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reg is mitogenic to primary culture of pancreatic ductal cells
A402
AGA ABSTRACTS
GASTROENTEROLOGY, Vol. 108, No. 4
USEFULNESS OF CAERULEIN STIMULATED PLASMA AMINO ACIDS CONSUMPTION TEST AS TUBELESS PANCREATIC EXOCRINE FUNCTION TEST U.Yoshioka, Y.Satoh, T.Saotome, Y.Kateh, Y.Okumura, H.Inoue, Y.Fujiyama and T.Bamba, Second Department of Internal Medicine, Shiga University of Medical Science, Ohtsu, Shiga, 520-21, Japan ' Plasma amino acids consumption test after caerulein stimulation (AACT) is promising and not troublesome method of pancreatic exocrine function test. But there are controversial reports about usefulness of AACT. So We examined AACT in comparison with other pancreatic exocrine function test (secretin test and BTPABA test). METHODS 23 patients (chronic pancrcatitis 17, pancreas cancer 1, Sj6gren syndrome 2, primary biliary cirrhosis 1, pancreas divisum 1, multiple pancrgas cysts 1) and 1 healthy volunteer were examined after an overnight fast. Caerulcin 50 ng/kg was injected by intravenously for 30 seconds, and blood sample was taken before and every 10 minutes to 50 minutes after eaerulein injection. Plasma was deproteinized by 5% sulfosalicylie acid. Total plasma amino acids concentration (TAA) was measured by Ninhydrin method, and individual amino acid was analyzed by HPLC (Hitachi L-8500). We compared AACT with results of secrctin test (normal 11, mild dysfunction 6, and severe dysfunction 6). RESULTS There were no differences in TAA among normal, mild and severe groups before caerulein stimulation. TAA was significantly reduced by caerulein stimulation in normal and mild groups, but there wer~ no differences in TAA of severe group between before and after caerulein stimulation. Decrease rate of TAA after caerulein stimulation (%) was significantly low in the severe group, compared with normal and mild groups. 10 rain. 20 min. 30 rain. 40 rain. 50 min. normal 6.85-+1,16 9.04-+1.17 9.75+0.66 11.01+-1.70 956+-1.65 mild 4.75-+ 1.50 7.89+ 1.18 8.17+-0.41 7.27-+ 1.16 7.66 + 1.23 severe 1.38+-0.85 2.37+-0.23i 1.83-+1.67 2.98_+2.65 2.64-+1.23 ** **## ***## * normal vs. severe # mild vs. severe mean±SE * P<0.05 ** #4 P<0.01 *** P<0.001 There were significant positive correlations between decrease rate of TAA and each parameters of secretin test. There was no significant decrease of TAA in normal and mild groups after stimulation by secretin alone (100 U iv). Sensitivity of AACT was 100% in the severe group and 40% in the mild group, which were higher than the sensitivity of BTPABA test. CONCLUSION AACT is a more useful than BTPABA test. AACT can detect severe pancreatic exocrine dysfunction, but cannot detect mild dysfunction. •
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PRESENCE OF OGF AND ZETA (~) OPIOID RECEPTOR IN HT-29 HUMAN COLON CANCER. I.S Zagon, S. Hytrek, C.M Lang, J.P. Smith P.J. McLaughlin. Depts. Neuroscience and Anatomy, Comparative Medicine, and Medicine, Penn State University College of Medicine Hershey, Pennsylvahia The endogenous opio~d system (EOS) consisting of opioid growth factor (OGF), [Met~}-enkephalin, and zeta ([) receptor has been shown to modulate the growth of animal and human tumors. Manipulation of this EOS in HT-29 human colon cancer cells xenografted to nude mice has suggested that both OGF and the f receptor are present in the HT-29 tumors. To identify and characterize the [ opioid receptor which mediates the action o~ OGF, bending assays using homogenates of HT-29 tumors and [~H]-[Met~]-enkephalin were performed Specific and saturable binding was detected, and data were consistent with a single binding site Scatehard analysis yielded a binding affinity (Kd) of 15.4 ± 2.0 nM and a binding capacity (Bmax) of 364.7 ± 25.7 fmol/mg protein Binding was dependent on protein concentration, time and temperature of assay conditions, and p~ of the h~ffer; binding was also sensitive to Na 2+ , Caz+ , and Mg 2+ ions, hut not to GppNHp. Subcellular fractionation studies revealed that binding was restricted to the fraction enriched in nuclei, with little or no specific binding detected in the membrane, mierosomal or soluble fractions. Competition assays with cold ligands for ~, 6, ~, o, and e opiold receptors demonstrated that OGF was at least 3-fold more competitive for the [ receptor. The presence of OGF was determined using radioimmunoassays for [Met ]-enkephalin Levels of OGF in tumor tissue were 59.3 ± 6.2 pg/mg tissue. Northern blot analysis showed that preproenkephalin A (PPE) mRNA expression was present in HT-29 tumors. These studies reveal, for the first time the presence of OGF and the opioid receptor the autocrine nature of OGF, and the characteristics of the [ receptor in a human colon cancer• Supported by the Laverty Foundation.
• INTERCELLULAR CALCIUM WAVES: MECHANISM OF TRANSMISSION AND RELATIONSHIP TO SECRETION IN RAT PANCREATIC AC1NI. D.I. Yule, E.L. Stuenkel and J.A. Williams. Departments o f Physiology and Internal Medicine, University of Michigan, Ann Arbor, MI 48109-0622. It is known that stimulation of acinar cells with secretagogues such as cholecystokinin (CCK) or carbachol (CCh) results in at low concentrations, the generation of Ca waves that appear to propagate through an acinus while at higher concentrations a uniform single peak m [Ca ]i followed by an elevated plateau is evoked. The role gapunctional communication plays in intercellular calcium signaling and the ubsequent secretion of digestive enzymes was studied. Intracelhilar calcium concentration [Ca2÷]i measurements were performed by digital imaging o f acini loaded with the fluorescent Ca 2÷ indicator fura,2. Imperrneant messenger and marker molecules were introduced into cells by microinjection. The extent of gag-junctional communication was measured simultaneously with [Ca~÷]~ signals (by fura-2) under physiologic conditions or supramaximal stimulation with secretagogues by monitoring diffusion of microinjected lissarhodamine fluorescence, (pipette concentration (PC) 0.25 rmM) from the injected cell to neighboring cells in the acinus. In cells stimulated with oscillatory (physiologic) concentrations of CCK the [CaZ+]i signal was characterized by the spread of Ca 2+ between cells in a wave-like fashion and the spread of lissarhodamine fluorescence was not significantly different from unstimulated cells. In contrast at supramaximal concentrations of CCK (1 riM) or CCh (0.1 mM), where secretion is diminished and oscillations are absent, no dye transfer occurred. These data indicate that under physiologic stimulation, but not supramaximal stimulation, gap-junctional communication can occur. When one cell in an acinus was injected with a relatively low concentration of inositol 1,4,5 trisphosphate (1,4,5-IP3 ; PC 50 mM ) a calcium signal could be recorded in adjoining cells. In contrast, ' injection of CaCI2 to yield Similar increases in [Ca2+]. failed to increase • cells other .than that rejected, • • * [Ca2+]i m even though co-injected lissarhodamine could be visualized in neighboring cells, indicating the presence of junctional communication. Injection of the 1,4,5-IP3 antagonist, heparin (PC 3 mg/ml) inhibited the spread or generation of the calcium signal On stimulation with oscillatory concentrations of agonist in the injected cell. These data indicate that 1,4,5-IP3 is a likely mediator of intercellular [Ca ]i sgnalmg m panereatzc aclnar cells. Furthermore gap.junctional communication may contribute to the pattern of [Ca ]i signal!ng and the extent of secretion in aeinar cells. •
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HUMAN PTP/reg IS M I T O G E N I C
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TO PRIMARY CULTURE OF PANCREATIC D U C T A L "CELLS M__EE Z e n i l m a n , J chen, T H Magnuson, and AR Shuldiner. Dept of Surgery, Albert Einstein College of M e d i c i n e , Bronx, NY, and Johns Hopkins University, Baltimore, MD Human pancreatic thread protein (hPTP) a n d t h e regenerating (reg) p r o t e i n a r e d e r i v e d f r o m a s i n g l e g e n e a n d is i n v o l v e d in i s l e t r e g e n e r a t i o n . We recently documented a trophic effect of hPTP/reg o n the rat A R I P d u c t u l a r c e l l line, a n d wished to test it o n primary d u c t cells in Culture• Rat pancreatic duct Cells were isolated by collagenase digestion, isolated colonies were s h o w n m o r p h o l o g i c a l l y a n d i m m u n o h i s t o c h e m i C a l l y to be d u c t u l a r , hPTP was isolated from panc±eatic extracts by sequential ammonium sulfate precipitation and acid precipitation. Purity was c o n f i r m e d b y P A G E a n d HPLC. Cells were cultured w i t h 1 0 n M of h P T P f o r 72 hr, p u l s e d w i t h 1 0 u M B ~ U for 2 hr. A f t e r f i x a t i o n , n u c l e i w e r e s t a i n e d w i t h propidium iodide and BrdU monoclonal antibody. Percent nuclei positive for BrdU was calculated f o r at l e a s t 5 c o l o n i e s p e r g r o u p ; r e s u l t s f r o m t w o e x p e r i m e n t s are shown. E f f e c t on A R I P c e l l s (IX105 c e l l s / w e l l ) was documented by thymidine i n c o r p o r a t i o n , a n d d a t a e x p r e s s e d in C P M (XI03, n=4 per group). 0mM PTP 10mM PTP l ° c u l t u r e (#i) 8.7±1.1% 25.0±6.0%* l ° c u l t u r e (#2) 3.3±0.5 9.9±0.9 * ARIP 43.1±0.5 81.4±12.0" "P<0.05 u n p a i r e d t - t e s t c o m p a r e d to c o n t r o l hPTP/reg is m i t o g e n i c to p r i m a r y c u l t u r e s o f d u c t u l a r cells, as it is to the A R I P c e l l line. A l t h o u g h p r i n c i p l y a p r o d u c t of the a c i n a r cell, hPTP/reg a p p e a r s to be an i m p o r t a n t m e d i a t o r of d u c t u l a r g r o w t h a n d p e r h a p s is i n v o l v e d in the differentiation of ductular precursors into islets.
AGA ABSTRACTS
GASTROENTEROLOGY, Vol. 108, No. 4
USEFULNESS OF CAERULEIN STIMULATED PLASMA AMINO ACIDS CONSUMPTION TEST AS TUBELESS PANCREATIC EXOCRINE FUNCTION TEST U.Yoshioka, Y.Satoh, T.Saotome, Y.Kateh, Y.Okumura, H.Inoue, Y.Fujiyama and T.Bamba, Second Department of Internal Medicine, Shiga University of Medical Science, Ohtsu, Shiga, 520-21, Japan ' Plasma amino acids consumption test after caerulein stimulation (AACT) is promising and not troublesome method of pancreatic exocrine function test. But there are controversial reports about usefulness of AACT. So We examined AACT in comparison with other pancreatic exocrine function test (secretin test and BTPABA test). METHODS 23 patients (chronic pancrcatitis 17, pancreas cancer 1, Sj6gren syndrome 2, primary biliary cirrhosis 1, pancreas divisum 1, multiple pancrgas cysts 1) and 1 healthy volunteer were examined after an overnight fast. Caerulcin 50 ng/kg was injected by intravenously for 30 seconds, and blood sample was taken before and every 10 minutes to 50 minutes after eaerulein injection. Plasma was deproteinized by 5% sulfosalicylie acid. Total plasma amino acids concentration (TAA) was measured by Ninhydrin method, and individual amino acid was analyzed by HPLC (Hitachi L-8500). We compared AACT with results of secrctin test (normal 11, mild dysfunction 6, and severe dysfunction 6). RESULTS There were no differences in TAA among normal, mild and severe groups before caerulein stimulation. TAA was significantly reduced by caerulein stimulation in normal and mild groups, but there wer~ no differences in TAA of severe group between before and after caerulein stimulation. Decrease rate of TAA after caerulein stimulation (%) was significantly low in the severe group, compared with normal and mild groups. 10 rain. 20 min. 30 rain. 40 rain. 50 min. normal 6.85-+1,16 9.04-+1.17 9.75+0.66 11.01+-1.70 956+-1.65 mild 4.75-+ 1.50 7.89+ 1.18 8.17+-0.41 7.27-+ 1.16 7.66 + 1.23 severe 1.38+-0.85 2.37+-0.23i 1.83-+1.67 2.98_+2.65 2.64-+1.23 ** **## ***## * normal vs. severe # mild vs. severe mean±SE * P<0.05 ** #4 P<0.01 *** P<0.001 There were significant positive correlations between decrease rate of TAA and each parameters of secretin test. There was no significant decrease of TAA in normal and mild groups after stimulation by secretin alone (100 U iv). Sensitivity of AACT was 100% in the severe group and 40% in the mild group, which were higher than the sensitivity of BTPABA test. CONCLUSION AACT is a more useful than BTPABA test. AACT can detect severe pancreatic exocrine dysfunction, but cannot detect mild dysfunction. •
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PRESENCE OF OGF AND ZETA (~) OPIOID RECEPTOR IN HT-29 HUMAN COLON CANCER. I.S Zagon, S. Hytrek, C.M Lang, J.P. Smith P.J. McLaughlin. Depts. Neuroscience and Anatomy, Comparative Medicine, and Medicine, Penn State University College of Medicine Hershey, Pennsylvahia The endogenous opio~d system (EOS) consisting of opioid growth factor (OGF), [Met~}-enkephalin, and zeta ([) receptor has been shown to modulate the growth of animal and human tumors. Manipulation of this EOS in HT-29 human colon cancer cells xenografted to nude mice has suggested that both OGF and the f receptor are present in the HT-29 tumors. To identify and characterize the [ opioid receptor which mediates the action o~ OGF, bending assays using homogenates of HT-29 tumors and [~H]-[Met~]-enkephalin were performed Specific and saturable binding was detected, and data were consistent with a single binding site Scatehard analysis yielded a binding affinity (Kd) of 15.4 ± 2.0 nM and a binding capacity (Bmax) of 364.7 ± 25.7 fmol/mg protein Binding was dependent on protein concentration, time and temperature of assay conditions, and p~ of the h~ffer; binding was also sensitive to Na 2+ , Caz+ , and Mg 2+ ions, hut not to GppNHp. Subcellular fractionation studies revealed that binding was restricted to the fraction enriched in nuclei, with little or no specific binding detected in the membrane, mierosomal or soluble fractions. Competition assays with cold ligands for ~, 6, ~, o, and e opiold receptors demonstrated that OGF was at least 3-fold more competitive for the [ receptor. The presence of OGF was determined using radioimmunoassays for [Met ]-enkephalin Levels of OGF in tumor tissue were 59.3 ± 6.2 pg/mg tissue. Northern blot analysis showed that preproenkephalin A (PPE) mRNA expression was present in HT-29 tumors. These studies reveal, for the first time the presence of OGF and the opioid receptor the autocrine nature of OGF, and the characteristics of the [ receptor in a human colon cancer• Supported by the Laverty Foundation.
• INTERCELLULAR CALCIUM WAVES: MECHANISM OF TRANSMISSION AND RELATIONSHIP TO SECRETION IN RAT PANCREATIC AC1NI. D.I. Yule, E.L. Stuenkel and J.A. Williams. Departments o f Physiology and Internal Medicine, University of Michigan, Ann Arbor, MI 48109-0622. It is known that stimulation of acinar cells with secretagogues such as cholecystokinin (CCK) or carbachol (CCh) results in at low concentrations, the generation of Ca waves that appear to propagate through an acinus while at higher concentrations a uniform single peak m [Ca ]i followed by an elevated plateau is evoked. The role gapunctional communication plays in intercellular calcium signaling and the ubsequent secretion of digestive enzymes was studied. Intracelhilar calcium concentration [Ca2÷]i measurements were performed by digital imaging o f acini loaded with the fluorescent Ca 2÷ indicator fura,2. Imperrneant messenger and marker molecules were introduced into cells by microinjection. The extent of gag-junctional communication was measured simultaneously with [Ca~÷]~ signals (by fura-2) under physiologic conditions or supramaximal stimulation with secretagogues by monitoring diffusion of microinjected lissarhodamine fluorescence, (pipette concentration (PC) 0.25 rmM) from the injected cell to neighboring cells in the acinus. In cells stimulated with oscillatory (physiologic) concentrations of CCK the [CaZ+]i signal was characterized by the spread of Ca 2+ between cells in a wave-like fashion and the spread of lissarhodamine fluorescence was not significantly different from unstimulated cells. In contrast at supramaximal concentrations of CCK (1 riM) or CCh (0.1 mM), where secretion is diminished and oscillations are absent, no dye transfer occurred. These data indicate that under physiologic stimulation, but not supramaximal stimulation, gap-junctional communication can occur. When one cell in an acinus was injected with a relatively low concentration of inositol 1,4,5 trisphosphate (1,4,5-IP3 ; PC 50 mM ) a calcium signal could be recorded in adjoining cells. In contrast, ' injection of CaCI2 to yield Similar increases in [Ca2+]. failed to increase • cells other .than that rejected, • • * [Ca2+]i m even though co-injected lissarhodamine could be visualized in neighboring cells, indicating the presence of junctional communication. Injection of the 1,4,5-IP3 antagonist, heparin (PC 3 mg/ml) inhibited the spread or generation of the calcium signal On stimulation with oscillatory concentrations of agonist in the injected cell. These data indicate that 1,4,5-IP3 is a likely mediator of intercellular [Ca ]i sgnalmg m panereatzc aclnar cells. Furthermore gap.junctional communication may contribute to the pattern of [Ca ]i signal!ng and the extent of secretion in aeinar cells. •
•
•
•
-.
2+
2+
'
2+
•
•
.
•
HUMAN PTP/reg IS M I T O G E N I C
•
'
•
TO PRIMARY CULTURE OF PANCREATIC D U C T A L "CELLS M__EE Z e n i l m a n , J chen, T H Magnuson, and AR Shuldiner. Dept of Surgery, Albert Einstein College of M e d i c i n e , Bronx, NY, and Johns Hopkins University, Baltimore, MD Human pancreatic thread protein (hPTP) a n d t h e regenerating (reg) p r o t e i n a r e d e r i v e d f r o m a s i n g l e g e n e a n d is i n v o l v e d in i s l e t r e g e n e r a t i o n . We recently documented a trophic effect of hPTP/reg o n the rat A R I P d u c t u l a r c e l l line, a n d wished to test it o n primary d u c t cells in Culture• Rat pancreatic duct Cells were isolated by collagenase digestion, isolated colonies were s h o w n m o r p h o l o g i c a l l y a n d i m m u n o h i s t o c h e m i C a l l y to be d u c t u l a r , hPTP was isolated from panc±eatic extracts by sequential ammonium sulfate precipitation and acid precipitation. Purity was c o n f i r m e d b y P A G E a n d HPLC. Cells were cultured w i t h 1 0 n M of h P T P f o r 72 hr, p u l s e d w i t h 1 0 u M B ~ U for 2 hr. A f t e r f i x a t i o n , n u c l e i w e r e s t a i n e d w i t h propidium iodide and BrdU monoclonal antibody. Percent nuclei positive for BrdU was calculated f o r at l e a s t 5 c o l o n i e s p e r g r o u p ; r e s u l t s f r o m t w o e x p e r i m e n t s are shown. E f f e c t on A R I P c e l l s (IX105 c e l l s / w e l l ) was documented by thymidine i n c o r p o r a t i o n , a n d d a t a e x p r e s s e d in C P M (XI03, n=4 per group). 0mM PTP 10mM PTP l ° c u l t u r e (#i) 8.7±1.1% 25.0±6.0%* l ° c u l t u r e (#2) 3.3±0.5 9.9±0.9 * ARIP 43.1±0.5 81.4±12.0" "P<0.05 u n p a i r e d t - t e s t c o m p a r e d to c o n t r o l hPTP/reg is m i t o g e n i c to p r i m a r y c u l t u r e s o f d u c t u l a r cells, as it is to the A R I P c e l l line. A l t h o u g h p r i n c i p l y a p r o d u c t of the a c i n a r cell, hPTP/reg a p p e a r s to be an i m p o r t a n t m e d i a t o r of d u c t u l a r g r o w t h a n d p e r h a p s is i n v o l v e d in the differentiation of ductular precursors into islets.