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W1697 Estrogen Potentiates Prostaglandin E2-Stimulated Duodenal Mucosal Bicarbonate Secretion in Mice
mucosa of aging rats exhibited hypoxia, increased apoptosis and reduced survivin and VEGF vs. young rats; all p<0.01. In placebo pretreated aging rats indomethacin and ethanol caused gastric injury involving 10.1±1.5% and 38.2±12% of mucosal area, respectively, and extensive endothelial and progenitor cell damage. HTL pretreatment significantly reduced ethanol and indomethacin-induced mucosal injury - area by 2.0- and 2.8-fold, respectively (both p<0.01), endothelial cell injury, microvascular damage and progenitor cell necrosis and apoptosis by 3.2 - 4.3-fold (p<0.01). In-vitro studies showed that in aging gastric endothelial cells HTL treatment activates VEGF, VEGFR2 and survivin genes and stimulates angiogenesis. Conclusions: 1) In aging gastric mucosa the endothelial cells of microvessels and progenitor cells are major targets of injury by indomethacin and ethanol. 2) Hydrotalcite administered orally effectively protects aging gastric mucosa against indomethacin and ethanol injury by preserving microvascular endothelial and progenitor cells. 3) The mechanisms of hydrotalcite protective action on aging gastric mucosa are mediated via activation of survivin and VEGF, which are major survival factors for endothelial and progenitor cells. 4) This study uncovered novel molecular mechanisms for mucosal protective action of hydrotalcite and suggests its potential therapeutic use for aging gastropathy. W1697 Estrogen Potentiates Prostaglandin E2-Stimulated Duodenal Mucosal Bicarbonate Secretion in Mice Biguang Tuo, Guorong Wen, Xue Wang, Xuemei Liu, Hui Dong Background&Aims: The cause of lower prevalence of duodenal ulcer in women than men is largely unknown. We recently found that gender differences in duodenal mucosal bicarbonate secretion exist in mice, indicating sex hormone is involved in the regulation of duodenal bicarbonate secretion. Prostaglandin E2 (PGE2) is an important endogenous mediator which is widely distributed in gastroduodenal mucosa and stimulates duodenal bicarbonate secretion. The aims of the present study were to investigate the effect of estrogen on PGE2stimulated duodenal bicarbonate secretion and the underlying mechanism. Methods: Murine duodenal mucosal bicarbonate secretion was examined In Vitro in Ussing chambers by pHstat titration. Phosphatidylinositol 3-kinase (PI3K) activity in duodenal mucosa was measured by immunoprecipitation of PI3K and ELISA, and Akt phosphorylation was measured by Western analysis with anti-phospho-Akt and anti-Akt antibodies. The cAMP content in the duodenal mucosa was determined by using a cAMP enzyme immunoassay kit. Results: 17βestradiol (10nM) slightly increased duodenal bicarbonate secretion without effect on short circuit current (Isc). However, the pretreatment of duodenal mucosa with 17β-estradiol (10 nM) at 20 minute before PGE2 markedly potentiated PGE2-stimulated duodenal bicarbonate secretion and Isc by 89% and 39% respectively (P<0.001 and P<0.05). Compared with addition of alone 17β-estradiol and alone PGE2, duodenal bicarbonate secretion and Isc were increased by 41% and 35% respectively. 17β-estradiol (0.01-100 nM) dose-dependently increased the activity of PI3K and induced the phosphorylation of Akt, a signaling molecule downstream of PI3K, in duodenal mucosa. 17β-estradiol (10nM) increased duodenal mucosal cAMP levels. Conclusion: Estrogen not only stimulates duodenal bicarbonate secretion, but also potentiates PGE2-stimulated duodenal bicarbonate secretion, which is possibly mediated through PI3K- and cAMP-dependent mechanisms. This may partially explain estrogen protection of the duodenum in the females.
W1695 Effect of Allyl Isothiocyanate, a Pungent Ingredient of Wasabi and Mustard Oil, on Gastric Mucosal Blood Flow and Ulcerogenesis in Rats: Comparison With Capsaicin Kimihito Tashima, Masaki Raimura, Kenjiro Matsumoto, Atsushi Chino, Takao Namiki, Katsutoshi Terasawa, Syunji Horie BACKGROUND & AIM: The excess intake of wasabi and mustard oil has been implicated as a cause of gastric mucosal injury. Recently, we have shown that allyl isothiocyanate (AITC), a pungent ingredient of wasabi and mustard oil, impairs gastric paracellular barrier In Vitro (J. Physiol. Pharmacol. 60 (Suppl 2) 105, 2009). However, no consensus as to the effect of AITC, which is known to stimulate transient receptor potential subtype A1 (TRPA1) co-expressed with TRPV1 in sensory nerves, on the mucosal defense has been reached In Vivo. In the present study, we examined the effect of AITC on gastric mucosal blood flow (GMBF) and ulcerogenesis in rats as compared with capsaicin. METHODS: Male SD rats (160~180 g) were used after 18 hr-fasting. Stomachs mounted on ex-vivo chambers under urethane-anesthetized rats, were perfused with 50 mM HCl. GMBF (a laser Doppler flowmeter) and luminal acid loss (H+ back-diffusion) were measured simultaneously before and after exposure to AITC (10~100 mM) or capsaicin (100 mM) for 30 min with omeprazole, and the mucosa was examined for lesions 90 min after either AITC or capsaicin exposure. Histological examination was performed by hematoxylin-eosin staining of the rat stomach tissues subjected to AITC (10~100 mM) for 30 min. In separate experiments, AITC (1~10 mM) was topically applied for 10 min to the rat stomach, and changes in GMBF were monitored. HC-030031 (14.1 mM), a TRPA1 antagonist, was co-applied with AITC (10 mM) for 10 min to the stomach. RESULTS: Mucosal application of AITC (above 30 mM) caused luminal acid loss, followed by an increase of GMBF, resulting in minimal damage in the mucosa. However, chemical ablation of TRPV1-expressed sensory nerves attenuated the GMBF response to AITC without affecting luminal acid loss, and resulted in severe lesions. In contrast, capsaicin (100 mM) did not induce luminal acid loss, resulting in no damage in the mucosa. Histological study showed that the middle of gastric glands were obviously cleaved by AITC (above 30 mM), although no lesion was visible in mucosa. AITC (1~10 mM) increased GMBF in a concentration-dependent manner, and those responses were completely abolished by HC-030031. CONCLUSION: It is suggested that AITC from wasabi and mustard oil exerts a protective effect on gastric mucosa In Vivo due to an increase of GMBF, yet this action differs in terms of concentration of AITC, but not capsaicin. It is likely that AITC in the low concentration (less than 10 mM) stimulates TRPA1 directly, whereas AITC in the high concentration (above 30 mM) stimulates TRPV1- and TRPA1expressed nerves via H+ back-diffusion following epithelial barrier disruption.
W1698 Role of CB1 in Erosive and Non-Erosive Gastroesophageal Reflux Disease Carlo Calabrese, Enzo Spisni, Fernando Rizzello, Giuseppina Liguori, Maria Chiara Valerii, Giorgia Lazzarini, Paolo Gionchetti, Massimo Campieri Background: Cannabinoid (CB) receptors have been located in brain areas involved in the triggering of TLESRs as well as in the nodose ganglion from which vagal afferents emanate. Tetrahydrocannabinol significantly inhibits the increase in TLESRs evoked by meal ingestion and reduces basal LES pressure. Aim: evaluate the presence and the activity of CB1 receptors in the esophageal epithelium in healthy subjects and in patients with erosive or non-erosive GERD. Material and methods: We enrolled 85 consecutive subjects that undergo both esophageal pH monitoring and endoscopy. 10 were healthy voluntary controls, 38 were non-erosive (NERD) and 37 had erosive esophagitis (ERD). In each subject, 8 specimens were taken. Four biopsies were fixed in 4% buffered formalin and embedded in paraffin. Tissue sections were mounted on slides, sections were deparaffinised with xylene and rehydrated through a series of graded alcohols, then were blocked in 5% bovine serum albumin in Phosphate-buffered saline (PBS) for 1h and incubated overnight at 4°C with CB1 antibody (Cayman Chemicals, USA) at a 1:500 dilution in PBS. Sections then were incubated in rabbit-specific secondary antibody for 30 minutes at room temperature and then in 3,3-diaminobenzidine tetrahydrochloride (DAKO) for 4 minutes. Four biopsies were immediately put in liquid nitrogen, and stored at -80° C until the RNA extraction for real-time PCR analysis or until protein extraction for WB examination. Results: Immunocytochemical staining shows the presence of CB1 receptor in esophageal mucosa of both control and GERD patients. Semi-quantitative analysis, performed with WB, shows a differential expression of CB1 receptor with the higher expression (40%) in NERD patients respect to ERD patients. Real-Time analysis shows a decrease of CB1 receptor RNA in normal esophagus respect to colon, taken as positive control, of about 30%. NERD patients show lower levels of CB1 receptor RNA compared with colon (-62%) and 2 fold higher respect to ERD patients. The results are the mean ± SEM of three independent experiments. Mean expression of CB1 in NERD mucosa is higher, but show elevated variability between patients, with respect to ERD mucosa. Conclusions: Immunocytochemical staining, real-time PCR and WB analyses confirm that CB1 receptor is expressed in esophageal mucosa. In ERD and NERD patients, the expression of CB1 is increased, in comparison to control mucosa. The mean expression of CB1 in NERD mucosa is higher than ERD mucosa. These findings confirm that CB1 receptors are also involved in the esophageal mucosal defense mechanisms.
W1696 Hydrotalcite Protects Aging Gastric Mucosa Against NSAID- And EthanolInduced Injury by Preserving Endothelial and Progenitor Cells. Underlying Molecular Mechanisms Include Activation of Survivin and VEGF. Andrzej S. Tarnawski, Amrita Ahluwalia, Vipal Gandhi, Xiaoming Deng, Ximing Xiong Background: Gastric mucosa of patients over age of 65 years and gastric mucosa of aging rats has increased susceptibility to injury by NSAIDs, ethanol, ischemia and impaired healing of injury. Aims: to determine whether hydrotalcite (HTL-Talcid, unique layer-lattice compound developed in Japan and in clinical use in Europe) can protect gastric mucosa of aging rats against indomethacin and/or ethanol injury and to identify molecular mechanisms involved. Material and Methods: Fisher F344 rats 24 months old (H. pylori free) received intragastrically placebo or HTL (400mg/kg) and 2 hrs later either: indomethacin 25 mg/kg i.p., ethanol 8 ml 50% /kg b.w. intragastrically or placebo. Untreated rats, 3month (young) and 24 months old (aging) served as baseline controls. Studies at 24 hrs: 1) the extent of mucosal injury, 2) quantitative histology: deep and superficial injury, microvascular damage, and injury of specific mucosal cells, 3) expression of survivin (anti-apoptosis protein), VEGF and its receptor 2, VEGFR2; 4) In studies performed in-vitro on endothelial cells isolated from gastric mucosa of aging and young rats we examined the effect of HTL on expressions of VEGF, VEGFR2 and survivin, and angiogenesis on Matrigel. Results: At baseline gastric
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AGA Abstracts
AGA Abstracts
compared the effect of ADMA (10-200 μg/kg i.g.) applied alone or combined with 1) Larginine (200 mg/kg i.g.); 2) calcitonin gene related peptide (CGRP 5 μg/kg s.c.) and low dose of capsaicin (0.5 mg/kg i.g.) capable of stimulating sensory afferents on ischemia/ reperfusion (I/R, 30 min of ischemia to celiac artery followed by 3 hrs of reperfusion) injury (series A)and the healing of chronic acetic acid ulcers (ulcer area = 28 mm2) in rats with intact and capsaicin-deactivated sensory nerves (series B). The area of gastric lesions and ulcers was determined by planimetry, the gastric blood flow (GBF) was assessed by H2-gas clearance method, NOx gastric content, plasma MPO activity and the mucosal activity of antioxidazing enzymes SOD, GPx and MDA concentration as an index of oxygen radicallipid peroxidation were assessed. The gastric content of ADMA was determined by HPLC method and gastric mucosal expression of transcription factor NFκB, IL-1β and TNF-α mRNAs their plasma levels were analyzed by RT-PCR and ELISA. I/R produced gastric lesions (series A) accompanied by an increase in plasma ADMA, MDA, MPO contents, the fall in the GBF by 31% and the suppression of SOD and GPx activities. ADMA which significantly reduced the luminal NOx content, dose-dependently aggravated I/R damage and significantly delayed the healing of acetic acid ulcers (series B); both effects being worsened by capsaicin denervation. These effects were accompanied by further fall in the GBF and a significant decrease in the SOD and GPx activities and the significant increase in MDA content. The ADMA delay in ulcer healing was accompanied by the upregulation of NFκB, IL-1β and TNF-α mRNAs at ulcer margin followed by the rise in plasma IL-1β, TNF-α and ADMA levels and these effects were markedly diminished by addition of L-Arg, CGRP or capsaicin to ADMA. We conclude that: 1) ADMA plays an important role in the pathogenesis of acute gastric lesions induced by I/R and delays ulcer healing due to the inhibition of NO generation, NFκB activation, overexpression of proinflammatory cytokines IL-1β and TNF-α and enhancement in lipid peroxidation; 2) L-Arg, a donor of NO or sensory nerve vasodilatory neuropeptide CGRP counteract the ADMA-induced facilitation of mucosal lesions and the impairment of the healing of preexisting gastric ulcers.
W1695 Effect of Allyl Isothiocyanate, a Pungent Ingredient of Wasabi and Mustard Oil, on Gastric Mucosal Blood Flow and Ulcerogenesis in Rats: Comparison With Capsaicin Kimihito Tashima, Masaki Raimura, Kenjiro Matsumoto, Atsushi Chino, Takao Namiki, Katsutoshi Terasawa, Syunji Horie BACKGROUND & AIM: The excess intake of wasabi and mustard oil has been implicated as a cause of gastric mucosal injury. Recently, we have shown that allyl isothiocyanate (AITC), a pungent ingredient of wasabi and mustard oil, impairs gastric paracellular barrier In Vitro (J. Physiol. Pharmacol. 60 (Suppl 2) 105, 2009). However, no consensus as to the effect of AITC, which is known to stimulate transient receptor potential subtype A1 (TRPA1) co-expressed with TRPV1 in sensory nerves, on the mucosal defense has been reached In Vivo. In the present study, we examined the effect of AITC on gastric mucosal blood flow (GMBF) and ulcerogenesis in rats as compared with capsaicin. METHODS: Male SD rats (160~180 g) were used after 18 hr-fasting. Stomachs mounted on ex-vivo chambers under urethane-anesthetized rats, were perfused with 50 mM HCl. GMBF (a laser Doppler flowmeter) and luminal acid loss (H+ back-diffusion) were measured simultaneously before and after exposure to AITC (10~100 mM) or capsaicin (100 mM) for 30 min with omeprazole, and the mucosa was examined for lesions 90 min after either AITC or capsaicin exposure. Histological examination was performed by hematoxylin-eosin staining of the rat stomach tissues subjected to AITC (10~100 mM) for 30 min. In separate experiments, AITC (1~10 mM) was topically applied for 10 min to the rat stomach, and changes in GMBF were monitored. HC-030031 (14.1 mM), a TRPA1 antagonist, was co-applied with AITC (10 mM) for 10 min to the stomach. RESULTS: Mucosal application of AITC (above 30 mM) caused luminal acid loss, followed by an increase of GMBF, resulting in minimal damage in the mucosa. However, chemical ablation of TRPV1-expressed sensory nerves attenuated the GMBF response to AITC without affecting luminal acid loss, and resulted in severe lesions. In contrast, capsaicin (100 mM) did not induce luminal acid loss, resulting in no damage in the mucosa. Histological study showed that the middle of gastric glands were obviously cleaved by AITC (above 30 mM), although no lesion was visible in mucosa. AITC (1~10 mM) increased GMBF in a concentration-dependent manner, and those responses were completely abolished by HC-030031. CONCLUSION: It is suggested that AITC from wasabi and mustard oil exerts a protective effect on gastric mucosa In Vivo due to an increase of GMBF, yet this action differs in terms of concentration of AITC, but not capsaicin. It is likely that AITC in the low concentration (less than 10 mM) stimulates TRPA1 directly, whereas AITC in the high concentration (above 30 mM) stimulates TRPV1- and TRPA1expressed nerves via H+ back-diffusion following epithelial barrier disruption.
W1698 Role of CB1 in Erosive and Non-Erosive Gastroesophageal Reflux Disease Carlo Calabrese, Enzo Spisni, Fernando Rizzello, Giuseppina Liguori, Maria Chiara Valerii, Giorgia Lazzarini, Paolo Gionchetti, Massimo Campieri Background: Cannabinoid (CB) receptors have been located in brain areas involved in the triggering of TLESRs as well as in the nodose ganglion from which vagal afferents emanate. Tetrahydrocannabinol significantly inhibits the increase in TLESRs evoked by meal ingestion and reduces basal LES pressure. Aim: evaluate the presence and the activity of CB1 receptors in the esophageal epithelium in healthy subjects and in patients with erosive or non-erosive GERD. Material and methods: We enrolled 85 consecutive subjects that undergo both esophageal pH monitoring and endoscopy. 10 were healthy voluntary controls, 38 were non-erosive (NERD) and 37 had erosive esophagitis (ERD). In each subject, 8 specimens were taken. Four biopsies were fixed in 4% buffered formalin and embedded in paraffin. Tissue sections were mounted on slides, sections were deparaffinised with xylene and rehydrated through a series of graded alcohols, then were blocked in 5% bovine serum albumin in Phosphate-buffered saline (PBS) for 1h and incubated overnight at 4°C with CB1 antibody (Cayman Chemicals, USA) at a 1:500 dilution in PBS. Sections then were incubated in rabbit-specific secondary antibody for 30 minutes at room temperature and then in 3,3-diaminobenzidine tetrahydrochloride (DAKO) for 4 minutes. Four biopsies were immediately put in liquid nitrogen, and stored at -80° C until the RNA extraction for real-time PCR analysis or until protein extraction for WB examination. Results: Immunocytochemical staining shows the presence of CB1 receptor in esophageal mucosa of both control and GERD patients. Semi-quantitative analysis, performed with WB, shows a differential expression of CB1 receptor with the higher expression (40%) in NERD patients respect to ERD patients. Real-Time analysis shows a decrease of CB1 receptor RNA in normal esophagus respect to colon, taken as positive control, of about 30%. NERD patients show lower levels of CB1 receptor RNA compared with colon (-62%) and 2 fold higher respect to ERD patients. The results are the mean ± SEM of three independent experiments. Mean expression of CB1 in NERD mucosa is higher, but show elevated variability between patients, with respect to ERD mucosa. Conclusions: Immunocytochemical staining, real-time PCR and WB analyses confirm that CB1 receptor is expressed in esophageal mucosa. In ERD and NERD patients, the expression of CB1 is increased, in comparison to control mucosa. The mean expression of CB1 in NERD mucosa is higher than ERD mucosa. These findings confirm that CB1 receptors are also involved in the esophageal mucosal defense mechanisms.
W1696 Hydrotalcite Protects Aging Gastric Mucosa Against NSAID- And EthanolInduced Injury by Preserving Endothelial and Progenitor Cells. Underlying Molecular Mechanisms Include Activation of Survivin and VEGF. Andrzej S. Tarnawski, Amrita Ahluwalia, Vipal Gandhi, Xiaoming Deng, Ximing Xiong Background: Gastric mucosa of patients over age of 65 years and gastric mucosa of aging rats has increased susceptibility to injury by NSAIDs, ethanol, ischemia and impaired healing of injury. Aims: to determine whether hydrotalcite (HTL-Talcid, unique layer-lattice compound developed in Japan and in clinical use in Europe) can protect gastric mucosa of aging rats against indomethacin and/or ethanol injury and to identify molecular mechanisms involved. Material and Methods: Fisher F344 rats 24 months old (H. pylori free) received intragastrically placebo or HTL (400mg/kg) and 2 hrs later either: indomethacin 25 mg/kg i.p., ethanol 8 ml 50% /kg b.w. intragastrically or placebo. Untreated rats, 3month (young) and 24 months old (aging) served as baseline controls. Studies at 24 hrs: 1) the extent of mucosal injury, 2) quantitative histology: deep and superficial injury, microvascular damage, and injury of specific mucosal cells, 3) expression of survivin (anti-apoptosis protein), VEGF and its receptor 2, VEGFR2; 4) In studies performed in-vitro on endothelial cells isolated from gastric mucosa of aging and young rats we examined the effect of HTL on expressions of VEGF, VEGFR2 and survivin, and angiogenesis on Matrigel. Results: At baseline gastric
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AGA Abstracts
AGA Abstracts
compared the effect of ADMA (10-200 μg/kg i.g.) applied alone or combined with 1) Larginine (200 mg/kg i.g.); 2) calcitonin gene related peptide (CGRP 5 μg/kg s.c.) and low dose of capsaicin (0.5 mg/kg i.g.) capable of stimulating sensory afferents on ischemia/ reperfusion (I/R, 30 min of ischemia to celiac artery followed by 3 hrs of reperfusion) injury (series A)and the healing of chronic acetic acid ulcers (ulcer area = 28 mm2) in rats with intact and capsaicin-deactivated sensory nerves (series B). The area of gastric lesions and ulcers was determined by planimetry, the gastric blood flow (GBF) was assessed by H2-gas clearance method, NOx gastric content, plasma MPO activity and the mucosal activity of antioxidazing enzymes SOD, GPx and MDA concentration as an index of oxygen radicallipid peroxidation were assessed. The gastric content of ADMA was determined by HPLC method and gastric mucosal expression of transcription factor NFκB, IL-1β and TNF-α mRNAs their plasma levels were analyzed by RT-PCR and ELISA. I/R produced gastric lesions (series A) accompanied by an increase in plasma ADMA, MDA, MPO contents, the fall in the GBF by 31% and the suppression of SOD and GPx activities. ADMA which significantly reduced the luminal NOx content, dose-dependently aggravated I/R damage and significantly delayed the healing of acetic acid ulcers (series B); both effects being worsened by capsaicin denervation. These effects were accompanied by further fall in the GBF and a significant decrease in the SOD and GPx activities and the significant increase in MDA content. The ADMA delay in ulcer healing was accompanied by the upregulation of NFκB, IL-1β and TNF-α mRNAs at ulcer margin followed by the rise in plasma IL-1β, TNF-α and ADMA levels and these effects were markedly diminished by addition of L-Arg, CGRP or capsaicin to ADMA. We conclude that: 1) ADMA plays an important role in the pathogenesis of acute gastric lesions induced by I/R and delays ulcer healing due to the inhibition of NO generation, NFκB activation, overexpression of proinflammatory cytokines IL-1β and TNF-α and enhancement in lipid peroxidation; 2) L-Arg, a donor of NO or sensory nerve vasodilatory neuropeptide CGRP counteract the ADMA-induced facilitation of mucosal lesions and the impairment of the healing of preexisting gastric ulcers.