Possible involvement of host defense mechanism in the suppression of rat acute reflux esophagitis by the particular histamine H2 receptor antagonist lafutidine.

AIMS: Gastroesophageal reflux disease is considered to be caused primarily by gastric juice refluxed into the esophagus. Here, we investigated the possible involvement of host defense mechanisms in the development of acute reflux esophagitis using lafutidine, a histamine H(2) receptor antagonist (H(2)RA) with proven gastric mucosal protective effects. METHODS AND RESULTS: The ligation of both the pylorus and the forestomach of SD rats under anesthesia caused hemorrhagic lesions in the esophageal mucosa at 6 h. Lesion formation was significantly inhibited by treatment with H(2)RAs, including the conventional H(2)RAs famotidine and cimetidine as well as lafutidine. The maximum suppressive abilities of these agents were similar to that of the proton pump inhibitor lansoprazole. Interestingly, unlike famotidine, lafutidine at low doses significantly suppressed esophagitis without inhibiting gastric acid secretion. Note that neither lafutidine nor famotidine inhibited hexosamine output in gastric juice samples obtained 3 h after ligation. Additionally, the protective effect of lafutidine, but not of famotidine, was partly attenuated by the denervation of capsaicin-sensitive afferent nerves with a large dose of capsaicin. CONCLUSION: The present results indicate that esophageal host-defense via capsaicin-sensitive afferent nerves may contribute to the therapeutic action of lafutidine.

Lafutidine-induced increase in intracellular ca(2+) concentrations in PC12 and endothelial cells.

Lafutidine, a histamine H(2) receptor antagonist, exerts gastroprotective effects in addition to gastric antisecretory activity. The gastrointestinal protective effects of lafutidine are mediated by capsaicin-sensitive neurons, where capsaicin excites neurons by opening a member of the transient receptor potential channel family (TRPV1). Since the effect of lafutidine on the intracellular Ca(2+) concentration ([Ca(2+)](i)) in cells has not been elucidated, we investigated the lafutidine response to [Ca(2+)](i) in rat pheochromocytoma PC12 and human endothelial cells. Lafutidine at pharmacological concentrations greater than 1 mM induced a sustained increase in [Ca(2+)](i) in the presence of extracellular CaCl(2) in PC12 cells, while capsaicin showed dual effects on [Ca(2+)](i) in PC12 cells, where it activated TRPV1 and inhibited store-operated Ca(2+) entry. The thapsigargin (an activator of store-operated Ca(2+) entry)-induced increase in [Ca(2+)](i) in PC12 cells was inhibited by capsaicin and SKF96365, an inhibitor of store-operated Ca(2+) entry, and the lafutidine response was inhibited by capsaicin but not by SKF96365. In endothelial cells, lafutidine induced an increase in [Ca(2+)](i) in a SKF96365-insensitive manner. These results suggest that lafutidine stimulates Ca(2+) entry via the capsaicin-sensitive pathway but not the SKF96365-sensitive pathway. The possible role of store-operated Ca(2+) entry induced by lafutidine on gastrointestinal function is also discussed.

Lafutidine inhibits Helicobacter pylori-induced interleukin-8 production in human gastric epithelial cells.

BACKGROUND AND AIM: Attachment of Helicobacter pylori to gastric epithelial cells leads to the production of chemokines, such as interleukin-8 (IL-8), which in turn activate and recruit neutrophils to the site of infection. Lafutidine [(+/-)-2-(furfurylsulfinyl)-N-(4-(4-(piperidinomethyl)-2-pyridyl)oxy-(Z)-2-butenyl)acetamide] is a new type of antiulcer drug that possesses an antisecretory action as well as gastroprotective activity, independent of its antisecretory action. In the present study, we examined the effects of lafutidine on H. pylori-induced IL-8 release and H. pylori adhesion to MKN45 cells. METHODS: MKN45 cells were stimulated with H. pylori, tumor necrosis factor (TNF)-alpha, or IL-1beta, then IL-6 and IL-8 levels in the culture supernatants were determined with a specific enzyme-linked immunosorbent assay kit. RESULTS: Lafutidine significantly inhibited both the release of IL-8 induced by H. pylori and the adhesion of H. pylori to cells in a dose-dependent manner. These properties of lafutidine are unrelated to the blockade of histamine H(2)-receptors, because the same effects have not been observed with other H(2)-receptor antagonists, such as cimetidine and famotidine. Lafutidine also significantly inhibited H. pylori-induced IL-6 release. Both TNF-alpha and IL-1beta-induced IL-8 releases, conversely, were little affected by lafutidine up to a concentration of 10(-5) M. CONCLUSIONS: These results suggest that lafutidine inhibits IL-8 release by inhibiting H. pylori adherence to gastric epithelial cells, indicating a novel mechanism by which lafutidine protects against the mucosal inflammation associated with H. pylori infection.

Protein kinase C activation by Helicobacter pylori in human gastric epithelial cells limits interleukin-8 production through suppression of extracellular signal-regulated kinase.

Helicobacter pylori (H. pylori) infection of gastric epithelial cells has been shown to induce interleukin (IL)-8 production, but the signal transduction mechanism leading to IL-8 production has not been clearly defined. Here, we investigate the role of protein kinase C (PKC) in the mechanism of induction of IL-8 release by H. pylori in human gastric epithelial cells. In MKN45 cells, H. pylori-induced IL-8 release was enhanced by treatment with PKC inhibitors (GF109203X and calphostin C) and PKC depletion, which completely inhibited PKC activity. Moreover, PKC inhibitors and PKC depletion increased extracellular signal-regulated kinase (ERK) activity and phosphorylation, but not calcium/calmodulin-dependent protein kinase II (CaMK II) activity, in response to H. pylori infection. PKC activated by H. pylori inhibited activation of ERK induced by H. pylori without affecting the CaMK II activity and negatively regulated IL-8 production in human gastric epithelial cells.

Identification of a signaling cascade for interleukin-8 production by Helicobacter pylori in human gastric epithelial cells.

Infecting gastric epithelial cells with Helicobacter pylori (H. pylori) has been shown to induce interleukin-8 (IL-8) production, but the signal transduction mechanism leading to IL-8 production is not defined clearly. In the present study, we investigated the molecular mechanism responsible for H. pylori-induced IL-8 release in human gastric epithelial cells. IL-8 levels in culture supernatants were determined by an enzyme linked-immunosorbent assay. Extracellular signal-regulated kinase (ERK) activity was tested using an in vitro kinase assay, which measured the incorporation of [gamma-33P]ATP into a synthetic peptide that is a specific ERK substrate. ERK phosphorylation and IkappaBalpha degradation by H. pylori infection were assessed by western blotting. In MKN45 cells, H. pylori-induced IL-8 release in a time-dependent manner. This IL-8 release was abolished by treatment with intracellular Ca2+ chelators (BAPTA-AM and TMB-8) but not by EGTA or nifedipine. The Ca2+ ionophore A23187 also induced IL-8 release to an extent similar to that of H. pylori infection. Calmodulin inhibitors (W7 and calmidazolium) and tyrosine kinase inhibitors (genistein and ST638) completely blocked IL-8 release by H. pylori and A23187. PD98059, an ERK pathway inhibitor, completely abolished H. pylori-induced IL-8 release. Moreover, BAPTA-AM, calmidazolium, and genistein, but not nifedipine, suppressed the ERK activation induced by H. pylori infection. PD98059 as well as MG132, an NF-kappaB pathway inhibitor, blocked both IL-8 production and degradation of IkappaBalpha induced by H. pylori infection, whereas only PD98059 inhibited ERK activity in response to H. pylori. There was no significant difference between IL-8 production induced by the cagA positive wild-type strain and the cagA negative isogenic mutant strain of H. pylori; therefore, CagA is not involved in the IL-8 production pathway. H. pylori-induced IL-8 production is dominantly regulated by Ca2+/calmodulin signaling, and ERK plays an important role in signal transmission for the efficient activation of H. pylori-induced NF-kappaB activity, resulting in IL-8 production.

Sensitizing effects of lafutidine on CGRP-containing afferent nerves in the rat stomach.

1. Capsaicin sensitive afferent nerves play an important role in gastric mucosal defensive mechanisms. Capsaicin stimulates afferent nerves and enhances the release of calcitonin gene-related peptide (CGRP), which seems to be the predominant neurotransmitter of spinal afferents in the rat stomach, exerting many pharmacological effects by a direct mechanism or indirectly through second messengers such as nitric oxide (NO). 2. Lafutidine is a new type of anti-ulcer drug, possessing both an antisecretory effect, exerted via histamine H(2) receptor blockade, and gastroprotective activities. Studies with certain antagonists or chemical deafferentation techniques suggest the gastroprotective actions of lafutidine to be mediated by capsaicin sensitive afferent nerves, but this is an assumption based on indirect techniques. In order to explain the direct relation of lafutidine to afferent nerves, we conducted the following studies. 3. We determined CGRP and NO release from rat stomach and specific [(3)H]-resiniferatoxin (RTX) binding to gastric vanilloid receptor subtype 1 (VR1), which binds capsaicin, using EIA, a microdialysis system and a radioreceptor assay, respectively. 4. Lafutidine enhanced both CGRP and NO release from the rat stomach induced by a submaximal dose of capsaicin, but had no effect on specific [(3)H]-RTX and capsaicin binding to VR1. 5. In conclusion, our findings demonstrate that lafutidine modulates the activity of capsaicin sensitive afferent nerves in the rat stomach, which may be a key mechanism involved in its gastroprotective action.