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Effects of suramin on gastric ulceration and EGF content of submandibular glands
A58
AGA ABSTRACTS
• HELICOBACTER PYLORI AND ITS FATTY ACID CIS-9,10METHYLENEOCTADECANOIC ACID STIMULATE PARIETAL CELL PROTEIN KINASE C. W~ Beil, C. Birkholz. Department of General Pharmacology, Medizinische Hochschule Hannover, Hannover, FRG We have recently reported that Helicobacter pylori (H.p.) affects parietgl ~ell acid production in a biphasic manner; i0 -i0 bacteria potentiated histamine- and dbcAMP-stimulated acid production, higher bacteria concentrations inhibited acid production (Beil e t al. Gut 35: 1176-80, 1994). H.p. produces the unusual fatty acid cis9,10-methyleneoctadecanoic acid (MOA). Cis-unsaturated fatty acids have been shown to be activators of protein kinase C (PK C):. In the present study we investigated whether H.p. and the H.p. fatty acid MOA activate parietal cell PK C. Results: In a cell-free cytosolic system derived from parietal cells PK C was activated in the presence of Ca2 ÷ by the H.p. culture medium extract and MOA i n a concentration-dependent manner. Kinetic analysis of PK C activation with respect to Ca 2+ showed that diolein increased the stimulatory effect of the culture medium extract and MOA at low Ca 2+ concentrations. Incubation of intact parietal cells with H.p. culture medium extract and MOA induced translocation of PK C activity from the cytosol to the membrane-bound fraction. The H.p. culture medium extract (from 3x10" bacteria) stimulated basal and potentiated histamine- and dbcAMP-stimulated acid secretion in parietal cells. C o n c l u s i o n : H.p. culture medium extract stimulates parietal cell acid production and activates parietal cell PK C. The PK C activating factor is cis-9,10-methyleneoctadecanoic acid. We suggest that stimulation and potentiation of parietal cell acid secretion by H.p. is caused by PK C activation.
• CHOLECYSTOKININ GENE EXPRESSION IN HUMAN DUODENAL BIOPSY SPECIMENS. N. Bentouimou, V. Leray, S. Bruley des Varannes, F. Zerbib, J.P. Galmiche. Equipe Fonctions Digestives et Nutrition, H6pital G e t R La~nnec BP1005, 44035 Nantes cedex. France. CCK is produced by duodenal "1" cells. Ingested nutrients, especially lipids and proteins, stimulate the secretion of CCK. Due to technical limitations, CCK gene expression was only studied n killed animals on mucosa scrappea Off. The aim of this study was to detect and assess the levels of CCK-mRNA from duodenal biopsy specimens obtained in humans undergoing routine gastrointestinal endoscopy. Methods. Six biopsy specimens were taken in 6 healthy subjects on two occasions. Endoscopy was performed without sedation on two different days, a) after a 12 h-fasting (time 0) and b) 6 or 12 h after feeding a fat liquid meal (600 kcal, 73% fat 20% proteins, 7% carbohydrates). In order to control the mea- n~duced CCK release, plasma CCK concentrations were determined in 6 age-matched healthy subjects by a highly specific and sensitive bioassay, which is based on the ability of CCK to stimulate amylase release from isolated rat pancreatic acini (J Clin Invest 1985;75:1144) 0~ 5, 10, 15, 30, 45, 60 and 120 min alter feeding. CCK gene expression was analysed by Northern-blotting using a specific probe. Normalization of CCK-mRNA values was performed with respect to phosphoglycerate kinase mRNA. Results. Plasma CCK increased from fasting values of 0.5:1:0.1 pmole/I (meat + SEM, n=6) to 8.2 + 1.5 pmole/I (P<0.01) 10 min after the beginning of the meaL, and then decreased to 1.2 + 0.5 pmole/I (P<0.02 compared to fasting) by 120 rain. CCK-mRNAs increased from 44 + 1% in fasting conditions to 74 + 6%, 6 h after the test meal (P<0.01), and then decreased to 47 + 1%, 12 I~ after the test meal. ¢0nclusions. These results suggest that a) detection and measure of CCK gene expression in human duodenal biopsy specimens is feasible, and b) the release of CCK is rapidly followed by an increase in the synthesis of CCK.
GASTROENTEROLOGY, VOl. 108, NO. 4
• OMEPRAZOLE AND COLLOIDAL BISMUTH SUBCITRATE INHIBIT HELICOBACTER PYLORI ATPASE. W. Beil. Department Of General Pharmacology, Medizinische Hochschule Hannover, Hannover, F R G Helicobacter pylori (H.p.) is Sensitive to the anti-ulcer agents colloidal bismuth subcitrate (CBS) and omeprazole in vitro. The mechanism of the anti-microbial action of CBS and omeprazole is not well understood. In bacteria a membrane-bound ATPase participates in energy transformation. The enzyme serves as a coupling factor in oxidative phosphorylation and facilitates proton transport across the bacterial membrane. In the present study we have examined the properties of H.p. ATPase (solubilized FI-ATPase ) and the reaction pathway of the enzyme with CBS and omeprazole. Results: 2+H'P" FI-ATPase was actlvated' by Mg 2+ but not by Ca . optlmum activation was observed at an ATP/Mg 2+ ratlo of 1/5 and pH 7.5. Azide, a known inhibitor of the FI-ATPase of bacteria, inhibited the enzyme. The anti-ulcer agents CBS and omeprazole inhibited in a concentration-dependent manner H.p. FI-ATPase with ICso values of 73 and 90 ~mol/l, respectively. Ranitidine, pirenzepine and sucralfate were devoid of such an effect. The inhibitory action of CBS and omeprazole was prevented and reversed by glutathione, indicating that both drugs interfere with SH-groups of the enzyme. Conclusions: The data indicate that CBS and Omeprazole owe their anti-bacterial activity against H.p., at least in par~, to inhbition of FI-ATPase , an enzyme involved in bacterial energy metabolism.
EFFECTS OF'SURAMIN ON GASTRIC ULCERATION AND EGF CONTENT OF SUBMANDIBULAR GLANDS. N. Bernardini I , C. Blandizzi2, F. Bianehi 1, G. Natale 2, M. Del Tacca 2. 1Institute of Anatomy and 2Institute of Medical Pharmacology, University of Pisa, Pisa, Italy The exocrine secretion of submandibular epidermal growth factor (EGF) reduces the susceptibility of gastric mucosa to the induction of acute necrotic damage [Tepperman et al., Gastroenterology 97, 123, 1989]. It is also known that suramin exerts anticancer effects by interfering with the biological activity of several growth factors, including EGF [Stein, Cancer Res. 53, 223, 1993]. In the present study, the influence exerted by suramin on ethanol-induced gastric mueosal damage as well as on immunohistochemically detectable content of EGF in the submandibular glands were investigated. Male Wistar rats (200-250 g b.w.) were treated with suramin (18 mg/kg every other day) or its vehicle intraperitorleally for 14 days. At the 15th day the animals, 24-h fasting, were acutely treated with absolute ethanol (1 ml/200 g b.w.) by intragastric gavage. After 1, 2, 4, 8, 12 and 24 h from ethanol administration, the stomachs were dissected out and used either for histological evaluation of mucosal damage (percent of necrotic mucosa v e r s u s total extension of mucosa) or for measuring the amount of mucus bound to the epithelial surface (/.tg of Alcian blue recovered per gram of wet glandular tissue). Histological sections, obtained from submandibular glands, were immunostained for EGF (anti-peroxidase method). Positive areas for EGF were quantitated by computer-aided image analysis. Suramin markedly enhanced ethanol-induced mucosal damage at all times tested, the peak effect being observed at 1 h (control: 9.7:£0.6%, n=8; suramin: 18.3i-0.3%, n=8; P<0.05). Bound gastric mucus was significantly increased following ethanol injection (basal: 116.6+16.6 Ixg of Alcian blue/g, n=8; peak at 1 h: 223.3+14.9 ~tg of Alcian blue/g, n=8; P<0.05). The increase in adherent mucus lasted up to 12 h and it was fully prevented by suramin. In addition, suramin caused a marked reduction (45.6%, n=30) of immunohistochemically detectable content of EGF in submandibular glands. It is concluded that a short-term treatment with suramin causes an impairment of protective mechanisms at the level of gastric mucosa. This ulcer-promoting effect might depend, at least in part, on a decrease in exocrine secretion of EGF from submandibular glands. The present findings may have significant implications in clinical settings.
AGA ABSTRACTS
• HELICOBACTER PYLORI AND ITS FATTY ACID CIS-9,10METHYLENEOCTADECANOIC ACID STIMULATE PARIETAL CELL PROTEIN KINASE C. W~ Beil, C. Birkholz. Department of General Pharmacology, Medizinische Hochschule Hannover, Hannover, FRG We have recently reported that Helicobacter pylori (H.p.) affects parietgl ~ell acid production in a biphasic manner; i0 -i0 bacteria potentiated histamine- and dbcAMP-stimulated acid production, higher bacteria concentrations inhibited acid production (Beil e t al. Gut 35: 1176-80, 1994). H.p. produces the unusual fatty acid cis9,10-methyleneoctadecanoic acid (MOA). Cis-unsaturated fatty acids have been shown to be activators of protein kinase C (PK C):. In the present study we investigated whether H.p. and the H.p. fatty acid MOA activate parietal cell PK C. Results: In a cell-free cytosolic system derived from parietal cells PK C was activated in the presence of Ca2 ÷ by the H.p. culture medium extract and MOA i n a concentration-dependent manner. Kinetic analysis of PK C activation with respect to Ca 2+ showed that diolein increased the stimulatory effect of the culture medium extract and MOA at low Ca 2+ concentrations. Incubation of intact parietal cells with H.p. culture medium extract and MOA induced translocation of PK C activity from the cytosol to the membrane-bound fraction. The H.p. culture medium extract (from 3x10" bacteria) stimulated basal and potentiated histamine- and dbcAMP-stimulated acid secretion in parietal cells. C o n c l u s i o n : H.p. culture medium extract stimulates parietal cell acid production and activates parietal cell PK C. The PK C activating factor is cis-9,10-methyleneoctadecanoic acid. We suggest that stimulation and potentiation of parietal cell acid secretion by H.p. is caused by PK C activation.
• CHOLECYSTOKININ GENE EXPRESSION IN HUMAN DUODENAL BIOPSY SPECIMENS. N. Bentouimou, V. Leray, S. Bruley des Varannes, F. Zerbib, J.P. Galmiche. Equipe Fonctions Digestives et Nutrition, H6pital G e t R La~nnec BP1005, 44035 Nantes cedex. France. CCK is produced by duodenal "1" cells. Ingested nutrients, especially lipids and proteins, stimulate the secretion of CCK. Due to technical limitations, CCK gene expression was only studied n killed animals on mucosa scrappea Off. The aim of this study was to detect and assess the levels of CCK-mRNA from duodenal biopsy specimens obtained in humans undergoing routine gastrointestinal endoscopy. Methods. Six biopsy specimens were taken in 6 healthy subjects on two occasions. Endoscopy was performed without sedation on two different days, a) after a 12 h-fasting (time 0) and b) 6 or 12 h after feeding a fat liquid meal (600 kcal, 73% fat 20% proteins, 7% carbohydrates). In order to control the mea- n~duced CCK release, plasma CCK concentrations were determined in 6 age-matched healthy subjects by a highly specific and sensitive bioassay, which is based on the ability of CCK to stimulate amylase release from isolated rat pancreatic acini (J Clin Invest 1985;75:1144) 0~ 5, 10, 15, 30, 45, 60 and 120 min alter feeding. CCK gene expression was analysed by Northern-blotting using a specific probe. Normalization of CCK-mRNA values was performed with respect to phosphoglycerate kinase mRNA. Results. Plasma CCK increased from fasting values of 0.5:1:0.1 pmole/I (meat + SEM, n=6) to 8.2 + 1.5 pmole/I (P<0.01) 10 min after the beginning of the meaL, and then decreased to 1.2 + 0.5 pmole/I (P<0.02 compared to fasting) by 120 rain. CCK-mRNAs increased from 44 + 1% in fasting conditions to 74 + 6%, 6 h after the test meal (P<0.01), and then decreased to 47 + 1%, 12 I~ after the test meal. ¢0nclusions. These results suggest that a) detection and measure of CCK gene expression in human duodenal biopsy specimens is feasible, and b) the release of CCK is rapidly followed by an increase in the synthesis of CCK.
GASTROENTEROLOGY, VOl. 108, NO. 4
• OMEPRAZOLE AND COLLOIDAL BISMUTH SUBCITRATE INHIBIT HELICOBACTER PYLORI ATPASE. W. Beil. Department Of General Pharmacology, Medizinische Hochschule Hannover, Hannover, F R G Helicobacter pylori (H.p.) is Sensitive to the anti-ulcer agents colloidal bismuth subcitrate (CBS) and omeprazole in vitro. The mechanism of the anti-microbial action of CBS and omeprazole is not well understood. In bacteria a membrane-bound ATPase participates in energy transformation. The enzyme serves as a coupling factor in oxidative phosphorylation and facilitates proton transport across the bacterial membrane. In the present study we have examined the properties of H.p. ATPase (solubilized FI-ATPase ) and the reaction pathway of the enzyme with CBS and omeprazole. Results: 2+H'P" FI-ATPase was actlvated' by Mg 2+ but not by Ca . optlmum activation was observed at an ATP/Mg 2+ ratlo of 1/5 and pH 7.5. Azide, a known inhibitor of the FI-ATPase of bacteria, inhibited the enzyme. The anti-ulcer agents CBS and omeprazole inhibited in a concentration-dependent manner H.p. FI-ATPase with ICso values of 73 and 90 ~mol/l, respectively. Ranitidine, pirenzepine and sucralfate were devoid of such an effect. The inhibitory action of CBS and omeprazole was prevented and reversed by glutathione, indicating that both drugs interfere with SH-groups of the enzyme. Conclusions: The data indicate that CBS and Omeprazole owe their anti-bacterial activity against H.p., at least in par~, to inhbition of FI-ATPase , an enzyme involved in bacterial energy metabolism.
EFFECTS OF'SURAMIN ON GASTRIC ULCERATION AND EGF CONTENT OF SUBMANDIBULAR GLANDS. N. Bernardini I , C. Blandizzi2, F. Bianehi 1, G. Natale 2, M. Del Tacca 2. 1Institute of Anatomy and 2Institute of Medical Pharmacology, University of Pisa, Pisa, Italy The exocrine secretion of submandibular epidermal growth factor (EGF) reduces the susceptibility of gastric mucosa to the induction of acute necrotic damage [Tepperman et al., Gastroenterology 97, 123, 1989]. It is also known that suramin exerts anticancer effects by interfering with the biological activity of several growth factors, including EGF [Stein, Cancer Res. 53, 223, 1993]. In the present study, the influence exerted by suramin on ethanol-induced gastric mueosal damage as well as on immunohistochemically detectable content of EGF in the submandibular glands were investigated. Male Wistar rats (200-250 g b.w.) were treated with suramin (18 mg/kg every other day) or its vehicle intraperitorleally for 14 days. At the 15th day the animals, 24-h fasting, were acutely treated with absolute ethanol (1 ml/200 g b.w.) by intragastric gavage. After 1, 2, 4, 8, 12 and 24 h from ethanol administration, the stomachs were dissected out and used either for histological evaluation of mucosal damage (percent of necrotic mucosa v e r s u s total extension of mucosa) or for measuring the amount of mucus bound to the epithelial surface (/.tg of Alcian blue recovered per gram of wet glandular tissue). Histological sections, obtained from submandibular glands, were immunostained for EGF (anti-peroxidase method). Positive areas for EGF were quantitated by computer-aided image analysis. Suramin markedly enhanced ethanol-induced mucosal damage at all times tested, the peak effect being observed at 1 h (control: 9.7:£0.6%, n=8; suramin: 18.3i-0.3%, n=8; P<0.05). Bound gastric mucus was significantly increased following ethanol injection (basal: 116.6+16.6 Ixg of Alcian blue/g, n=8; peak at 1 h: 223.3+14.9 ~tg of Alcian blue/g, n=8; P<0.05). The increase in adherent mucus lasted up to 12 h and it was fully prevented by suramin. In addition, suramin caused a marked reduction (45.6%, n=30) of immunohistochemically detectable content of EGF in submandibular glands. It is concluded that a short-term treatment with suramin causes an impairment of protective mechanisms at the level of gastric mucosa. This ulcer-promoting effect might depend, at least in part, on a decrease in exocrine secretion of EGF from submandibular glands. The present findings may have significant implications in clinical settings.