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Methimazole slows hepatocyte streaming in rats
HEPATOLOGY Vol. 22, No. 4, Pt. 2, 1995
769
AASLD A B S T R A C T S
EXPERIMENTAL COUTIS INCREASES HEPATOCYTE TIGHT JUNCTIONAL PERMEABILITY. L Lore, M. Mazzon*, W. Fries, MT. Vilei, C. Cadotto, M. Murasa, A. D'Odorico, R. Naccarato, D. Martines. Department of Internal Medicine, Department of Biology*-, University of Padua, Padua. Hepatobiliary complications are frequently observed in patients with inflammatory bowel disease (IBD). Translocation of intestinal bacteda and their toxins through the gut wall and invasion of the hepatobiliary system has been suggested as pathogenic mechanism. The integrity of hepatocellular tight junctions (-IJs) is thought to play an important role in preventing the entry of bacteria from the portal blood in the biliary tract. AIM of this study was to evaluate liver TJ morphology and function in experimental colitis in the rat. METHODS: male Sprague-Dawley rats (200250 g) were used. Experimental colitis was induced instilling in the colon 25 mg trinitrobenzene sulphonic acid in 1 ml 50% ethanol (TNB rats). Control group consisted of untreated animals (CON rats). At one week of TNBinduced colitis, morphological study of TJs was performed after perfusing the livers with lanthanum nitrate (4%) (n=3 per group) and analyzing its penetration in the junctional complexes and bile cacalicular lumen by Transmission Electron Microscopy, In a second sedes of experiments, to examine the biliary paracellular permeability, horseradish peroxidase (HRP) was pulsed, within 1 rain, into perfused rat livers (5 per groups) under single-pass conditions. Bile was collected before peffusion for analysis of basal bile flow rate and bile salt excretion and during peffusion to examine the pattern of HRP biliary excretion. RESULTS:Basal bile flow rate and bile salt excretion were unchanged in TNB rats. Electron microscopic examination (35.000 X) showed lanthanum accumulation in the 85.6+ 1.4% of 335 _+35 TJs observed in TNB rats compared to 9.4+ 1.3% of 392 +84 TJs analyzed in CON rats, p<0.0001. Tracer of lanthanum in the bile canalicular lumen was observed in 66.9__.6.7% and 5.2+2.1% in TNB and CON rats, respectively, p<0.001. The area under the first peak of HRP excretion curve (peraceUular permeability) was significantly increased in TNB rats (.86---.12 vs .37+ .04 ng/g bw .10 rain, p<0.02). CONCLUSIONS: 1.Experimental colitis is associated with an increased TJ permeability. 2. This alteration may represent part of the mechanisms involved in the hepatobiliary injudes in IBD.
77 1 IDENTIFICATION OF CELL-VOLUME-RESPONSIVE ELEMENTS IN THE PROMOTER OF THE PHOSPHOENOLPYRUVATE CARBOXYKINASE (PCK) GENE S. Kaiser, GI & Liver Division, Dept. of Medicine, University of Ttibingen, FRG Liver cell volume changes are increasingly recognized as an important regulator of liver metabolism. The expression of the gene coding for the ratelimiting enzyme of gluconeogenesis, phosphoenolpyruvate earboxykinase (PCK), is also markedly regulated by cell volume in H 35 rat hepatoma cells and in cultured primary human hepatocytes. Cell shrinkage increases, whereas cell swellingdecreasesPCK mRNA. These effects are largely transcriptional. To identify the potential signal tmnsduction pathway of this novel type of liver gene regulation, transfection studies of chimeric PCK promoter/CAT constructs were performed in primary human hepatocytes. Subconfluent cultures of primary human hepatocytes exhibited only a 2-fold increase in PCK mKNA levels when exposed to hypertonic medium (400 mOsm). However, well differentiated cultures (12-14d post splitting) showed a 6-fold increase in PCK mRNA under hypertonic conditions. Likewise, subconfluant ceils transfected with a -490bp PCK/CAT construct showed no increase in CAT activity when shifted to hypertonic medium for 48 h. In contrast, CAT activity expressed in differentiated cells transfected with the -490bp PCK/CAT construct was increased 4-fold by hypertonie medium. Thus, the complete signal transductionpathway which mediates the hypertonieresponse appearsto be expressed only in fully differentiated cells. Mutation or deletion of the CRE-1 element and a site which has previouslybeen shown to bind HNF-3 and mediate effects of Jun and Fos on PCK expression each produced a 50% decrease in the hypertonic responsiveness. Mutations in all of the other characterized regions of the PCK promoter had no effect on the activity. In liver cells so far only Fos and Jan have been shown to act through both the above described elements, suggestingthat changes in the activities or levels of these proteins may mediate increased transcriptionof the PCK gene during cell shrinkage. Further characterization of these elements and their associated trans-acting factors should reveal the molecular mechanism involved in the effect of cell volume on liver gene expression.
299A
770 METHIMAZOLE SLOWS HEPATOCYTE STREAMING IN RATS. R Oren*. G Zajicek**, Y Maarvi*, G Kenet***, and N Arber**** Dept of Medicine*, and the H.H.Humphrey Center for Experimental Medicine and Cancer Research, Hebrew University Hadassah Medical School**, lerusalem, Dept. of Gastroenterology, Wolfson Medical Center, Holon***, and Dept. of Gastroenterology****, Ichilov Hospital, Tel-Aviv, Israd. Previous studies showed that hepatocytes stream from the portal tract to the terminal hepatic vein and that some degree of hypothyroidism could prevent hepatic damage and decrease portal pressure in the rat. We evaluated the effect of hypothyroidism induced by methimazole (0.04% - two weeks prior to and throughout the study) on the migration of liver cells. Thirty male adult rats, random brand, were. injected with with 0.5 ~tCi (3H)-thymidine, specific activity 5 Ci/mmol/g body weight. The rats were killed, ha groups of 5 at the following times: 1 h, 14 and 28 days. The livers were processed histologically and dipped into liquid emulsion for autoradiography. In each animal 50 labeled hepatoeytes and 50 lltoral cells were randomly selected and their distances from the nearest terminal portal tract rim were measured. Hypothyroidism was confirmed by a significant TSH elevation (1.44:t0.28 vs 0.25:t:0.03, p<0.001). Hepatocyte derived from the methimazole treated groups streamed significantly slower than those from normal untreated rats (p<0.00001). Hepatocytes and littoral cells streamed at the same velocity. Since streaming velocity is proportional to cell turnover we conclude that methimazole could decrease hepatoeytes proliferation in the rat.
772 OXYGEN MODLrLATES THE GLUCAGON-DEPENDENT ACTIVATION OF 'i"1~ PHOSPHOENOLPYRUVATE CARBOXYKINASE GENE IN TIIE FIRST 277 BP OF ITS PROMOTER BY SEQUENCES DIFFERENT FROM THE CRE VIA A IIEME PROTEIN-MEDIATED ~MECHANISM IN ltEPATOCYTES. T Kietmmm, J Bratke and K lnstitut Fur Biodaemte and Molekulare ZeUbiologle, Humboldtallee 23,1)-37073 ~ Germany Oxygen modulates ~ glucagon-depondent activation of the phosphoenolpyruvate carboxykinase (PCK) gene in rat hepatocytes. The PCK gene may contain eider 5" or 3" DNA sequences as is the case with the erythmpoietth gene, which are regulated by Oz. This modulation could be effected either directly via an O2/CO sensitive transcnption factor or indirectly by the glucegon signal chain via the cAMP responsive element binding protein (CREB). To test for an upstream (5") DNA sequence and the involvement of a heine protein as O2-sensor primary hepatocytes were t~ansfacted with 5"-deleted PCK promoter chloramphenicol acetyltransferase (CAT) constructs (pPCKSCAT) under different 02 tensions ~ and without carbon monoxide (CO). To prove the involvement of the glucagon signal chain a CRE-thymidine Idnase promoter CAT construct (pCRETKCAT) was tested. A SV40 promoter CAT construct (pCATprom) was used as independent conb'oL Gel shift assays were used to test the PCK promoter DNA protein interactions and its modulation by Oz. The pPCKSCAT constructs were transfected into primary hepatocytas using cationic liposomes. CAT activity was determined after induc~on with 10nM glucagon under arterial (16%) 02 and venous (8%) O2 as well as with pPCKSCAT-2500 after addition of 2% CO. W ~ pPCKSCAT-2500, pPCKSCAT-490 and pPCKSCAT-277 CAT activities were increased with different strength maximally under arterial Oz and half maximally under venous 02. CO counteracted the reduced induction at venous 02. The induction by glucegon of pCRETKCAT was not modulated by 02. Nuclear extracts from hepatocytes precultured for 24 h under venous 02 showed a higher DNA binding activity to the -1491.42 fragment of the PCK promoter as those from cells cultured under arterial Oz. These results indicate that the promoter of the PCK gene contains DNA elements which are regulated by (3= via a farro-heme protein as an Oz sensor without the direct involvement of CREB.
769
AASLD A B S T R A C T S
EXPERIMENTAL COUTIS INCREASES HEPATOCYTE TIGHT JUNCTIONAL PERMEABILITY. L Lore, M. Mazzon*, W. Fries, MT. Vilei, C. Cadotto, M. Murasa, A. D'Odorico, R. Naccarato, D. Martines. Department of Internal Medicine, Department of Biology*-, University of Padua, Padua. Hepatobiliary complications are frequently observed in patients with inflammatory bowel disease (IBD). Translocation of intestinal bacteda and their toxins through the gut wall and invasion of the hepatobiliary system has been suggested as pathogenic mechanism. The integrity of hepatocellular tight junctions (-IJs) is thought to play an important role in preventing the entry of bacteria from the portal blood in the biliary tract. AIM of this study was to evaluate liver TJ morphology and function in experimental colitis in the rat. METHODS: male Sprague-Dawley rats (200250 g) were used. Experimental colitis was induced instilling in the colon 25 mg trinitrobenzene sulphonic acid in 1 ml 50% ethanol (TNB rats). Control group consisted of untreated animals (CON rats). At one week of TNBinduced colitis, morphological study of TJs was performed after perfusing the livers with lanthanum nitrate (4%) (n=3 per group) and analyzing its penetration in the junctional complexes and bile cacalicular lumen by Transmission Electron Microscopy, In a second sedes of experiments, to examine the biliary paracellular permeability, horseradish peroxidase (HRP) was pulsed, within 1 rain, into perfused rat livers (5 per groups) under single-pass conditions. Bile was collected before peffusion for analysis of basal bile flow rate and bile salt excretion and during peffusion to examine the pattern of HRP biliary excretion. RESULTS:Basal bile flow rate and bile salt excretion were unchanged in TNB rats. Electron microscopic examination (35.000 X) showed lanthanum accumulation in the 85.6+ 1.4% of 335 _+35 TJs observed in TNB rats compared to 9.4+ 1.3% of 392 +84 TJs analyzed in CON rats, p<0.0001. Tracer of lanthanum in the bile canalicular lumen was observed in 66.9__.6.7% and 5.2+2.1% in TNB and CON rats, respectively, p<0.001. The area under the first peak of HRP excretion curve (peraceUular permeability) was significantly increased in TNB rats (.86---.12 vs .37+ .04 ng/g bw .10 rain, p<0.02). CONCLUSIONS: 1.Experimental colitis is associated with an increased TJ permeability. 2. This alteration may represent part of the mechanisms involved in the hepatobiliary injudes in IBD.
77 1 IDENTIFICATION OF CELL-VOLUME-RESPONSIVE ELEMENTS IN THE PROMOTER OF THE PHOSPHOENOLPYRUVATE CARBOXYKINASE (PCK) GENE S. Kaiser, GI & Liver Division, Dept. of Medicine, University of Ttibingen, FRG Liver cell volume changes are increasingly recognized as an important regulator of liver metabolism. The expression of the gene coding for the ratelimiting enzyme of gluconeogenesis, phosphoenolpyruvate earboxykinase (PCK), is also markedly regulated by cell volume in H 35 rat hepatoma cells and in cultured primary human hepatocytes. Cell shrinkage increases, whereas cell swellingdecreasesPCK mRNA. These effects are largely transcriptional. To identify the potential signal tmnsduction pathway of this novel type of liver gene regulation, transfection studies of chimeric PCK promoter/CAT constructs were performed in primary human hepatocytes. Subconfluent cultures of primary human hepatocytes exhibited only a 2-fold increase in PCK mKNA levels when exposed to hypertonic medium (400 mOsm). However, well differentiated cultures (12-14d post splitting) showed a 6-fold increase in PCK mRNA under hypertonic conditions. Likewise, subconfluant ceils transfected with a -490bp PCK/CAT construct showed no increase in CAT activity when shifted to hypertonic medium for 48 h. In contrast, CAT activity expressed in differentiated cells transfected with the -490bp PCK/CAT construct was increased 4-fold by hypertonie medium. Thus, the complete signal transductionpathway which mediates the hypertonieresponse appearsto be expressed only in fully differentiated cells. Mutation or deletion of the CRE-1 element and a site which has previouslybeen shown to bind HNF-3 and mediate effects of Jun and Fos on PCK expression each produced a 50% decrease in the hypertonic responsiveness. Mutations in all of the other characterized regions of the PCK promoter had no effect on the activity. In liver cells so far only Fos and Jan have been shown to act through both the above described elements, suggestingthat changes in the activities or levels of these proteins may mediate increased transcriptionof the PCK gene during cell shrinkage. Further characterization of these elements and their associated trans-acting factors should reveal the molecular mechanism involved in the effect of cell volume on liver gene expression.
299A
770 METHIMAZOLE SLOWS HEPATOCYTE STREAMING IN RATS. R Oren*. G Zajicek**, Y Maarvi*, G Kenet***, and N Arber**** Dept of Medicine*, and the H.H.Humphrey Center for Experimental Medicine and Cancer Research, Hebrew University Hadassah Medical School**, lerusalem, Dept. of Gastroenterology, Wolfson Medical Center, Holon***, and Dept. of Gastroenterology****, Ichilov Hospital, Tel-Aviv, Israd. Previous studies showed that hepatocytes stream from the portal tract to the terminal hepatic vein and that some degree of hypothyroidism could prevent hepatic damage and decrease portal pressure in the rat. We evaluated the effect of hypothyroidism induced by methimazole (0.04% - two weeks prior to and throughout the study) on the migration of liver cells. Thirty male adult rats, random brand, were. injected with with 0.5 ~tCi (3H)-thymidine, specific activity 5 Ci/mmol/g body weight. The rats were killed, ha groups of 5 at the following times: 1 h, 14 and 28 days. The livers were processed histologically and dipped into liquid emulsion for autoradiography. In each animal 50 labeled hepatoeytes and 50 lltoral cells were randomly selected and their distances from the nearest terminal portal tract rim were measured. Hypothyroidism was confirmed by a significant TSH elevation (1.44:t0.28 vs 0.25:t:0.03, p<0.001). Hepatocyte derived from the methimazole treated groups streamed significantly slower than those from normal untreated rats (p<0.00001). Hepatocytes and littoral cells streamed at the same velocity. Since streaming velocity is proportional to cell turnover we conclude that methimazole could decrease hepatoeytes proliferation in the rat.
772 OXYGEN MODLrLATES THE GLUCAGON-DEPENDENT ACTIVATION OF 'i"1~ PHOSPHOENOLPYRUVATE CARBOXYKINASE GENE IN TIIE FIRST 277 BP OF ITS PROMOTER BY SEQUENCES DIFFERENT FROM THE CRE VIA A IIEME PROTEIN-MEDIATED ~MECHANISM IN ltEPATOCYTES. T Kietmmm, J Bratke and K lnstitut Fur Biodaemte and Molekulare ZeUbiologle, Humboldtallee 23,1)-37073 ~ Germany Oxygen modulates ~ glucagon-depondent activation of the phosphoenolpyruvate carboxykinase (PCK) gene in rat hepatocytes. The PCK gene may contain eider 5" or 3" DNA sequences as is the case with the erythmpoietth gene, which are regulated by Oz. This modulation could be effected either directly via an O2/CO sensitive transcnption factor or indirectly by the glucegon signal chain via the cAMP responsive element binding protein (CREB). To test for an upstream (5") DNA sequence and the involvement of a heine protein as O2-sensor primary hepatocytes were t~ansfacted with 5"-deleted PCK promoter chloramphenicol acetyltransferase (CAT) constructs (pPCKSCAT) under different 02 tensions ~ and without carbon monoxide (CO). To prove the involvement of the glucagon signal chain a CRE-thymidine Idnase promoter CAT construct (pCRETKCAT) was tested. A SV40 promoter CAT construct (pCATprom) was used as independent conb'oL Gel shift assays were used to test the PCK promoter DNA protein interactions and its modulation by Oz. The pPCKSCAT constructs were transfected into primary hepatocytas using cationic liposomes. CAT activity was determined after induc~on with 10nM glucagon under arterial (16%) 02 and venous (8%) O2 as well as with pPCKSCAT-2500 after addition of 2% CO. W ~ pPCKSCAT-2500, pPCKSCAT-490 and pPCKSCAT-277 CAT activities were increased with different strength maximally under arterial Oz and half maximally under venous 02. CO counteracted the reduced induction at venous 02. The induction by glucegon of pCRETKCAT was not modulated by 02. Nuclear extracts from hepatocytes precultured for 24 h under venous 02 showed a higher DNA binding activity to the -1491.42 fragment of the PCK promoter as those from cells cultured under arterial Oz. These results indicate that the promoter of the PCK gene contains DNA elements which are regulated by (3= via a farro-heme protein as an Oz sensor without the direct involvement of CREB.