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A380 AGA ABSTRACTS GASTROENTEROLOGY, Vol. 108, No. 4 • CYSTIC FIBROSIS HETEROZYGOSITY AND ALCOHOLIC PANCREATITIS. ID Norton 1, MV Apte 1, H Dixson 1, RJ Trent2, RC Pirola 1, JS WilsonL 1Dept. of Gastroenterology, Prince of Wales Hospital and 2Dept. of Molecular Genetics, Royal Prince Alfred Hospital, Sydney, Australia. Background: Only 1 in 20 alcoholics develop pancreatitis. 1 Predisposing factors for this disease have not been determined. It was hypothesized that hemmrozygosityfor cystic fibrosis (CF) predisposes heavy drinkers to pancreatitis because: a. The frequency of pancreatitis amongst alcoholics is similar to the frequency of CF heterozygosity (approximately 1 in 20); b. Ductal obstruction is thought to be an Important mechanism Of pancreatic disease in both cystic fibrosis and alcoholic pancreatitis; c. Secretory abnormalities have been demonstrated in CF heterozygotes.2 A i m : To determine whether patients with alcoholic pancreatitis are heterozygotes for cystic fibrosis. Method: Twenty-one patients with alcoholic pancreatitis were examined for the AF-508, G551D and G542X mutations. These account for approximately 70-75% of CF mutations in Western Caucasian populations. DNA was extracted from leukocytes by the phenol- chloroform extraction method. Relevant regions of interest of the CF gene were amplified via PCR using appropriate primers. Each gel included control lanes with: i) no DNA; ii) normal CF genes; iii) heterozygote and iv) homozygote for the mutation of interest. Heterozygotes for AF-508 were identified by heteroduplex formation when the PCR product was run on SDS PAGE. For G542X and G551D mutations, the PCR product was dotted onto a nylon membrane and was probed with allele specific oligonucleotides complementary to normal and mutant alleles. After washing, normal and mutant hybrids were detected by enhanced chemiluminescence. Results: None of the 21 patients with alcoholic pancreatitis showed evidence of the AF-508, G551D or G542X mutations: Conclusion: Heterozygosity for cystic fibrosis does not appear to be a risk factor for alcoholic pancreatitis. References: 1. Steinberg W and Tenner S. New Engl J Med, 330:1198, 1994. 2. Behm J K, Hagiwara G, Lewiston NJ, Quinton PM and Wine JJ Pediatric Research, 22" 271, 1987. RAT PANCREATIC CYTOCHROME P-450 2E1 (CYP2E1) MESSENGER RNA IS NOT INCREASED BY CHRONIC ETHANOL FEEDING. ID Norton. MV Apte, M Veronese, GW MeCaughan, RC Pirola, JS Wilson. Department of Gastroenterology, Prince of Wales Hospital, Sydney, Australia. Background: Oxidative stress is increasingly recognized as a mechanism of pancreatic injury in pancreatitis. With respect to alcoholic pancreatitis, the metabolism of ethanol in the pancreas by an ethanol-inducible form of cytochrome P-450 (CYP2E1) may lead to formation of reactive oxygen species with subsequent intracellular damage. Induction of CYP2E1 has been demonstrated in many parts of the gastrointestinal tract, but the pancreas has not been examined in this regard. Aims: i) to examine the rat pancreas for presence of CYP2E1 mRNA ii) to determine the effect of chronic ethanol administration on rat pancreatic CYP2E1 mRNA levels Methods: Male Sprague,Dawley rats (n=16) were pair-fed nutritionally adequate diets containing ethanol (as 36% of energy) or an isocaloric amount of carbohydrate for 4 weeks. Ethanol feeding of this duration is known cause CYP2E1 induction in other tissues. Total pancreatic mRNA was analyzed by Northern and d0t blotting using a eDNA probe for CYP2E1 and an oligonucleotide probe for 13-actin (internal control). Messenger RNA levels were determined by electronic autoradiography of dot blots. Probing for 8-actin confirmed equal loading of RNA on membranes. Data were analyzed by Student's paired t-test. Results: The Table indicates mRNA levels expressed as total counts for the period of scanning. Control Ethanol CYP2E1 894.6i95.1 829.3+64.9 p = 0.504 Conclusions: i) In rat pancreas, mRNA for CYP2E1 can be detected, ii) Chronic ethanol consumption has no effect on mRNA levels for CYP2E1. Implication: If CYP2E1 induction occurs in rat pancreas following chronic ethanol consumption, it is not regulated via increased mRNA levels. REGULATION OF THE SODIUM/HYDROGEN EXCHANGER SUBTYPE 1 (NHE-1) GENE IN IMMORTALIZED PANCREATIC DUCTAL(PD) CELLS. S. Novak, R. Marsick and LR. Marino. The Cystic Fibrosis Center & Dept. of Pediatrics, Case Western Reserve University, Cleveland, Ohio. NHE-1 is critical for the regulation of intracellular pH in the base secreting cells of the pancreatic duct. Although the mechanisms for NHE- 1 protein activation are well described, little is known about the genetic regulation of NHE-1. Specifically, there is ambiguity as to whether protein kinase-A and protein kinase-C increase NHE-! expression. To begin our understanding of how the NHE-1 gene is controlled in the pancreas, we used an immortalized pancreatic ductal cell line. To determine if cAMP and protein kinase-A played any role in regulating NHE-1 gene expression, we stimulated PD cells with forskolin(F) 10-5M and extracted total RNA from the cells after 60'. We used Northern blotting to determine if there was a change in NHE-1 expression. An ~1000bp eDNA probe made to human NHE-1 was used for hybridization. Inaccuracies in loading were corrected by measuring the density of ethidium bromide stained 18S and 28S ribosomal bands. The density of the NHE-1 hybridization signals were normalized using the control gene ubiquitin. After forskolin, NHE-1 mRNA increased two fold over control, p<0.03. The NHE-1 promoter contains three non-consensus CREB or Apl simms.To determine if these sites regulated a transcriptional response in NHE-1 following an increase in protein kinase-A or protein kinase-C, we transfected PD ceils with chloramphenicol acetyl transferase(CAT) reporter gene constructs containing the 1500bp NHE-1 promoter(a gift ofR. T. Miller) with the sites of interest. After the transfecmmd cells were serum starved for 24 hours we stimulated them with forskolin(F) 10-5M or phorbol ester(TPA) 10-6M for ]2 hours and measured CAT activity. Transcription from the promoter increased after F and TPA stimulation to 230% control(p<0.005) and 170% control(p<0.02), respectively. Although the increase in NHE-1 expression after F may reflect changes in transcriptional and post-transcriptional regulation, the results from our reporter gene assays indicate that a portion of the increase is due to a rise in transcription of the gene. Furthermore, agents that evoke protein kinase-C mediated events also increase the transcription of NHE-1. We speculate that the non-consensus CREB/Apl sites on the NHE-1 promoter play an active role in regulating these responses and that the sequences of these sites may allow for transcriptional cross-talk between the protein kinase-A and protein kinase-C signal transduetion pathways. FINAL RESULTS OF THE PROSPECTIVE, RANDOMIZED, CONTROLLED STUDY ON ENDOSCOPIC SPHINCTEROTOMY VERSUS CONVENTIONAL MANAGEMENT IN ACUTE BILIARY PANCREATITIS. A.Nowak, E.Nowakowska-Dulawa, T.AMarek; J.Rybicka. Department of Gastroenterology, Silesian University School of Medical Sciences, Katowice, Poland. Back,qround: There are still no convincing data that endoscopic sphincterotomy (ES) should be performed in every case of acute biliary pancreatitis (ABP). Our knowledge concerning how urgent should ES be done is also scanty. Methods: 280 consecutive patients with ABP underwent duodenoscopy within 24 hours of admission. 75 patients (group I) with stone impacted in the papilla of Vater were treated by immediate ES. 205 patients with normal papilla were randomized to be treated by immediate ES (group II -- 103 patients) or to be managed conventionally (CM; group Ill -- 102 patients). The groups were well matched according to predicted severity, age and sex. Results: There were 17% of complications in ES group (l+ll) in. comparison to 36% in CM group; (95%Cl of difference: 0.10 to 0.32; p=0.000). Corresponding values for mortality were 2% and 13% respectively (95%C1: 0.04 to 0.17; p=0.000). These differences were true for predicted severe as well as for predicted mild cases. Among patients treated endoscopically, the best results were obtained, when the interval between onset of ABP and ES was shorter than 24 hours (complications -- 7%, mortality -- 0%); the worst, when the delay exceeded 72 hours (complications -- 22%, mortality -- 8%). This trend was much more significant in predicted severe than in predicted mild cases. Conclusions: 1. Urgent ES applied for ABP significantly decreases complication and mortality rates, in predicted severe as well as in predicted mild cases. 2. For best results ES should be performed during the first 24 hours of the disease. 3. ES should 'be the method of choice in every case of acute biliary pancreatitis.

Rat pancreatic cytochrome P-450 2E1 (CYP2E1) messenger RNA is not increased by chronic ethanol feeding

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A380 AGA ABSTRACTS GASTROENTEROLOGY, Vol. 108, No. 4

• CYSTIC FIBROSIS HETEROZYGOSITY AND ALCOHOLIC PANCREATITIS. ID Norton 1, MV Apte 1, H Dixson 1, RJ Trent 2, RC Pirola 1, JS WilsonL 1Dept. of Gastroenterology, Prince of Wales Hospital and 2Dept. of Molecular Genetics, Royal Prince Alfred Hospital, Sydney, Australia.

Background: Only 1 in 20 alcoholics develop pancreatitis. 1 Predisposing factors for this disease have not been determined. It was hypothesized that hemmrozygosity for cystic fibrosis (CF) predisposes heavy drinkers to pancreatitis because: a. The frequency of pancreatitis amongst alcoholics is similar to the

frequency of CF heterozygosity (approximately 1 in 20); b. Ductal obstruction is thought to be an Important mechanism Of

pancreatic disease in both cystic fibrosis and alcoholic pancreatitis; c. Secretory abnormalities have been demonstrated in CF heterozygotes. 2 A i m : To determine whether patients with alcoholic pancreatitis are heterozygotes for cystic fibrosis. M e t h o d : Twenty-one patients with alcoholic pancreatitis were examined for the AF-508, G551D and G542X mutations. These account for approximately 70-75% of CF mutations in Western Caucasian populations. DNA was extracted from leukocytes by the phenol- chloroform extraction method. Relevant regions of interest of the CF gene were amplified via PCR using appropriate primers. Each gel included control lanes with: i) no DNA; ii) normal CF genes; iii) heterozygote and iv) homozygote for the mutation of interest. Heterozygotes for AF-508 were identified by heteroduplex formation when the PCR product was run on SDS PAGE. For G542X and G551D mutations, the PCR product was dotted onto a nylon membrane and was probed with allele specific oligonucleotides complementary to normal and mutant alleles. After washing, normal and mutant hybrids were detected by enhanced chemiluminescence. Resu l t s : None of the 21 patients with alcoholic pancreatitis showed evidence of the AF-508, G551D or G542X mutations: Conc lus ion: Heterozygosity for cystic fibrosis does not appear to be a risk factor for alcoholic pancreatitis. References: 1. Steinberg W and Tenner S. New Engl J Med, 330:1198, 1994. 2. Behm J K, Hagiwara G, Lewiston NJ, Quinton PM and Wine JJ Pediatric Research, 22" 271, 1987.

RAT PANCREATIC CYTOCHROME P-450 2E1 (CYP2E1) MESSENGER RNA IS NOT INCREASED BY CHRONIC ETHANOL FEEDING. ID Norton. MV Apte, M Veronese, GW MeCaughan, RC Pirola, JS Wilson. Department of Gastroenterology, Prince of Wales Hospital, Sydney, Australia. Background: Oxidative stress is increasingly recognized as a mechanism of pancreatic injury in pancreatitis. With respect to alcoholic pancreatitis, the metabolism of ethanol in the pancreas by an ethanol-inducible form of cytochrome P-450 (CYP2E1) may lead to formation of reactive oxygen species with subsequent intracellular damage. Induction of CYP2E1 has been demonstrated in many parts of the gastrointestinal tract, but the pancreas has not been examined in this regard.

Aims: i) to examine the rat pancreas for presence of CYP2E1 mRNA ii) to determine the effect of chronic ethanol administration on

rat pancreatic CYP2E1 mRNA levels

Methods: Male Sprague,Dawley rats (n=16) were pair-fed nutritionally adequate diets containing ethanol (as 36% of energy) or an isocaloric amount of carbohydrate for 4 weeks. Ethanol feeding of this duration is known cause CYP2E1 induction in other tissues. Total pancreatic mRNA was analyzed by Northern and d0t blotting using a eDNA probe for CYP2E1 and an oligonucleotide probe for 13-actin (internal control). Messenger RNA levels were determined by electronic autoradiography of dot blots. Probing for 8-actin confirmed equal loading of RNA on membranes. Data were analyzed by Student's paired t-test.

Results: The Table indicates mRNA levels expressed as total counts for the period of scanning.

Control Ethanol

CYP2E1 894.6i95.1 829.3+64.9

p = 0.504

Conclusions: i) In rat pancreas, mRNA for CYP2E1 can be detected, ii) Chronic ethanol consumption has no effect on mRNA levels for CYP2E1.

Implication: If CYP2E1 induction occurs in rat pancreas following chronic ethanol consumption, it is not regulated via increased mRNA levels.

• REGULATION OF THE SODIUM/HYDROGEN EXCHANGER SUBTYPE 1 (NHE-1) GENE IN IMMORTALIZED PANCREATIC DUCTAL(PD) CELLS. S. Novak, R. Marsick and LR. Marino. The Cystic Fibrosis Center & Dept. of Pediatrics, Case Western Reserve University, Cleveland, Ohio.

NHE-1 is critical for the regulation of intracellular pH in the base secreting cells of the pancreatic duct. Although the mechanisms for NHE- 1 protein activation are well described, little is known about the genetic regulation of NHE-1. Specifically, there is ambiguity as to whether protein kinase-A and protein kinase-C increase NHE-! expression. To begin our understanding of how the NHE-1 gene is controlled in the pancreas, we used an immortalized pancreatic ductal cell line. To determine if cAMP and protein kinase-A played any role in regulating NHE-1 gene expression, we stimulated PD cells with forskolin(F) 10-5M and extracted total RNA from the cells after 60'. We used Northern blotting to determine if there was a change in NHE-1 expression. An ~1000bp eDNA probe made to human NHE-1 was used for hybridization. Inaccuracies in loading were corrected by measuring the density of ethidium bromide stained 18S and 28S ribosomal bands. The density of the NHE-1 hybridization signals were normalized using the control gene ubiquitin. After forskolin, NHE-1 mRNA increased two fold over control, p<0.03. The NHE-1 promoter contains three non-consensus CREB or Apl simms. To determine if these sites regulated a transcriptional response in NHE-1 following an increase in protein kinase-A or protein kinase-C, we transfected PD ceils with chloramphenicol acetyl transferase(CAT) reporter gene constructs containing the 1500bp NHE-1 promoter(a gift ofR. T. Miller) with the sites of interest. After the transfecmmd cells were serum starved for 24 hours we stimulated them with forskolin(F) 10-5M or phorbol ester(TPA) 10-6M for ]2 hours and measured CAT activity. Transcription from the promoter increased after F and TPA stimulation to 230% control(p<0.005) and 170% control(p<0.02), respectively. Although the increase in NHE-1 expression after F may reflect changes in transcriptional and post-transcriptional regulation, the results from our reporter gene assays indicate that a portion of the increase is due to a rise in transcription of the gene. Furthermore, agents that evoke protein kinase-C mediated events also increase the transcription of NHE-1. We speculate that the non-consensus CREB/Apl sites on the NHE-1 promoter play an active role in regulating these responses and that the sequences of these sites may allow for transcriptional cross-talk between the protein kinase-A and protein kinase-C signal transduetion pathways.

FINAL RESULTS OF THE PROSPECTIVE, RANDOMIZED, CONTROLLED STUDY ON ENDOSCOPIC SPHINCTEROTOMY VERSUS CONVENTIONAL MANAGEMENT IN ACUTE BILIARY PANCREATITIS. A.Nowak, E.Nowakowska-Dulawa, T.AMarek; J.Rybicka. Department of Gastroenterology, Silesian University School of Medical Sciences, Katowice, Poland.

Back,qround: There are still no convincing data that endoscopic sphincterotomy (ES) should be performed in every case of acute biliary pancreatitis (ABP). Our knowledge concerning how urgent should ES be done is also scanty.

Methods: 280 consecutive patients with ABP underwent duodenoscopy within 24 hours of admission. 75 patients (group I) with stone impacted in the papilla of Vater were treated by immediate ES. 205 patients with normal papilla were randomized to be treated by immediate ES (group II -- 103 patients) or to b e managed conventionally (CM; group Ill -- 102 patients). The groups were well matched according to predicted severity, age and sex.

Results: There were 17% of complications in ES group (l+ll) in. comparison to 36% in CM group; (95%Cl of difference: 0.10 to 0.32; p=0.000). Corresponding values for mortality were 2% and 13% respectively (95%C1: 0.04 to 0.17; p=0.000). These differences were true for predicted severe as well as for predicted mild cases.

Among patients treated endoscopically, the best results were obtained, when the interval between onset of ABP and ES was shorter than 24 hours (complications -- 7%, mortality -- 0%); the worst, when the delay exceeded 72 hours (complications -- 22%, mortality -- 8%). This trend was much more significant in predicted severe than in predicted mild cases.

Conclusions: 1. Urgent ES applied for ABP significantly decreases complication and mortality rates, in predicted severe as well as in predicted mild cases. 2. For best results ES should be performed during the first 24 hours of the disease. 3. ES should 'be the method of choice in every case of acute biliary pancreatitis.