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Helicobacter-extract stimulates production of nitric oxide, an endogenous carcinogen
April 1995
• EFFECTS OF NS-398, A SPECIFIC INHIBITOR FOR CYCLOOXYGENASE-2, ON GASTRIC CELL PROLIFERATION. S.Tsuii. H.Sawaoka, S.Kawano, Y.Takei, K.Nagano, H.Fusamoto and T.Kamada. Dept. of MOd., Osaka Univ. Sch. of Med., Suita, Japan. Background and Aims: Although their products affect peptic ulcer healing and gastric cytoprotection, the roles of cyclcoxygenases (COXs), constitutive COX-1 and mitogen-inducible COX-2, in gastric cell kinetics are not known. NS-398 is a novel sulfonamide derivative which inhibits COX-2 activities by 50% at the concentrations less than lif e M, but did not affect COX-I activities at 10-4 M. In this study, we examined the effects of NS-398 on cell proliferation of RGM1 (Matsui & Ohno, 1989, RCB-0876), a cell-line established from rat gastric mucosal epithelium. Gene expression of COX-2 was also examined in this cell-line. Materials and Methods: RGM1, a gift from Matsui and Ohno, had cytokeratin, scanty secretary granules and mierovilli. The cells were deprived from fetal calf serum (FCS) for 3 days. The quiescent cells were stimulated by changing media containing 0, 2, 5, 20% FCS, or FCS plus 10"9-10-5 M of NS-398. Gastric cell proliferation was assessed by M'Fr assay and tripan-blue dye exclusion test 3 days later. Total RNA were extracted by acid guanidium-phenol-ehloroform method and analyzed by Northern hybridization with COX-2 eDNA. Results: RGM1 proliferated with FCS in a dose-dependent manner. The shortest doubling time was 15.8 h. after stimulation with 20% FCS. NS-398 strongly suppressed the proliferation of RGMI in a dose-dependent manner. Northern analysis demonstrated increased expression of COX-2 mRNA 20-40 rain. after the addition of FCS. Discussion and conclusion: The expression of COX-2 mRNA increases in RGM1 cells after the mitogenic stimulation. NS-398, a COX-2 specific inhibitor, inhibits the cell proliferation. These results indicates that expression of COX-2 and COX-2 products play a pivotal role in gastric cell replication.
GASTROESOPHAGEAL MOTILITY IN INFANTS WITH GASTRIC VOLVULUS. K.Tsakada, K. Kosuge, M. Hosokawa, R. Umezu, M. Mnrata. Dept. of Pediatrics, Tokyo Women's Medical College, Daini Hospital Tokyo, Japan. Gastric volvulus (GV) is an unusual clinical problem in infancy. In the complete type there should be little difficulty in clinical recognition. However, in tlw incomplete type the symptoms may be sufficiently mild and infrequent as to be overlooked, or may be suspected gastroesophageal reflux (GER) until diagnostic studies disclose the lesion. IncompleteGV may be responsible for gastroesophageal dysmotility or GER because it likely induces gastric distension. We analyzed the results of studies of gastroesophageal motility in infants with GV to investigate the influence. Subjects and Methods: In 10 infants with GV (6 days- 8 months old. symptoms; reccarrent vomiting, wheezing, poor weight gain. apnea, and choking) esophageal motility and lower esophageal sphincter pressure (LESP) were evaluated by esophageal manometry using a catheter with 3 solid state pressure transducers. Gastroesophageal reflux was evaluated by 24 hour pH monitoring (pn-metry) with 2 flexible antimony pH electrodes. Upper GI series with barium (UGt) was also performed recorded with video-film to obtain esophagogra~ radiologic evidence of GER, and gastric emptying time. The positive cases of GER on pU-metry were treated with not only positional therapy for GV but also antacids and/or prokinetics. Results and Conclusions: In manometric study hypotonie LES revealed in 2 out of the I0 cases, incomplete relaxation of LES in 2 cases. On pH-motry GER was positive in 5 cases. Upper GI series detected GER in 7 cases, esophageal spasm or to-and-fro movement of intraesophageal barium in 8 cases, and significant delay of the gastric emptying time in none of the 10 cases. We concluded that GV might induce esophageal dysmotility and/or GER, and gastroesophageal function should be evaluated in the eases of GV.
Esophageal, Gastric, and Duodenal Disorders
A245
HELICOBACTER-EXTRACT S ~ U L A ~
PRODUCTION OF NITRIC OXIDE, AN EINrDOGENOUS CARCINOGEN. $.Tsuii, S.I,"awano, Y.Takei, E.Masuda, H.Fusamoto, T.I~Lamada. 1st Dept. of MOd., Osaka Univ. School of Med. Suita, Japan. Background and Aims: A growing body of evidence indicates that Helicobacter pylori flip) infection is associated with gastric carcinogenesis. However, precise mechanism for Hp-related carcinogenesis remains unclear. Nitric oxide (NO), a free radical originated from inflammatory cells, is an endogenous precursor of nitroso-compounds as well as a direct mutagen. In this study, we examined the effects of Hp-extract on rat macrophages and neutrophils. Materials and Methods: Five clinical strains of Hp were isolated from Japanese patients, grown in Skirrow's agar under a microaerobic condition, and suspended in phosphate buffered saline (10s e.f.u./ml). The cell suspension was sonicated, ultracentrifuged and unsepticaily filtrated. Aliquots were also heated at 98°C or treated with protease K for 2 hrs. Peritoneal macrophages and neutrophils were obtained from specific pathogen-free Sprague Dawley rats after stimulation with thioglycolate medium and sodium caseinate, respectively. The cells were incubated with Rt'MI-1640 medium with or without L-arginine (200 mg/L) containing 0-25% of the Hpextract. L-arginine-dependent NO production was assessed by Griess reaction. Results: The Hp-extract stimulated NO-production in rat peritoneal maerophages and neutrophils in a dose-dependent manner. The heat-treatment and the protease-digestion significantly decreased the effect of the Hp-extraet on the NO production in these cells. Discussion and conclusion: Recent studies indicate that inflammatory~ NO is an endogenous precursor of nitrosocompounds in humans and animals. In vitro studies also demonstrate that NO deaminates DNA and causes mutation in bacteria and mammarian cells in culture. The present result support the hypothesis that Hp-infeetion induces gastric inflammation and stimulates production of NO, an endogenous carcinogen.
INFLUENCE OF AGING ON THE HEALING OF CHRONIC GASTRIC ULCERS IN RATS. Y. Tsukimi S. Okabe. Department of Applied Pharmacology, Kyoto Pharmaceutical University, Kyoto, Japan Aging is known to cause gastric dysfunction. The present study was designed to investigate the influence of aging on healing of chronic gastric ulcers as well as gastric functions related to ulcer healing. [Method] Male Fischer-344 rats (young; 2 mo, aged; 24-26 mo) were used. Gastric ulcers were induced by luminal application of acetic acid (60%, 0.2ml, 45sec). At 1, 10 and 20 days after ulceration, ulcerated area and histological parameters (the length of regenerated mucosa and ruptured muscularis mucesa) were measured. Gastric acid secretion, mucosal blood flow, gastrin levels, mucosal DNA synthesis and motility (contraction and relaxation) were compared between young and aged rats. [Results] One day after acid application, the penetrated ulcers were the same size in both groups (110 mm2). These ulcers healed with time in a similar manner, but the healing was slightly delayed at 20 days in aged animals, the ulcerated area of young and aged animals being 6.3_+0.3 ram2 and 8.6_+0.5 mm2, respectively. In aged animals, regeneration of ulcerated mueosa was significantly lower, whereas the tissue contraction proceeded as determined by the length of the ruptured musculalris mucosa. Basal and histamine-stimulated gastric acid secretions of aged animals were significantly lower than those of young animals. The increase in mucosal blood flow at ulcer margin and mucosal cell proliferation was reduced in aged animals. However, serum gastrin levels were almost the same in both groups. Gastric contractile activity induced by electric vagal stimulation was almost the same in both groups, while gastric relaxative response via NANC neurons was significantly reduced in aged animals. [Conclusion] We conclude that 1) aging had little or no effect on healing of chronic gastric ulcers in rats, and 2) the influence of aging on healing of gastric ulcers may be due to various gastric dysfunctions, including positive and negative components in the ulcer healing.
• EFFECTS OF NS-398, A SPECIFIC INHIBITOR FOR CYCLOOXYGENASE-2, ON GASTRIC CELL PROLIFERATION. S.Tsuii. H.Sawaoka, S.Kawano, Y.Takei, K.Nagano, H.Fusamoto and T.Kamada. Dept. of MOd., Osaka Univ. Sch. of Med., Suita, Japan. Background and Aims: Although their products affect peptic ulcer healing and gastric cytoprotection, the roles of cyclcoxygenases (COXs), constitutive COX-1 and mitogen-inducible COX-2, in gastric cell kinetics are not known. NS-398 is a novel sulfonamide derivative which inhibits COX-2 activities by 50% at the concentrations less than lif e M, but did not affect COX-I activities at 10-4 M. In this study, we examined the effects of NS-398 on cell proliferation of RGM1 (Matsui & Ohno, 1989, RCB-0876), a cell-line established from rat gastric mucosal epithelium. Gene expression of COX-2 was also examined in this cell-line. Materials and Methods: RGM1, a gift from Matsui and Ohno, had cytokeratin, scanty secretary granules and mierovilli. The cells were deprived from fetal calf serum (FCS) for 3 days. The quiescent cells were stimulated by changing media containing 0, 2, 5, 20% FCS, or FCS plus 10"9-10-5 M of NS-398. Gastric cell proliferation was assessed by M'Fr assay and tripan-blue dye exclusion test 3 days later. Total RNA were extracted by acid guanidium-phenol-ehloroform method and analyzed by Northern hybridization with COX-2 eDNA. Results: RGM1 proliferated with FCS in a dose-dependent manner. The shortest doubling time was 15.8 h. after stimulation with 20% FCS. NS-398 strongly suppressed the proliferation of RGMI in a dose-dependent manner. Northern analysis demonstrated increased expression of COX-2 mRNA 20-40 rain. after the addition of FCS. Discussion and conclusion: The expression of COX-2 mRNA increases in RGM1 cells after the mitogenic stimulation. NS-398, a COX-2 specific inhibitor, inhibits the cell proliferation. These results indicates that expression of COX-2 and COX-2 products play a pivotal role in gastric cell replication.
GASTROESOPHAGEAL MOTILITY IN INFANTS WITH GASTRIC VOLVULUS. K.Tsakada, K. Kosuge, M. Hosokawa, R. Umezu, M. Mnrata. Dept. of Pediatrics, Tokyo Women's Medical College, Daini Hospital Tokyo, Japan. Gastric volvulus (GV) is an unusual clinical problem in infancy. In the complete type there should be little difficulty in clinical recognition. However, in tlw incomplete type the symptoms may be sufficiently mild and infrequent as to be overlooked, or may be suspected gastroesophageal reflux (GER) until diagnostic studies disclose the lesion. IncompleteGV may be responsible for gastroesophageal dysmotility or GER because it likely induces gastric distension. We analyzed the results of studies of gastroesophageal motility in infants with GV to investigate the influence. Subjects and Methods: In 10 infants with GV (6 days- 8 months old. symptoms; reccarrent vomiting, wheezing, poor weight gain. apnea, and choking) esophageal motility and lower esophageal sphincter pressure (LESP) were evaluated by esophageal manometry using a catheter with 3 solid state pressure transducers. Gastroesophageal reflux was evaluated by 24 hour pH monitoring (pn-metry) with 2 flexible antimony pH electrodes. Upper GI series with barium (UGt) was also performed recorded with video-film to obtain esophagogra~ radiologic evidence of GER, and gastric emptying time. The positive cases of GER on pU-metry were treated with not only positional therapy for GV but also antacids and/or prokinetics. Results and Conclusions: In manometric study hypotonie LES revealed in 2 out of the I0 cases, incomplete relaxation of LES in 2 cases. On pH-motry GER was positive in 5 cases. Upper GI series detected GER in 7 cases, esophageal spasm or to-and-fro movement of intraesophageal barium in 8 cases, and significant delay of the gastric emptying time in none of the 10 cases. We concluded that GV might induce esophageal dysmotility and/or GER, and gastroesophageal function should be evaluated in the eases of GV.
Esophageal, Gastric, and Duodenal Disorders
A245
HELICOBACTER-EXTRACT S ~ U L A ~
PRODUCTION OF NITRIC OXIDE, AN EINrDOGENOUS CARCINOGEN. $.Tsuii, S.I,"awano, Y.Takei, E.Masuda, H.Fusamoto, T.I~Lamada. 1st Dept. of MOd., Osaka Univ. School of Med. Suita, Japan. Background and Aims: A growing body of evidence indicates that Helicobacter pylori flip) infection is associated with gastric carcinogenesis. However, precise mechanism for Hp-related carcinogenesis remains unclear. Nitric oxide (NO), a free radical originated from inflammatory cells, is an endogenous precursor of nitroso-compounds as well as a direct mutagen. In this study, we examined the effects of Hp-extract on rat macrophages and neutrophils. Materials and Methods: Five clinical strains of Hp were isolated from Japanese patients, grown in Skirrow's agar under a microaerobic condition, and suspended in phosphate buffered saline (10s e.f.u./ml). The cell suspension was sonicated, ultracentrifuged and unsepticaily filtrated. Aliquots were also heated at 98°C or treated with protease K for 2 hrs. Peritoneal macrophages and neutrophils were obtained from specific pathogen-free Sprague Dawley rats after stimulation with thioglycolate medium and sodium caseinate, respectively. The cells were incubated with Rt'MI-1640 medium with or without L-arginine (200 mg/L) containing 0-25% of the Hpextract. L-arginine-dependent NO production was assessed by Griess reaction. Results: The Hp-extract stimulated NO-production in rat peritoneal maerophages and neutrophils in a dose-dependent manner. The heat-treatment and the protease-digestion significantly decreased the effect of the Hp-extraet on the NO production in these cells. Discussion and conclusion: Recent studies indicate that inflammatory~ NO is an endogenous precursor of nitrosocompounds in humans and animals. In vitro studies also demonstrate that NO deaminates DNA and causes mutation in bacteria and mammarian cells in culture. The present result support the hypothesis that Hp-infeetion induces gastric inflammation and stimulates production of NO, an endogenous carcinogen.
INFLUENCE OF AGING ON THE HEALING OF CHRONIC GASTRIC ULCERS IN RATS. Y. Tsukimi S. Okabe. Department of Applied Pharmacology, Kyoto Pharmaceutical University, Kyoto, Japan Aging is known to cause gastric dysfunction. The present study was designed to investigate the influence of aging on healing of chronic gastric ulcers as well as gastric functions related to ulcer healing. [Method] Male Fischer-344 rats (young; 2 mo, aged; 24-26 mo) were used. Gastric ulcers were induced by luminal application of acetic acid (60%, 0.2ml, 45sec). At 1, 10 and 20 days after ulceration, ulcerated area and histological parameters (the length of regenerated mucosa and ruptured muscularis mucesa) were measured. Gastric acid secretion, mucosal blood flow, gastrin levels, mucosal DNA synthesis and motility (contraction and relaxation) were compared between young and aged rats. [Results] One day after acid application, the penetrated ulcers were the same size in both groups (110 mm2). These ulcers healed with time in a similar manner, but the healing was slightly delayed at 20 days in aged animals, the ulcerated area of young and aged animals being 6.3_+0.3 ram2 and 8.6_+0.5 mm2, respectively. In aged animals, regeneration of ulcerated mueosa was significantly lower, whereas the tissue contraction proceeded as determined by the length of the ruptured musculalris mucosa. Basal and histamine-stimulated gastric acid secretions of aged animals were significantly lower than those of young animals. The increase in mucosal blood flow at ulcer margin and mucosal cell proliferation was reduced in aged animals. However, serum gastrin levels were almost the same in both groups. Gastric contractile activity induced by electric vagal stimulation was almost the same in both groups, while gastric relaxative response via NANC neurons was significantly reduced in aged animals. [Conclusion] We conclude that 1) aging had little or no effect on healing of chronic gastric ulcers in rats, and 2) the influence of aging on healing of gastric ulcers may be due to various gastric dysfunctions, including positive and negative components in the ulcer healing.