Protein kinase C isozymes in TNFα-induced cytotoxicity to rat intestinal epithelial cells

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    Role of ECL-Cell Histamine in Acute Gastric Mucosal Damage Induced by ischemia- Reperfusion. Masayuki Kitano, Dept of Gastroenterol and Hepatol, Kinki Univ, Oeaka-Sayama Japan; 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 Gastroenterol and Hepatol, Kinki Univ, Osaka-Sayama Japan; 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 damage induced by ischemia- reperfusion. 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 access to 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 connected to a microinfusion pump and the outlet was allowed to drain into polystyrene vials. The microdialysis probes were perfused with degassed saline (1.2 p.I/min). Samples for histamine measurement were collected every 10 min. After collecting basal fractions, the celiac artery was occluded for 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 pretreated with ~-fluoromethylhistidine (a- FMH), histamine H1 antagonist or H2 antagonist. ~-FMH is known to deplete the 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 microdialysate histamine started to rise in Wister rats. After 30 rain of ischemla, the micrndialy- sate histamine increased 50-fold. The histamine H1 and H2 receptor antagonists dose-depen- dently 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 between Ws/Ws rats and wild type rats with respect to the total area of erosions. In both types of rats, ~-FMH prevented the rise in microdialysate histamine and protected the gastric mucosa from damage. CONCLUSION: The histamine mobilized during ischemia seems to derive from ECL cells rather than mast cells and plays a pivotal role in the pathogenesis of gastric mucocal damage induced by ischemia-reperfusion.


    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 adaptive cytoprotection induced in the stomach by mild irritants, yet the EP-receptor subtype involved in this phenomenon remains unexplored. In the present study, we used taurocholate (TC) as a mild irritant in both rats and EP-receptor knockout mice, and examined which EP receptor subtype is responsible for the adaptive gastric 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.3 mg/kg) or TC (5-20 mM) was administered PO 30 min before HCI/ethanol. Results: HCI/ethanol-induced gastric lesions were dose-dependently prevented by TC, and the effect at 20 mM was equivalent to that induced by PGE2at 0.03 mg/ kg. The protective effect of TC was significantly attenuated by indomethacin as well as ONO- AE-829 the EP1 antagonist, but not affected by either NS-398 the selective COX-2 inhibitor or chemical ablation of capsaicin-sensitive sensory neurons, Likewise, the protective action of PGE2against HCI/ethanol-induced damage was also antagonized by ONO-AE-829 but not chemical deafferentation. TC significantly increased the mucocal PGE2contents in 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 PGE2contents similarly in both wild-type and knockout mice lacking EP1- or EP3-receptors, yet the protective action of TC against HCVethanol was 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 capsaicin protection was totally attenuated in the animals lacking IP receptors. Conclusion: The present findings confirmed a critical role for endogenous PGE2 produced mainly by COX- 1 in adaptive gastric cytoprotection and suggested that this action is mediated by activation of EPl-receptors but not associated with capsaicin-sensifive afferent neurons. This study also suggests the involvement of IP-receptor in gastric protection induced by capsaicin.


    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: Gastric preconditioning (GPC) refers to an increased resistance of gastric mucosa which is subjected to repeated brief episodes of ischaemia that limit the mucosal damage caused by subsequent more prolonged ischeamic insult. Aim: We have demonstrated recently that COX-derived prostaglandins and nitric oxide are involved in the mechanism of GPC but the role in this phenomenon of genetically programmed cell death (apoptosis)and proinflammatory

    cytokines remains unknown. Methods: GPC was 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~-gas clearance technique and blood was collected for plasma IL-1/3 and TNF~x levels. Gastric mucosal samples were taken for determination of IL-1/3 and TNFc~ mRNA and Bax and Bcl-2 expression assessed by transferase-mediated dUPT-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 accompanied by 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~z levels 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 undetectable in 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. Expression of Bax mRNA was detected in first 6 h while Bcl-2 mRNA was significantly decreased up to 12 h after I/R and these effects were significantly attenuated by GPC. Conclusions: 1) I/R-induced lesions involve an overexpression of inflammatory cytokines, the impairment in the GBF and enhancement in apoptosis triggered 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.


    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 necrosis factor (TNF,~) can induce cytotoxicity 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