. Expression of IL-lp and TNFa mRNA rose significantly up to 12 h after I/R and this was significandy inhibited by ICE-1 and PTX. Bax and Bcl-2 mRNA were undetectablein the intact gastric mucosa but after the end of I/R at 3h and 12h, a significant imbalance between Bax and Bcl-2 (Bax/Bcl-2 ratio>l) was observed. Expressionof Bax mRNA was detected in first 6 h while Bcl-2 mRNA was significantly decreasedup to 12 h after I/R and these effects were significantly attenuatedby GPC.Conclusions:1) I/R-inducedlesions involve an overexpression of inflammatory cytokines,the impairment in the GBFand enhancementin apoptosistriggered from the shift from cell death effector Bax to the cell death repressor Bcl-2 and 2) GPC renders the gastric mucosa more tolerant to prolonged ischaemic insult due to attenuation of apoptosis and suppression of cytokine expression and release.
726 Protein Kimice C Isozymes In TNPa-lnduced Cytotoxicity to Rat Intestinal Epithelial Ceils Oing Chang, Barry L. Tepperman, Univ of Western Ontario, London Canada Background:Tumour necrosisfactor (TNF,~)can inducecytotoxicity and apoptosis in a number of cell types and has been implicated in the regulation of many inflammatory processes. It has been suggested that protein kinase C (PKC) is one of the intracellular mediators of the actions of TNF<=.In the presentstudy, the role of PKCisoforms in TNF,~--mediatedcytotoxicity and apoptosisin rat intestinalepithelialcells was investigated.Methods:The intestinal epithelial cell line, IEC-18 were used in these studies. Cells were incubated with either TNFe or the PKC activator, phorbol myristate acetate(PMA)in the presenceor absenceof the transcription inhibitor, actinomycin D (AMD). Cytoxicity was assessedby a formazan-basedspectrophotometric assay.Apoptosis was determined by fluorescent microscopic examinationfor nuclear condensation and fragmentation as well as determination of DNA fragmentation by ELISA. Results: The extent of cell damage in responseto TNF<~treatment was enhancedwhen AMD was added to the incubation medium suggesting new protein synthesis plays a role in the cytotoxic actions of this cytokine. TNFe also induced the translocation of the PKC-~,8 and • isoforms from cytosol to the membrane fraction of the intestinal cells. Furthermore, the cytotoxic and apoptotic effects of TNF were reduced selectively by pretreating cells with the PKC-e translocation inhibitor, PKC-eV1-2. In contrast, while cells incubated with PMA also displayedan increase in cell injury, the extent of cytotoxicity and apoptosis was not enhanced by AMD. Furthermore. PMA-inducedcell damaoewas reduced by rottlerin, a PKC-~inhibitor. Caspase-3, an enzyme implicated in the mediation of apoptosis, was activated in cells in response to either TNF,,or PMA challenge to the cells and their effects on caspeseactivity were reduced by selective inhibition of PKC-• and 5 respectively. Furthermore, inhibition of caspase-3 activity reduced apoptosis in these cells. Conclusions: TNFa--and PMA-induced apoptosisand cytotoxicity in intestinalepithelial cells may be mediatedby differential regulation of selective PKC isoforms and these processes may occur through a caspase-dependent pathway.
Role of Herpes Simplex Virus Type I (HSV-1) in the Pathogenesis of Peptic Ulcer Disease Klisthenis X. Tsamakides, Saint Sawas Anticancer Hosp, Athens Greece; Evi Panotopoulou, G PapanikolaouResearch Unit, Athens Greece; Dimitrios A. Dimitrouldpoulos, Saint Sawas Anticancer Hosp, Athens Greece; Maria Christopouldu, G PapanikolaouResearch Unit, Athens Greece; Dimitrios Xinopoulos, Saint Sawas Anticancer Hosp, Athens Greece; E Goula, G PapanikolaouResearch Unit, Athens Greece; Marina Kontou, EmmanouU Arcbavlis, Saint Sawas Anticancer Hosp, Athens Greece; Stavros Kottaridis, G Papanikolaou Research Unit, Athens Greece; Emmanuel Paraskevas,Saint Sawas Anticancer Hosp, Athens Greece Aim of the study was the identification of the herpes simplex virus type 1 (HSV-t) from biopsy specimens of patients with active peptic ulcer as well as the investigation of the hypothesisthat HSV-1 may be associatedwith peptic ulcer disease.PATIENTSAND METHODS: 90 patients, 34 with prepyloric and 56 with duodenal ulcer as well as 50 patients with no evidenceof peptic ulcer consideredas a control group, were examined. Biopsies were taken from the crater and the rim of the ulcer, 3 cm away from the ulcer, as well as from endoscopicallyhealthy mucosa area of the patients and control group. Malignancyin patients with prepyloric ulcer was excluded histologically. The methods used for the identification of HSV-1 were polymerasechain reaction (PCR), in situ hybridization analysis, direct isolation and HSV-1 indirect immunofluorescence. Moreover, the detection of the HP was achievedby CLO-testat samplestaken from the antrum of the stomach. We also proceededto the statistical analysis of the results of the experimentaldata, as well as those epidemiologicalparameters, which may be associated with the pathogenesis of peptic ulcer disease. RESULTS: Using PCR method for the presenceof HSV-1, positive results were found in 28 out of 90 patients with peptic ulcer (31%) and specifically in 17 out of 56 patients with duodenal ulcer (30.4%) and in 11 out of 34 patients with prepyloric ulcer (32.4%). Using the isolation method HSV1 was isolated and identified in 8 out of 90 patients with peptic ulcer (8.9%) and more specifically in 7 out of 56 patients with duodenal ulcer (12.5%) and 1 out of 34 patients with prepyloric ulcer (2.9%). Positive samples for HSV-1 were obtained only from the crater or