Yoshihisa Nozawa

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.

Distribution and characterization of vanilloid receptors in the rat stomach

The cloned vanilloid receptor-1 (VR1) is recognized as a common molecular target for protons, noxious heat, and vanilloids. The presence of VR1 in the dorsal root, trigeminal, and nodose ganglia has been firmly established, but it is unclear in the gut, despite this VR1 may be important for gastric mucosal homeostasis. In this study we used an antibody and a radioligand to show the distribution of vanilloid receptors (VRs) in rat stomach and to characterize it. The deafferentiation of capsaicin-sensitive nerves in rats was induced by consecutive injections of capsaicin. VR1-immunopositive nerve endings were predominantly found in the mucous neck cells of the proliferation zone, and around blood vessels in the submucosa. Radioreceptor assay using [3H]-resiniferatoxin (RTX) revealed the existence of high affinity and single-class binding site in the membrane fractions of the mucosa. Capsaicin completely inhibited the specific binding of [3H]-RTX. Both the VR1 immunoreactivity and the receptor density of [3H]-RTX binding sites significantly reduced by the application of capsaicin for prolonged periods of time in the mucosa of rats. Our results indicate that VRs are expressed in the rat stomach, and suggest that they may be involved in mucosal protection by increasing cell proliferation and blood flow.

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

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). Lafutidine is a new type of anti‐ulcer drug, possessing both an antisecretory effect, exerted via histamine H2 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. We determined CGRP and NO release from rat stomach and specific [3H]‐resiniferatoxin (RTX) binding to gastric vanilloid receptor subtype 1 (VR1), which binds capsaicin, using EIA, a microdialysis system and a radioreceptor assay, respectively. Lafutidine enhanced both CGRP and NO release from the rat stomach induced by a submaximal dose of capsaicin, but had no effect on specific [3H]‐RTX and capsaicin binding to VR1. 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.

Effects of TH-142177 on angiotensin II-induced proliferation, migration and intracellular signaling in vascular smooth muscle cells and on neointimal thickening after balloon injury

We investigated the effects of TH-142177 (N-n-butyl-N-[2-(1-H-tetrazole-5-yl) biphenyl-4-yl]-methyl-(N-carboxy methyl-benzylamino)-acetamide), a novel selective antagonist of angiotensin II type 1-receptor (AT1-R) on angiotensin II (AII)-induced proliferation and migration of vascular smooth muscle cells (VSMC), and on neointimal formation in the rat carotid artery after balloon injury, and on the intracellular signaling by the stimulation of AT1-R. High affinity AII receptor sites were detected in rat VSMC by the use of [125I]Sar1,Ile8-AII. TH-142177 and losartan competed with [125I]Sar1,Ile8-AII for the binding sites in VSMC in a monophasic manner, although PD123177, a selective antagonist of angiotensin II type 2-receptor (AT2-R), had little inhibitory effect, demonstrating the predominant existence of AT1-R in rat VSMC. TH-142177 prevented AII-induced DNA synthesis and migration, with a significant inhibition of 74 and 55%, respectively, at the concentration of 100 nM. AII-induced activation of p21ras, mitogen-activated protein kinase (p42MAPK and p44MAPK), and protein kinase C was significantly (50-78%) inhibited by TH-142177 (100 nM), suggesting that the activation of these enzymes is mediated through the stimulation of AT1-R. Balloon-injured left carotid arteries in rats showed extensive neointimal thickening, and TH-142177 (3 mg/kg) brought out a marked decrease in the neointimal thickening after balloon injury. In conclusion, TH-142177 inhibited AII-induced proliferation and migration of rat VSMC and neointimal formation in the carotid artery after balloon injury, and these effects may be related, in part, to the suppression of ras, p42MAPK and p44MAPK, and protein kinase C activities through the blockade of AT1-R. Thus, TH-142177 may have therapeutic potential for the treatment of vascular diseases such as atherosclerosis and restenosis.

DOWN-REGULATION OF ANGIOTENSIN II RECEPTORS IN HYPERTROPHIED HUMAN MYOCARDIUM

1. Specific [125I]‐angiotensin II (AngII) binding in normal and hypertrophied human myocardial membranes was saturable and of high affinity. Low concentrations of unlabelled AngII and saralasin competed with [125I]‐AngII for the binding sites in these tissues. Thus, saturable [125I]‐AngII binding in human myocardium exhibited pharmacological specificity that characterized high affinity receptors for AngII.

Loss of Muscarinic and Purinergic Receptors in Urinary Bladder of Rats With Hydrochloric Acid-induced Cystitis

OBJECTIVESnTo clarify the basic mechanism involved in the pathophysiology of cystitis by characterizing the urodynamic parameters, pharmacologically relevant (muscarinic and purinergic) receptors, and the in vivo release of adenosine triphosphate (ATP) in the bladder of hydrochloric acid (HCl)-treated rats.nnnMETHODSnThe muscarinic and purinergic receptors in rat tissue were measured by radioreceptor assays using (N-methyl-³H) scopolamine methyl chloride ([³H]NMS) and αβ-methylene-ATP (2,8-³H) tetrasodium salt ([³H]αβ-MeATP), respectively. The urodynamic parameters and ATP levels were measured using a cystometric method and the luciferin-luciferase assay, respectively.nnnRESULTSnIn the HCl-treated rats, the micturition interval and micturition volume were significantly (48% and 55%, respectively, P <.05) decreased and the number of micturitions was significantly (3.2-fold, P <.05) increased compared with those of the control rats. The maximal number of binding sites for [³H]NMS and [³H]αβ-MeATP was significantly (55% and 72%, respectively, P <.001) decreased in the bladder of HCl-treated rats, suggesting downregulation of both muscarinic and purinergic receptors. In the HCl-treated rats, the inhibition constant, K(i), values for oxybutynin, solifenacin, and darifenacin were significantly (1.3-1.4-fold, P <.05) increased, but those for tolterodine and AF-DX116 were unchanged. Similarly, the inhibition constant for A-317491, pyridoxal-phosphate-6-azophenyl-2,4-disulfonic acid tetrasodium, and MRS2273 was significantly (5.5, 11, and 7.6-fold, respectively, P <.001) increased. Furthermore, the in vivo release of ATP was significantly (P <.05) enhanced in the HCl-treated rat bladder.nnnCONCLUSIONSnBoth muscarinic and purinergic mechanisms might be, at least in part, associated with the urinary dysfunction due to cystitis.

Receptor Occupancy in Myocardium, Adrenal Cortex, and Brain by TH-142177, a Novel AT1 Receptor Antagonist in Rats, in Relation to Its Plasma Concentration and Hypotensive Effect

AbstractPurpose. To study the relationship between angiotensin II (All) receptor occupancy ex vivo in tissues plasma concentration and hypotensive effect of a novel All receptor antagonist, TH-142177 and losartan in rats.nMethods. At 2, 8 and 24 hr after oral administration of TH-142177 and losartan in rats, All receptors in myocardium, adrenal cortex and cerebral cortex were determined by radioligand binding assay using [125I]Sar1,Ile8-AII. Plasma concentrations of both drugs and metabolite in rats were also measured using validated HPLC assays. Further, systolic blood pressure (SBP) in conscious renal hypertensive rats treated orally with TH-142177 and losartan were measured by using a tail cuff plethysmographic method.nResults. Oral administration of TH-142177 (1.8 and 5.5 μmol/kg) and losartan (6.5 and 21.7 μmol/kg) in rats brought about dose-dependent decreases in [125I]Sar1,Ile8-AII binding sites (Bmax) in myocardium and adrenal cortex. The extent of receptor occupancy by both drugs in adrenal cortex was maximal at 2 hr later but that in myocardium at 8 hr later. Further, the receptor occupancy was more sustained in myocardium than adrenal cortex. The ex vivo binding affinity of TH-142177 for All receptors in these tissues was roughly three times higher than that of losartan. Also, cerebral cortical [125I]Sar1,Ile8-AII binding was significantly reduced by oral administration of losartan but not by TH-142177. The time course of All receptor occupancy by both drugs in adrenal cortex appeared to be in parallel with that of their plasma concentrations, while the time course in myocardium correlated with that of their hypotensive effects rather than plasma concentrations.nConclusions. TH-142177 produced a relatively selective and sustained occupancy ex vivo of All receptors in myocardium and adrenal cortex of rats with approximately three times greater potency than losartan. Its time course of myocardial receptor occupancy was in parallel with that of hypotensive effect rather than plasma concentration.

Analysis of Helicobacter pylori binding site on HEp‐2 cells and three cell lines from human gastric carcinoma

Abstract— Helicobacter pylori (H. pylori) is a pathogen responsible for chronic gastritis and peptic ulcer diseases. It colonises the gastric mucus layer and adheres to the gastric epithelial cell surface. As this adherence is the first step of infection, it is important to study the adherence mechanism. The aim of this study was to analyse the specific binding assay of H. pylori to HEp‐2 cells and three gastric phenotype cell lines, AGS, MKN‐45 and AZ‐521. H. pylori NCTC 11637 grown on agar plates was harvested and used in experiments. H. pylori was inoculated to pre‐cultured cell monolayers. Adhered bacteria were labelled with an anti‐H. pylori antibody and an FITC‐conjugated secondary antibody and quantified by using a fluorescent plate reader. Microbial adherence to HEp‐2 cells increased with incubation time and incubated concentration of H. pylori. No further increase was obtained with four or more hours of incubation or with a concentration of 4 × 107 bacteria/well or more. Scatchard analysis revealed a linear plot and the Bmax value was 88.3. Similar adherence patterns were obtained when AGS, AZ‐521 and MKN‐45 cells were used for adherence assays, but they had a lower binding affinity than HEp‐2 cells and AZ‐521. MKN‐45 cells had less receptors than HEp‐2 and AGS cells. In conclusion, H. pylori adhered to the cell surface could be quantified by this assay method. H. pylori adhesion to cell surfaces has a single population of binding site and one type of binding site on HEp‐2, AGS, AZ‐521 and MKN‐45 cells.

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

Background and Aim:u2002 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.

Pharmacological profile of TH-142177, a novel orally active AT1-receptor antagonist

Summary— The pharmacological properties of TH‐142177 (N‐n‐butyl‐N‐[2′‐(1‐H‐tetrazole‐5‐yl)biphenyl‐4‐yl]‐methyl‐(N‐carboxymethyl‐benzylamino)‐acetamide), a novel antagonist of the angiotensin II (AII) AT1 receptor, were studied in vitro and in vivo, and compared to those of losartan. In the rat isolated aorta, TH‐142177 produced parallel shifts to the right of the concentration‐response curves for AII‐induced contractions without affecting the maximal response (pA2 = 9.07). The inhibitory potency of TH‐142177 in the aorta was about three times greater than that of losartan. TH‐142177 completely inhibited the specific binding of [125I]AII to AT1 receptor in rat aortic membranes (Ki = 1.6 × 10−8 M), whereas specific [125I]AH binding to AT2 receptor in bovine cerebellum and human myocardium was not affected by concentrations of TH‐142177 up to 10−5 M. Losartan also inhibited the [125I] AII binding to rat aortic membranes (Ki = 2.2 × 10−8 M). Following the intravenous administration to anesthetized normotensive rats, TH‐142177 dose‐dependently inhibited the increase in systolic blood pressure induced by an intravenous bolus injection of AII that was 1.5 times less potent than losartan. Furthermore, the oral administration of TH‐142177 to conscious renal hypertensive rats exerted a dose‐dependent reduction of systolic blood pressure without significantly effecting the heart rate. TH‐142177 was at least three times more potent than losartan. These results demonstrate that TH‐142177 is a potent and selective antagonist of AT1 receptors and by oral administration has a long‐lasting antihypertensive activity.