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Sequential assessment of nitric oxide synthase mRNA and protein expression in portal hypertensive esophageal mucosa
April 1 9 9 5
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Esophageal, Gastric, and Duodenal Disorders
R A N I T I D I N E B I S M U T H CITRATE PROTECTS GASTRIC M U C O S A FROM I N D O M E T H A C I N - I N D U C E D INJURY IN RATS BY ALTERING THE A C I D PERMEABILITY OF GASTRIC M U C U S S. Tanaka, Y. Nishizaki, G. Paulsen, P.H. Guth and J.D. Kaunitz. Res. Svc. and Dept. of Medicine, VAMC & UCLA, Los Angeles, CA Ranitidine bismuth citrate, GR12231 I X (GR) prevents aspirin-induced gastric injury in healthy volunteers. Aim: To determine the mechanism by which GR exerts its gastreprotective effect in a rat model. Methods: GR (0- 100mg p.o.) was given 30 min prior to the injection of Indo (60mg/kg s.c.). Six hrs after Indo, gross and histologic lesions were scored. In separate experiments (same drug schedule), intracetlular pH (pH~ and mucusgel thidmess (MGT) were measured by in rive micrc~copy. Gastric mucc~al blood flow (GMBF) was measured by laser-Doppler tlowrnelry and acid output (AO) by back-titration. Gastric mucosae were superfused with pH 7.4 buffer during time= 0-30 min and pH 1.0 buffer during time = 30-50 min. Initial acidification rate OAR), a measure of mucus permeability to acid, was calculated from the fall in pH~during time = 30-32 min. Results: "p<0.05 vs. control; tp<0.05 vs. Indo:
Lesion Score AO (Hraollmin/cm2) %GMBF gross I histologie 0 min 40 min 150 min Control 134_+10 33.4_+3.3 Control 0.39_+0.0791.9_+1.1 83.6_+1.4 GR 10 mg/kg 160_+5" 23.5_+7.8 GR 100mg/kg 0,07_+0.02 88.5_+2.378J_+4.9 GR30 mg/kg 106_+79" 8.0_+4.4" Indo 0.27_+0.0688.0_+3.979.1_+2.5 GR 100 mg/kg 77_+11" 1.6_+lJ" GR+lndo 0.11_+0.05'93.1_+1.5 90.8_+5.4 Nor(~m) IAR pHi 30 min (rnM/min) 30 min I 32 min I 50 min control 114.8_+5.4 7.5+--0.5 control 7.04--+0.046.69-+0.02 6.59-+0.04 GR 100mg/kg 108.9-+52 6.4-+0.6t GR 100mg/kgt 7.05-+0.05 6.75-+0.04 6.60-+0.04 Indo 933-+4.8° 11.8±0.5" Indo'" 7.04-+0.046.51_+0.03 6.20_+0.07 GR+lndu 9L0±5J" 8J-+0.5t GR+lndo "~ 7.05-+0.03 6.68-+0.02 6.41-+0.05 Conclusions: Despite a thinned mucus gel, GR+lndo-treated rats had a normal IAR and near normal pH~ recovery during an acid load, in contrast to an increased IAR and impaired pH~ recovery in Indo treated rats. This indicated a GR-associated decrease in the acid permeability of the mucus gel, probably as a result of Bi3+ binding to gastric mucus. These results can thus help explain the protective effect of GR against indomethacin-induced gastric injury. (Supported by Glaxo Pharmaceuticals).
• PROTECTIVE EFFECT OF TEPRENONE ON PORTAL HYPERTENSIVE GASTRIC MUCOSA. K Tanoue, A Taruawski, ICI Waldsb'om, JJ Lee, FL Irwin, Jr, TH Nguyen, M Haslaizume,K Sugimachi, and IJ Sarfeh., DVA Med Cent, Long Beach CA and Univ Calif',lrvine, and Dpt Sttrg II, Kynshu Univ, Fukuoka, Japan. Teprenone (geranylgeranyl acetone) (TP) is a gastric mucosal protective drug used clinicallyin Japan for treatment of gastric ulcers and gastritis. Its effect on the portal hypertensive (PHT) gastric mucosa (which has impaired mucosal defenses and increased susceptibilityto injury) has not been studied. The aims of this study were to determine whether: a) TP pre-treatment may protect PHT gastric mucosa against ethanol injury and b) explain mechanisms of its mucosal protective action. METHODS: Portal hypertension (PHT) was produced by staged portal vein ligation in 40 rats. TP (100 mg/kg) or placebo (PL) were administered twice daily p.o. for 7 days. Gastric mucosal injury was produced by intragastric instillation of 8 ml/kg ethanol, and assessed macroscopically with a video image analysis system. STUDIES: Standardized gastric specimens were analyzed morphometrieally. In separate groups of PHT rats (without ethanol injury)we studied the effect of TP on defensive mechanisms of gastric mucosa: 1) portal pressure; 2) gastric pH; 3) gastric mucosal blood flow (GMBF) using laser Doppler flowmetry; 4) HXM content in gastric mucosa; 5) basic fibroblast growth factor (bFGF) expression in the gastric mucosa. This was done by using a video image analysis system at~er immtmostaining of mueosal section with a specific antibody. RESULTS: In PL pretreated rats, ethanol produced extensive necrosis. Histology revealed extensive deep hemorrhagic necrosis involving > 45+_5%ofmucosal sections length. TP did not affect portal pressure, but did sigmficantly reduce ethanol induced damage to PHT gastric mucosa, gross necrosis by 4-fold, deep hemorrhagic necrosis by 5 fold (!0<0.05). In rats without ethanol, GMBF was significantly higher in the TP group than in the PL group (4884-104 and 3464-70 PU respectively, p<0.05). TP treatment significantlyincreased HXM (174-3 and 134-2 lag/rag respectively). Immunofluorescence intensity of bFGF in endothelia of the submucosal veins and gastric mucosa was significantly higher in the TP group than in the PL group (147±11 vs 1074-16 units, p<0.01, and 76±5 vs 644-6 units, p<0.05, respectively). CONCLUSIONS: 1) TP significantly protects PHT gastric mucosa against ethanol injury. 2) TP administration increases GMBF, mucosal content ofHXM (a mucus component) and expression ofbFGF (an angiogenic factor) in endothelia of mucosal microvessels and submucosal veins of PHT gastric mucosa. 3) These effects of TP, combined with its known prostaglandni-stimulating action, suggest an important potential role of this agent in prophylaxis of PHT gastric mucosal injury.
A235
SUBSTITUTE, INEXPENSIVE DIAGNOSIS OF H. PYLORI INFECTION BY INFRA-RED(IR) SPECTEOPHOTOHETER IN IsC-UREA BREATH TEST Y. Taniguchi, K. Kimura, K.Satoh, H.Kawada, H.Sohara, H. Yamamoto, K. Ido, Y. Ogihara*, Y. Goto*, K. Takatori*, K. lida*, M.Kadiwara # Dept. of Gastroenterology, Jichi Medical School, Tochigi, Japan *Exploratory Research Lab. ~, Daiichi R a D Center, Tokyo and ~Dept. of Medicinal Chemistry, Heiji College of Pharmacy, Tokyo Since the ~SC-urea breath test has been described to assess H. pylori infection, most of the a l t e r n a t i v e protocols have been successful, except the cost benefit, s i m p l i c i t i e s and timeoomsumption with Mass spectrophotometric analysis. In order to overcome the above shortages, we designed to u t i l i z e infra-red(IR) spectrometer as a s u b s t i t u t e for Mass spectrometer. [Methods] Total 82 p a t i e n t s suffered with peptic ulcers or nonulcer dyspepsia were investigated. Breath samples were c o l l e c t e d at before, 15, 30, 45 and 60 min post-ingestion of l~C-urea (100 mg in 30 ml water) for time-course study and a t 15 min for diagnostic evaluation. IR spectrophotometer (EX130, JASCO,Tokyo) was used to determine the concentration of 1~C02 in each expirate. In addition, 13C02/i2C02 r a t i o was also measured by Mass spectrometry in order to compare with IR spectrometric analysis. Direct detection of H.pylori was q u a l i f i e d according to our methods in biopsy specimens (Am J Gastroenterol 1991;66:285-9I). EResults] 65 p a t i e n t s were p o s i t i v e in H. pylori infection and 17 were negative. In the urea breath t e s t , the plateau responses of 13C02 excretion was achieved between 15 and 30 min in the timecourse study. The mean value in the p o s i t i v e group was s i g n i f i cantly higher than that in the negative group (0.057 ± 0.035 vs 0.006 ± 0.004, respectively). The c u t - o f f level was determined 0.01 as h ~3C atom%. The s e n s i t i v i t y of IR spectrometer was 96.9% (63/65) and the s p e c i f i c i t y was 88. 2% (15/17). Correlation between Hass and IR photometric a n a l y s i s revealed extremely high c o e f f i c i e n t (r=0.999). [Conclusions] I t is suggested t h a t IR spectrometry has high p o t e n t i a l s not only to diagnose H. pylori infection but also to estimate the treatment with several advantages including technical s i m p l i c i t y , cost e f f e c t i v e n e s s and high accuracy.
• SEQUENTIAL ASSESSMENT OF NITRIC OXIDE SYNTHASE mRNA AND PROTEIN EXPRESSION IN PORTAL HYPERTENSIVE ESOPHAGEAL MUCOSA. K Taneue, IJ Sarfeh, A Tamawski, KJ Wahlstrom, FL Irwin Jr, JJ Lee, TH Nguyen, and K Sugirnaehi. DVA Med Cent, Long Beach, CA, Univ of Calif, Irvine, and Kyushu Univ., Japan. The lower esophagus is predisposed to varieeal development and rupture in the portal hypertensive (PHT) state. A possible mechanism may be overexpression of nitric oxide synthase (NOS) resulting in a local hyperdynamic state and weakened esophageal wall. To test this hypothesis, we analyzed expression ofNOS mRNA and performed quantitative histologie assessment in PHT esophagus. METHODS: Portal hypertension (PHT) was produced by staged portal vein ligation. In control rats and in PHT rats on 3, 7, and 14 postoperative days (POD), the lower esophagus was removed and fixed in 4% paraformaldehyde or frozen in liquid nitrogen. STUDIES: 1) Portal pressure; 2) Quantitative histology; 3) Reverse transcription-polymerase chain reaction (RT/PCR) using specific primers for NOS; 4) Immunohistoehemieal staining for constitutive NOS (eNOS) and inducible NOS (iNOS) with specific antibodies and quantitation of fluorescence signal with video image system. RESULTS: Portal pressure at 3 POD was significantly higher than in controls (33.4_+3.7 and 14.6+9.9 em H20, respectively, p<0.01), and persisted elevated pressure at 7 and 14 POD (28.3+9.2 and 26.8+9.4 cm H20, respectively, p<0.05). The esophageal muscularis mucosae and epithelium in the areas overlying dilated submucosal veins in PHT rats at 14 POD were significantly thinner than in controls (museularis mucosae: 27.7±5.3 vs 36.64-6.0 pro, p<0.01; epithelium: 40.4*6.0 vs 52.54-9.8 pm, p<0.05). With RT/PCR, NOS mRNA expression at 3, 7, and 14 POD was significantly increased (vs controls) by 91, 90, 112%, respectively (p<0.01 ). Immunofluorescence signal intensity of eNOS in endothelia of submucosal veins of PHT esophagus was significantly increased from 77+J units in controls to 86+4, 94-+6units at 7 and 14POD (!0<0.01). In museularis mucosae of PHT rats at 14 POD, eNOS was also significantly increased from 62-+10 to 82-+10 units (p<0.01). Fluorescence signal for iNOS in endothelia and museularis mucosae on 3, 7,14 POD was significantly increased vs controls by 1223 % (p<0.05). CONCLUSIONS: 1) Portal hypertension enhances the NOS gene expression in esophageal mucosa with resulting overexpression of both eNOS and !NOS protein. 2) The excess NO generated by NOS overexpression may play an tmportant role in the development of esophageal varices in portal hypertension, 3) and, combined with the thinner esophageal museularis mucosae and epithelium, may contribute to variceal rupture.
•
Esophageal, Gastric, and Duodenal Disorders
R A N I T I D I N E B I S M U T H CITRATE PROTECTS GASTRIC M U C O S A FROM I N D O M E T H A C I N - I N D U C E D INJURY IN RATS BY ALTERING THE A C I D PERMEABILITY OF GASTRIC M U C U S S. Tanaka, Y. Nishizaki, G. Paulsen, P.H. Guth and J.D. Kaunitz. Res. Svc. and Dept. of Medicine, VAMC & UCLA, Los Angeles, CA Ranitidine bismuth citrate, GR12231 I X (GR) prevents aspirin-induced gastric injury in healthy volunteers. Aim: To determine the mechanism by which GR exerts its gastreprotective effect in a rat model. Methods: GR (0- 100mg p.o.) was given 30 min prior to the injection of Indo (60mg/kg s.c.). Six hrs after Indo, gross and histologic lesions were scored. In separate experiments (same drug schedule), intracetlular pH (pH~ and mucusgel thidmess (MGT) were measured by in rive micrc~copy. Gastric mucc~al blood flow (GMBF) was measured by laser-Doppler tlowrnelry and acid output (AO) by back-titration. Gastric mucosae were superfused with pH 7.4 buffer during time= 0-30 min and pH 1.0 buffer during time = 30-50 min. Initial acidification rate OAR), a measure of mucus permeability to acid, was calculated from the fall in pH~during time = 30-32 min. Results: "p<0.05 vs. control; tp<0.05 vs. Indo:
Lesion Score AO (Hraollmin/cm2) %GMBF gross I histologie 0 min 40 min 150 min Control 134_+10 33.4_+3.3 Control 0.39_+0.0791.9_+1.1 83.6_+1.4 GR 10 mg/kg 160_+5" 23.5_+7.8 GR 100mg/kg 0,07_+0.02 88.5_+2.378J_+4.9 GR30 mg/kg 106_+79" 8.0_+4.4" Indo 0.27_+0.0688.0_+3.979.1_+2.5 GR 100 mg/kg 77_+11" 1.6_+lJ" GR+lndo 0.11_+0.05'93.1_+1.5 90.8_+5.4 Nor(~m) IAR pHi 30 min (rnM/min) 30 min I 32 min I 50 min control 114.8_+5.4 7.5+--0.5 control 7.04--+0.046.69-+0.02 6.59-+0.04 GR 100mg/kg 108.9-+52 6.4-+0.6t GR 100mg/kgt 7.05-+0.05 6.75-+0.04 6.60-+0.04 Indo 933-+4.8° 11.8±0.5" Indo'" 7.04-+0.046.51_+0.03 6.20_+0.07 GR+lndu 9L0±5J" 8J-+0.5t GR+lndo "~ 7.05-+0.03 6.68-+0.02 6.41-+0.05 Conclusions: Despite a thinned mucus gel, GR+lndo-treated rats had a normal IAR and near normal pH~ recovery during an acid load, in contrast to an increased IAR and impaired pH~ recovery in Indo treated rats. This indicated a GR-associated decrease in the acid permeability of the mucus gel, probably as a result of Bi3+ binding to gastric mucus. These results can thus help explain the protective effect of GR against indomethacin-induced gastric injury. (Supported by Glaxo Pharmaceuticals).
• PROTECTIVE EFFECT OF TEPRENONE ON PORTAL HYPERTENSIVE GASTRIC MUCOSA. K Tanoue, A Taruawski, ICI Waldsb'om, JJ Lee, FL Irwin, Jr, TH Nguyen, M Haslaizume,K Sugimachi, and IJ Sarfeh., DVA Med Cent, Long Beach CA and Univ Calif',lrvine, and Dpt Sttrg II, Kynshu Univ, Fukuoka, Japan. Teprenone (geranylgeranyl acetone) (TP) is a gastric mucosal protective drug used clinicallyin Japan for treatment of gastric ulcers and gastritis. Its effect on the portal hypertensive (PHT) gastric mucosa (which has impaired mucosal defenses and increased susceptibilityto injury) has not been studied. The aims of this study were to determine whether: a) TP pre-treatment may protect PHT gastric mucosa against ethanol injury and b) explain mechanisms of its mucosal protective action. METHODS: Portal hypertension (PHT) was produced by staged portal vein ligation in 40 rats. TP (100 mg/kg) or placebo (PL) were administered twice daily p.o. for 7 days. Gastric mucosal injury was produced by intragastric instillation of 8 ml/kg ethanol, and assessed macroscopically with a video image analysis system. STUDIES: Standardized gastric specimens were analyzed morphometrieally. In separate groups of PHT rats (without ethanol injury)we studied the effect of TP on defensive mechanisms of gastric mucosa: 1) portal pressure; 2) gastric pH; 3) gastric mucosal blood flow (GMBF) using laser Doppler flowmetry; 4) HXM content in gastric mucosa; 5) basic fibroblast growth factor (bFGF) expression in the gastric mucosa. This was done by using a video image analysis system at~er immtmostaining of mueosal section with a specific antibody. RESULTS: In PL pretreated rats, ethanol produced extensive necrosis. Histology revealed extensive deep hemorrhagic necrosis involving > 45+_5%ofmucosal sections length. TP did not affect portal pressure, but did sigmficantly reduce ethanol induced damage to PHT gastric mucosa, gross necrosis by 4-fold, deep hemorrhagic necrosis by 5 fold (!0<0.05). In rats without ethanol, GMBF was significantly higher in the TP group than in the PL group (4884-104 and 3464-70 PU respectively, p<0.05). TP treatment significantlyincreased HXM (174-3 and 134-2 lag/rag respectively). Immunofluorescence intensity of bFGF in endothelia of the submucosal veins and gastric mucosa was significantly higher in the TP group than in the PL group (147±11 vs 1074-16 units, p<0.01, and 76±5 vs 644-6 units, p<0.05, respectively). CONCLUSIONS: 1) TP significantly protects PHT gastric mucosa against ethanol injury. 2) TP administration increases GMBF, mucosal content ofHXM (a mucus component) and expression ofbFGF (an angiogenic factor) in endothelia of mucosal microvessels and submucosal veins of PHT gastric mucosa. 3) These effects of TP, combined with its known prostaglandni-stimulating action, suggest an important potential role of this agent in prophylaxis of PHT gastric mucosal injury.
A235
SUBSTITUTE, INEXPENSIVE DIAGNOSIS OF H. PYLORI INFECTION BY INFRA-RED(IR) SPECTEOPHOTOHETER IN IsC-UREA BREATH TEST Y. Taniguchi, K. Kimura, K.Satoh, H.Kawada, H.Sohara, H. Yamamoto, K. Ido, Y. Ogihara*, Y. Goto*, K. Takatori*, K. lida*, M.Kadiwara # Dept. of Gastroenterology, Jichi Medical School, Tochigi, Japan *Exploratory Research Lab. ~, Daiichi R a D Center, Tokyo and ~Dept. of Medicinal Chemistry, Heiji College of Pharmacy, Tokyo Since the ~SC-urea breath test has been described to assess H. pylori infection, most of the a l t e r n a t i v e protocols have been successful, except the cost benefit, s i m p l i c i t i e s and timeoomsumption with Mass spectrophotometric analysis. In order to overcome the above shortages, we designed to u t i l i z e infra-red(IR) spectrometer as a s u b s t i t u t e for Mass spectrometer. [Methods] Total 82 p a t i e n t s suffered with peptic ulcers or nonulcer dyspepsia were investigated. Breath samples were c o l l e c t e d at before, 15, 30, 45 and 60 min post-ingestion of l~C-urea (100 mg in 30 ml water) for time-course study and a t 15 min for diagnostic evaluation. IR spectrophotometer (EX130, JASCO,Tokyo) was used to determine the concentration of 1~C02 in each expirate. In addition, 13C02/i2C02 r a t i o was also measured by Mass spectrometry in order to compare with IR spectrometric analysis. Direct detection of H.pylori was q u a l i f i e d according to our methods in biopsy specimens (Am J Gastroenterol 1991;66:285-9I). EResults] 65 p a t i e n t s were p o s i t i v e in H. pylori infection and 17 were negative. In the urea breath t e s t , the plateau responses of 13C02 excretion was achieved between 15 and 30 min in the timecourse study. The mean value in the p o s i t i v e group was s i g n i f i cantly higher than that in the negative group (0.057 ± 0.035 vs 0.006 ± 0.004, respectively). The c u t - o f f level was determined 0.01 as h ~3C atom%. The s e n s i t i v i t y of IR spectrometer was 96.9% (63/65) and the s p e c i f i c i t y was 88. 2% (15/17). Correlation between Hass and IR photometric a n a l y s i s revealed extremely high c o e f f i c i e n t (r=0.999). [Conclusions] I t is suggested t h a t IR spectrometry has high p o t e n t i a l s not only to diagnose H. pylori infection but also to estimate the treatment with several advantages including technical s i m p l i c i t y , cost e f f e c t i v e n e s s and high accuracy.
• SEQUENTIAL ASSESSMENT OF NITRIC OXIDE SYNTHASE mRNA AND PROTEIN EXPRESSION IN PORTAL HYPERTENSIVE ESOPHAGEAL MUCOSA. K Taneue, IJ Sarfeh, A Tamawski, KJ Wahlstrom, FL Irwin Jr, JJ Lee, TH Nguyen, and K Sugirnaehi. DVA Med Cent, Long Beach, CA, Univ of Calif, Irvine, and Kyushu Univ., Japan. The lower esophagus is predisposed to varieeal development and rupture in the portal hypertensive (PHT) state. A possible mechanism may be overexpression of nitric oxide synthase (NOS) resulting in a local hyperdynamic state and weakened esophageal wall. To test this hypothesis, we analyzed expression ofNOS mRNA and performed quantitative histologie assessment in PHT esophagus. METHODS: Portal hypertension (PHT) was produced by staged portal vein ligation. In control rats and in PHT rats on 3, 7, and 14 postoperative days (POD), the lower esophagus was removed and fixed in 4% paraformaldehyde or frozen in liquid nitrogen. STUDIES: 1) Portal pressure; 2) Quantitative histology; 3) Reverse transcription-polymerase chain reaction (RT/PCR) using specific primers for NOS; 4) Immunohistoehemieal staining for constitutive NOS (eNOS) and inducible NOS (iNOS) with specific antibodies and quantitation of fluorescence signal with video image system. RESULTS: Portal pressure at 3 POD was significantly higher than in controls (33.4_+3.7 and 14.6+9.9 em H20, respectively, p<0.01), and persisted elevated pressure at 7 and 14 POD (28.3+9.2 and 26.8+9.4 cm H20, respectively, p<0.05). The esophageal muscularis mucosae and epithelium in the areas overlying dilated submucosal veins in PHT rats at 14 POD were significantly thinner than in controls (museularis mucosae: 27.7±5.3 vs 36.64-6.0 pro, p<0.01; epithelium: 40.4*6.0 vs 52.54-9.8 pm, p<0.05). With RT/PCR, NOS mRNA expression at 3, 7, and 14 POD was significantly increased (vs controls) by 91, 90, 112%, respectively (p<0.01 ). Immunofluorescence signal intensity of eNOS in endothelia of submucosal veins of PHT esophagus was significantly increased from 77+J units in controls to 86+4, 94-+6units at 7 and 14POD (!0<0.01). In museularis mucosae of PHT rats at 14 POD, eNOS was also significantly increased from 62-+10 to 82-+10 units (p<0.01). Fluorescence signal for iNOS in endothelia and museularis mucosae on 3, 7,14 POD was significantly increased vs controls by 1223 % (p<0.05). CONCLUSIONS: 1) Portal hypertension enhances the NOS gene expression in esophageal mucosa with resulting overexpression of both eNOS and !NOS protein. 2) The excess NO generated by NOS overexpression may play an tmportant role in the development of esophageal varices in portal hypertension, 3) and, combined with the thinner esophageal museularis mucosae and epithelium, may contribute to variceal rupture.