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Helicobacter pylori broth culture supernantants activate protein kinase C
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AGA ABSTRACTS
GASTROENTEROLOGY, Vol. 1 0 8 , No. 4
INHIBITION OF GASTRIC MUCOSAL MUCIN RECEPTOR BY H; PYLORI LPS: EFFECT OF EBROTIDINE. B.L. Slomiany, J. Piotrowski, V.L.N. Murty and A. Slomiany. Res. Ctr., UMDNJ, Newark, NJ 07103.
S U L G L Y C O T I D E A F F E C T S T H E S U S C E P T I B I L I T Y OF H. PYLORI TO ANTIMICROBIAL AGENTS. B.L. Slomianv, J. Piotrowski and A. Slomiany. Res. Ctr., UMDNJ, Newark, NJ, USA
Helicobacter pylori, a Gram-negative bacterium c o l o n i z i n g the e p i t h e l i a l s u r f a c e s of g a s t r i c mucosa, is recognized as a major cause of acute and c h r o n i c g a s t r i t i s , a n d t h e d e v e l o p m e n t of g a s t r i c and d u o d e n a l ulcers. A m o n g the m o s t striking alterations in gastric mucosal defense is the loss of mucus coat continuity and its patchy appearance. Thus, the bacterium evokes severe d i s t u r b a n c e s in t h e a b i l i t y of m u c u s c o a t to m a i n t a i n its f u n c t i o n s as the p r e - e p i t h e l i a l e l e m e n t of g a s t r i c m u c o s a l defense. In t h i s study, we present evidence that H. pylori lipopolysaccharide (LPS) interferes with the mucin binding site on the mucosal receptor, and that antiulcer agent, ebrotidine, is capable of counteracting this untoward effect of the bacterium. The receptor for mucin was isolated from the solubilized gastric epithelial cell membrane by affinity chromatography on Sepharosebound wheat germ a g g l u t i n i n and, following iodination with 125I-used in the binding assays for mucin in the presence of H. pylori LPS and ebrotidine. The mucin binding to the receptor was susceptible to H. pylori LPS and reached a maximum inhibition of 91% at 30Bg/ml. The effect of H. pylori was countered by ebrotidine, preincubation of which with the LPS caused a dose-dependent relief of the inhibitory effect. The maximum restoration o f 51% in mucin-receptor binding was attained at 60~g/ml ebrotidine. The results show that H. pylori, through its lipopolysaccharide, is c a p a b l e of d i s r u p t i n g the i n t e g r i t y of m u c u s p e r i m e t e r of g a s t r i c m u c o s a l d e f e n s e a n d t h a t a n t i u l c e r agent, e b r O t i d i n e , c o u n t e r a c t s t h i s untoward effect.
Sulglycotide is a potent antiulcer agent d e r i v e d from pig duodenal mucin by a process of extensive p r o t e o l y s i s and c h e m i c a l e s t e r i f i c a t i o n of its carbohydrate chains with sulfate. The agent has been also shown to display a strong H. pylori aggregating activity and exerts a potent inhibitory effect on t h e m u c o l y t i c e n z y m e s elaborated by the bacterium. In this study, w e assessed the effect of sulglycotide on the in vitro activity of antibiotics commonly used in the treatment for H. pylori eradication. The experiments were conducted with H. pylori, strain ATCC 43504, cultured for 72h in B r u c e ~ a broth medium, yielding a viable count of 5 x 10" CFU/ml. Aliquots of inoculum (20#i) were transferred to the wells containing different concentrations of antibiotics; metronidazole, erythromycin, tetracycline o r amoxycillin, either alone or in the presence of various doses of sulglycotide, and incubated at 37°C for 3 days for MIC evaluation. The results of assays in the absence of sulglycotide gave MIC value 0.10mg/L for erythromycin, 0.12mg/L for amoxycillin, 0.15mg/L for tetracycline, and 14mg/L for metronidazole, w h i l e s u l g l y c o t i d e g a v e M I C v a l u e of 20mg/L. Inclusion of sulglycotide in the assay systems led to e n h a n c e m e n t in M I C of a n t i b i o t i c s . The sulglycotide at its optimal dose of 5mg/L evoked a 4-fold enhancement in the MIC of amoxycillin, 5fold enhancement in the MIC of tetracycline, and 8 . 3 - f o l d i n c r e a s e in the M I C of e r y t h r o m y c i n , while the MIC of metronidazole improved 4-fold a t 10mg/L sulglycotide. The results point towards the value of a combination therapy of sulglycotlde and antibiotics in the treatment of gastric disease caused by H. pylori.
• EXPRESSION OF H,K-ATPase SUBUNITS AND HISTAMINE H2 RECEPTOR IN INSECT CELLS. A. Smolkm P. Zwarych, I.D. Molano. Dept: of Medicine, Medical University of South Carolina, Charleston, SC We assessed recombinant baculovirus (BV)-infected insect cells as a potential acid-secretory model. Gastric acid secretion is mediated by H,K-ATPase, an integral protein of parietal cell (PC) apical membranes consisting of a 94 kDa non-glycosylated catalytic a subunit and a tightly-associated 60-80 kDa glycosylated .6 subunit. Acid secretion is regulated in part by histamine interacting with H2 receptors on PC basolateral membranes, leading to increased cAMP levels, initiation of a protein phosphorylation cascade, and stimulation of H + pump activity. In this study, lepidopteran ovarian (sfg) cells were infected with recombinant BV carrying pig H,K-ATPase a and B subunit cDNAs. Confocal microscopic immun0cytochemistry with H,K-ATPase-specific antibodies showed that both a and f~ subunits were targeted to Sf9 cell plasma membranes within 48 hours of infection. Immunoblotting showed that early in infection, 1~subunits were expressed as 34 kDa core peptides which over 48 hours were progressively transformed into mature 60-80 kDa species. Sf9 cells infected with canine H2 receptor recombinant BV expressed a protein of the size (40 kDa) predicted from receptor primary structure deduced from DNA sequence. In addition, infected Sf9 cells bound |methyl-3H]-tiotidine (receptor agonist), and this binding was dose-dependently displaced by cimetidine (antagonist) with an ED50 of 10"7 M. Diphenhydramine (HI receptor antagonist, up to 10-3 M) failed to displace tiotidine binding to these Sf9 cells. The results show that Sf9 cells express exogenous H,K-ATPase a and £ subunits, carry out posttranslational 1~subunit glycosylation, and direct both subunits to the appropriate cellular compartment. The cells also express exogenous histamine H2 receptor which is functional in terms of ligand binding. Thus, insect cell expression systems may allow studies of acid-secretory stimulus-secretion coupling, and of H + pump subunit assembly and functional interaction (DK 43138).
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HELICOBACTER PYLORI BROTH CULTURE SUPERNATANTS ACTIVATE PROTEIN KINASE C. D.T. Smoot, J.H. Resau, K.A. Eltiget, M.H. Eadington, A.W. Wood, A. Asseffa. Depts. of Medicine and Biochemistry, Howard Univ. College of Medicine, Washington, D.C.; Dept. of Pathology, Univ. of Maryland School of Medicine, Baltimore, MD & ABL-BRP, NCI-FCRDC, Frederick, MD I-t. pylori (HP) is the most common cause of active chronic gastritis. The mechanisms by which HP directly injures gastric epithelial cells is not well understood. HP secretes a protein (VacA) that stimulates reversible vacuolation in eukaryotic cells. The following studies were undertaken to determine if HP broth culture supernatants with VacA activity stimulate protein kinase C (PKC). AGS cells, human gastric cancer cells, and HAE cells, SV4O TAg transformed normal human gastric epithelial cells, were used in the these experiments. AGS and HAE cells were grown on chamber slides and exposed HP broth culture supematants with VacA activity diluted in culture medium. Controls were exposed to Brucella broth alone. After 15, 30 and 60 minutes of exposure, cells were fixed and stained using a monoclonal antibody specific for PKC. Analysis by laser scanning confocal microscopy (LSCM) showed increased PKC staining starting at 15 minutes with continued increasing staining intensity after 30 and 60 minutes in HAE cells. Similarly, analysis of AGS cells showed a dramatic increase in staining as eady as 15 minutes, with peak staining intensity at 30 minutes. In addition to increased stain intensity, LSCM also demonstrated translocation of PKC from the cytoplasm to the cell membrane in HP broth treated cells, which is an indicator of PKC activation. Biochemical assay for PKC activity demonstrated increased PKC activity after exposure to HP broth reaching maximal specific activity at 60 minutes. Analysis of intrecellular [Ca2*], by LSCM shows a 3 fold increase in cytosolic [Ca2.] in HP broth treated AGs calls peaking within 15 - 20 min of exposure. This Increase in cytosoUc [Ca2.] concentration may be required for the PKC activation we observed in these experiments. HP broth activates PKC, stimulating protein synthesis and translocation to the cytoplasmic membrane. The rise in cytosolic [Ca2.] and activation of PKC may be due to a common signaling pathway initiated by HP broth. Therefore, the data would suggest that HP broth-induced cytotoxicity to human gastric epithelial cells is mediated, in part, by the PKC/Ca2* signal transduction pathway. Further studies are needed to elucidate the role of Ca2÷and PKC in mediating H. pylori-inducad cell injury, and to determine if PKC may mediate host cell DNA damage, since PKC is known to alter gene expression. Supported by PHS Grant - SO6 GM08244.
AGA ABSTRACTS
GASTROENTEROLOGY, Vol. 1 0 8 , No. 4
INHIBITION OF GASTRIC MUCOSAL MUCIN RECEPTOR BY H; PYLORI LPS: EFFECT OF EBROTIDINE. B.L. Slomiany, J. Piotrowski, V.L.N. Murty and A. Slomiany. Res. Ctr., UMDNJ, Newark, NJ 07103.
S U L G L Y C O T I D E A F F E C T S T H E S U S C E P T I B I L I T Y OF H. PYLORI TO ANTIMICROBIAL AGENTS. B.L. Slomianv, J. Piotrowski and A. Slomiany. Res. Ctr., UMDNJ, Newark, NJ, USA
Helicobacter pylori, a Gram-negative bacterium c o l o n i z i n g the e p i t h e l i a l s u r f a c e s of g a s t r i c mucosa, is recognized as a major cause of acute and c h r o n i c g a s t r i t i s , a n d t h e d e v e l o p m e n t of g a s t r i c and d u o d e n a l ulcers. A m o n g the m o s t striking alterations in gastric mucosal defense is the loss of mucus coat continuity and its patchy appearance. Thus, the bacterium evokes severe d i s t u r b a n c e s in t h e a b i l i t y of m u c u s c o a t to m a i n t a i n its f u n c t i o n s as the p r e - e p i t h e l i a l e l e m e n t of g a s t r i c m u c o s a l defense. In t h i s study, we present evidence that H. pylori lipopolysaccharide (LPS) interferes with the mucin binding site on the mucosal receptor, and that antiulcer agent, ebrotidine, is capable of counteracting this untoward effect of the bacterium. The receptor for mucin was isolated from the solubilized gastric epithelial cell membrane by affinity chromatography on Sepharosebound wheat germ a g g l u t i n i n and, following iodination with 125I-used in the binding assays for mucin in the presence of H. pylori LPS and ebrotidine. The mucin binding to the receptor was susceptible to H. pylori LPS and reached a maximum inhibition of 91% at 30Bg/ml. The effect of H. pylori was countered by ebrotidine, preincubation of which with the LPS caused a dose-dependent relief of the inhibitory effect. The maximum restoration o f 51% in mucin-receptor binding was attained at 60~g/ml ebrotidine. The results show that H. pylori, through its lipopolysaccharide, is c a p a b l e of d i s r u p t i n g the i n t e g r i t y of m u c u s p e r i m e t e r of g a s t r i c m u c o s a l d e f e n s e a n d t h a t a n t i u l c e r agent, e b r O t i d i n e , c o u n t e r a c t s t h i s untoward effect.
Sulglycotide is a potent antiulcer agent d e r i v e d from pig duodenal mucin by a process of extensive p r o t e o l y s i s and c h e m i c a l e s t e r i f i c a t i o n of its carbohydrate chains with sulfate. The agent has been also shown to display a strong H. pylori aggregating activity and exerts a potent inhibitory effect on t h e m u c o l y t i c e n z y m e s elaborated by the bacterium. In this study, w e assessed the effect of sulglycotide on the in vitro activity of antibiotics commonly used in the treatment for H. pylori eradication. The experiments were conducted with H. pylori, strain ATCC 43504, cultured for 72h in B r u c e ~ a broth medium, yielding a viable count of 5 x 10" CFU/ml. Aliquots of inoculum (20#i) were transferred to the wells containing different concentrations of antibiotics; metronidazole, erythromycin, tetracycline o r amoxycillin, either alone or in the presence of various doses of sulglycotide, and incubated at 37°C for 3 days for MIC evaluation. The results of assays in the absence of sulglycotide gave MIC value 0.10mg/L for erythromycin, 0.12mg/L for amoxycillin, 0.15mg/L for tetracycline, and 14mg/L for metronidazole, w h i l e s u l g l y c o t i d e g a v e M I C v a l u e of 20mg/L. Inclusion of sulglycotide in the assay systems led to e n h a n c e m e n t in M I C of a n t i b i o t i c s . The sulglycotide at its optimal dose of 5mg/L evoked a 4-fold enhancement in the MIC of amoxycillin, 5fold enhancement in the MIC of tetracycline, and 8 . 3 - f o l d i n c r e a s e in the M I C of e r y t h r o m y c i n , while the MIC of metronidazole improved 4-fold a t 10mg/L sulglycotide. The results point towards the value of a combination therapy of sulglycotlde and antibiotics in the treatment of gastric disease caused by H. pylori.
• EXPRESSION OF H,K-ATPase SUBUNITS AND HISTAMINE H2 RECEPTOR IN INSECT CELLS. A. Smolkm P. Zwarych, I.D. Molano. Dept: of Medicine, Medical University of South Carolina, Charleston, SC We assessed recombinant baculovirus (BV)-infected insect cells as a potential acid-secretory model. Gastric acid secretion is mediated by H,K-ATPase, an integral protein of parietal cell (PC) apical membranes consisting of a 94 kDa non-glycosylated catalytic a subunit and a tightly-associated 60-80 kDa glycosylated .6 subunit. Acid secretion is regulated in part by histamine interacting with H2 receptors on PC basolateral membranes, leading to increased cAMP levels, initiation of a protein phosphorylation cascade, and stimulation of H + pump activity. In this study, lepidopteran ovarian (sfg) cells were infected with recombinant BV carrying pig H,K-ATPase a and B subunit cDNAs. Confocal microscopic immun0cytochemistry with H,K-ATPase-specific antibodies showed that both a and f~ subunits were targeted to Sf9 cell plasma membranes within 48 hours of infection. Immunoblotting showed that early in infection, 1~subunits were expressed as 34 kDa core peptides which over 48 hours were progressively transformed into mature 60-80 kDa species. Sf9 cells infected with canine H2 receptor recombinant BV expressed a protein of the size (40 kDa) predicted from receptor primary structure deduced from DNA sequence. In addition, infected Sf9 cells bound |methyl-3H]-tiotidine (receptor agonist), and this binding was dose-dependently displaced by cimetidine (antagonist) with an ED50 of 10"7 M. Diphenhydramine (HI receptor antagonist, up to 10-3 M) failed to displace tiotidine binding to these Sf9 cells. The results show that Sf9 cells express exogenous H,K-ATPase a and £ subunits, carry out posttranslational 1~subunit glycosylation, and direct both subunits to the appropriate cellular compartment. The cells also express exogenous histamine H2 receptor which is functional in terms of ligand binding. Thus, insect cell expression systems may allow studies of acid-secretory stimulus-secretion coupling, and of H + pump subunit assembly and functional interaction (DK 43138).
•
HELICOBACTER PYLORI BROTH CULTURE SUPERNATANTS ACTIVATE PROTEIN KINASE C. D.T. Smoot, J.H. Resau, K.A. Eltiget, M.H. Eadington, A.W. Wood, A. Asseffa. Depts. of Medicine and Biochemistry, Howard Univ. College of Medicine, Washington, D.C.; Dept. of Pathology, Univ. of Maryland School of Medicine, Baltimore, MD & ABL-BRP, NCI-FCRDC, Frederick, MD I-t. pylori (HP) is the most common cause of active chronic gastritis. The mechanisms by which HP directly injures gastric epithelial cells is not well understood. HP secretes a protein (VacA) that stimulates reversible vacuolation in eukaryotic cells. The following studies were undertaken to determine if HP broth culture supernatants with VacA activity stimulate protein kinase C (PKC). AGS cells, human gastric cancer cells, and HAE cells, SV4O TAg transformed normal human gastric epithelial cells, were used in the these experiments. AGS and HAE cells were grown on chamber slides and exposed HP broth culture supematants with VacA activity diluted in culture medium. Controls were exposed to Brucella broth alone. After 15, 30 and 60 minutes of exposure, cells were fixed and stained using a monoclonal antibody specific for PKC. Analysis by laser scanning confocal microscopy (LSCM) showed increased PKC staining starting at 15 minutes with continued increasing staining intensity after 30 and 60 minutes in HAE cells. Similarly, analysis of AGS cells showed a dramatic increase in staining as eady as 15 minutes, with peak staining intensity at 30 minutes. In addition to increased stain intensity, LSCM also demonstrated translocation of PKC from the cytoplasm to the cell membrane in HP broth treated cells, which is an indicator of PKC activation. Biochemical assay for PKC activity demonstrated increased PKC activity after exposure to HP broth reaching maximal specific activity at 60 minutes. Analysis of intrecellular [Ca2*], by LSCM shows a 3 fold increase in cytosolic [Ca2.] in HP broth treated AGs calls peaking within 15 - 20 min of exposure. This Increase in cytosoUc [Ca2.] concentration may be required for the PKC activation we observed in these experiments. HP broth activates PKC, stimulating protein synthesis and translocation to the cytoplasmic membrane. The rise in cytosolic [Ca2.] and activation of PKC may be due to a common signaling pathway initiated by HP broth. Therefore, the data would suggest that HP broth-induced cytotoxicity to human gastric epithelial cells is mediated, in part, by the PKC/Ca2* signal transduction pathway. Further studies are needed to elucidate the role of Ca2÷and PKC in mediating H. pylori-inducad cell injury, and to determine if PKC may mediate host cell DNA damage, since PKC is known to alter gene expression. Supported by PHS Grant - SO6 GM08244.