- Email: [email protected]
Cholecystokinin-responsive gastric vagal afferents in vitro in rats
April 1998
Hormones, Transmitters, Growth Factors, and their Receptors Al189
promoter (-373/+26)-luciferase plasmid was treated with SssI methylase and transfected into a human hepatoma cell line, HepG2; luciferase activity was measured. RESULTS. (1) Five CpG sites are located in this region (-340 to +61) of the rat NT/N gene: -340, -190 (consensus AP-1 site), -147 (CRE halfsite), -31 and +61. The number and percentage of clones methylated at these sites are listed below: -340 -190 -147 14 d fetus: 7/14 (51%) 6/14 (43%) 6/14 (43%) 16 d fetus: 12/16 (73%) 9/16 (57%) 5/16 (31%) 60 d adult: 15/15 (100%) 15/15(100%) 15/15(00%) 120 d adult: 17/17(100%) 17/17(100%) 17/17(100%) ..........................................................................................................
-31 +61 4/14 (29%) 0/14 (0) 5/16 (31%) 0/16 (0) 5/15(33%) 2/15(13%) 8/17(47%) 4/17(24%)
CpG dinucleotides located at -340, -190 and -147 sites were 100% methylated in 60 and 120 d adult livers in which the NT/N gene is silenced. In contrast, fetal (14 and 16 d) livers, which express NT/N, demonstrate varying degrees of demethylation at these sites. (2) In addition, in vitro methylation of the NT/N-luciferase construct resulted in a 67-fold reduction in luciferase activity in HepG2 cells. CONCLUSIONS. Our results of NT/N gene methylation strongly correlate with developmental expression of NT/N in the liver with upstream sites methylated in the adult livers in which the NT/N gene is not expressed. Moreover, our data suggest that DNA methylation plays a key role in the silencing of the gut hormone NT/N in the adult liver; demethylation of these sites may explain the low level of NT/N expression noted in the fetal liver. We speculate that DNA methylation, in combination with binding of certain transcription factors, accounts for the developmental pattern of NT/N expression. • G4860
CYCLIC NUCLEOTIDE-GATED CATION CHANNELS IN RAT INTESTINE: DISTRIBUTION AND FUNCTIONAL REGULATION. .X.-T. Wang. W. Qiu, B. Lee and S. Guggino. Division of Gastroenterology, Johns Hopkins School of Medicine, Baltimore, MD. Previous studies (Am. J. Physiol. 238:F261-268, 1980) have shown the existence of sodium conductances which cannot be attributed to the amiloride sensitive sodium channel (ENaC). Nucleotide-gated channels are a small family of highly related nonselective cation channels which have a primary (CNC1, CNC2, CNC3) and secondary subunit (~CNCab). Our previous studies demonstrated that CNCI and CNC3 mRNA is expressed in all segments of intestine, while the CNC2 and [3CNCab mRNA is mainly restricted to the ileum and colon respectively. In rats fed a low-salt diet or injected with aldosterone, conditions which increase the message for the 13ENaC and are well-known to increase ENaC mediated net transepithelial sodium absorption (which restores body sodium homeostasis), the message for CNC1 is also increased in rat ileum and colon. We have now found that the changes at the message level reflect similar changes in CNC proteins. Using immunofluorescence and confocal microscopy, a polyclonal peptide antibody specific to rat CNC1 intensely labeled the rat retinal photoreceptor layer, which is the positive control tissue for CNCI. In the rat intestine, positive staining was seen mainly in the ascending colon, and the labeling localized to the apical membrane of the epithelial cells. After low-sait diet, the labeling was more intense. To further evaluate the function of these channels, sodium-mediated short circuit current (Isc) from rat colon was recorded. The basal sodium-mediated Isc was partially blocked by 10 laM amiloride. In the continuous presence of amiloride, 8-Br-cGMP increased the Isc, which was inhibited by apical addition of 1-cis-diltiazem and dichlorobenzamil, blockers of CNC. The above results provide critical data to support our hypothesis that cyclic nucleotide-gated cation channels represent another sodium transepithelial transport pathway which may regulate blood volume. • G4861
FUNCTIONAL
EXPRESSION
OF
THE
HUMAN
EPIDERMAL
G R O W T H FACTOR RECEPTOR IN ESCHERICHIA COLI: A NEW METHOD FOR ANALYZING THE BIOLOGICAL ACTIVITY OF EGF
ISOFORMS*. Yutian Wang, Jalu Hu, Suming Cheng. Xijing Hospital, Xi'an 710033, CHINA; Shengli Yang. Shanghai Bioengineering Investigation Center, Shanghai 200233 CHINA. The members of EGF family, which include epidermal growth factor(EGF), transforming growth factor-~(TGF-c0, amphiregulin(AR), heparin-binding EGF:like growth factor(HB-EGF), betacellulin(BTC), and epiregulin, share the same receptor, the EGF-R, and a spectrum of biological activities. In order to investigate the physiologic and pathologic role of the EGF family in digestive diseases and cancers, we try to establish a system that could quantitatively analyze the integral biological function of the EGF family. The main strategy of the study is to obtain an E.coli strain which expresses the extracellular domain of the human EGF-RIII (EGF-RIII), which mainly contributes to ligand-binding, in its membrane, by which we might create a method to measure the biological activity of EGF family according to the theory that multi-ligands bind competitively to the same receptor. It contained three steps: firstly, to clone the DNA of EGF-RIII; secondly, to construct the expressing vector pLERIII in which the coding region of EGF-RIII was fused in phase to the 5' part of the Lamb gene(coding for the first 279 amino-acids of the LamB protein) under ptacl2 promoter control; thirdly, to obtain the stable expressing clone. The results of the study showed that after induction
with isopmpyl-13-D-galactopyranoside(IPTG), the strain of E.coli JM109 transformed with the plasmid pLERIII could express specific and saturable binding sites for 125I-EGF, which were discovered mainly on its membrane by immunoelectmnmicroscopy. By Scatchard analysis, the results suggested that the number of the sites on the stable expressing strain was about 738 sites per cell, and its dissociation constant was about 3.0x10 -11 tool/1. Basing on the stable expressing strain, competition experiments showed all the members of EGF family could bind competitively to these sites. The study indicated that system might become a useful tool for analyzing the biological activity of EGF family. * Funded by National Nature Science Research Foundation of China. G4862
REGULATION OF CHOLECYSTOKININ SECRETION IN STC-1 CELLS THROUGH pH-SENSITIVE POTASSIUM CHANNELS. Y. Wang, V. Prpic, J. G. Fitz, and R. A. Liddle. Departments of Medicine, Durham VA and Duke University Medical Centers, Durham, NC and University of Colorado, Denver, CO. Cholecystokinin (CCK) is secreted into the blood from cells of the upper small intestine upon ingestion of food. Previous studies have shown that the resting membrane potential is determined in large part by K + channel activity and that membrane depolarization produced by K÷ channel blockade by barium, disopyramide and other maneuvers is a stimulant of CCK secretion. Moreover, inhibition of Na+/H÷ exchange by amiloride analogs which would tend to acidify the intracellular compartment, has also been reported to stimulate CCK secretion. Since certain K÷ channels are pH-sensitive, the purpose of these studies was to determine whether alterations in intracellular pH (pHi) in STC-1 cells directly affect K + channel activity and CCK secretion. The effects of extraceUular pH (pile) and pHi on K+ channel activity and CCK secretion was examined in STC-1 cells. Basal secretion of CCK over 15 minutes at pH 7.4 and 37°C averaged 6.3 fmol/well (-106 cells). Substitution of buffer at pH 7.0 significantly stimulated CCK release by 232%. To examine whether pH directly affects membrane K+ permeability, outward currents carried by K+ were measured using whole cell patch clamp techniques. Under basal conditions (pHe 7.4, pHi 7.2), potassium current (IK) measured at 0 mV averaged 902 -+ 118 (mean -+ SE) pA. I K was significantly inhibited by lowering pHe to 7.0 (I K = 325 _+ 132 pA) or 6.5 OK = 180 _+ 152 pA). These effects appear to be mediated through changes in pH i since intracellular dialysis with acidic solutions (pHe 7.4, pH i 7.0) nearly eliminated current activity [6-+ 13 pA (p<0.002)]. These findings indicate that the K+ permeability of STC-1 cells is tightly regulated by changes in pHi. Consequently, inhibition of K+ efflux by effects of phi on K+ channels may serve as a potent stimulus for hormone secretion, linking cell metabolism and secretory functions. Q G4863 CItOLECYSTOKININ-RESPONSIVE GASTRIC VAGAL AFFERENTS IN VITRO IN RATS. Yu Hua Wang, Yvette Tach6 and Jen Yu Wek Dept. of
Med., CURE Digest. Dis. Res. Ctr. and BRI, UCLA Sch. of Med., Los Angeles, CA 90095. Back~,round: Although cholecystokinin (CCK)-responsive gastric vagal afferents (GVAs) have been studied in vivo for more than a decade, the terminal locations and properties remain the subject of investigation. To reduce the sample bias and the interference from different levels of reflexes, and to improve the accessibility of the receptive fields, CCK-responsive GVAs were further investigated on an in vitro isolated stomach. Purpos~ in vitro 1) to determine whether there are CCK-responsive GVAs and whether the terminal is polymodal ending; 2) to investigate if the response is doserelated and the action is CCK receptor mediated; and 3) to study whether the terminal's CCK sensitivity is effected by ambient CCK level. Methods~ Experiments were conducted on the rat in vitro gastric vagus-stomach preparation. The single and multi-unit activities of GVAs were recorded and analyzed from the ventral gastric branch of the vagus nerve strands. The left gastric artery was cannulated for intra-arterial injections (ia) of vehicle (0.1 ml), CCK-8 (0.1 to 100 pmol), CCK-A (devazepide) and CCK-B (L365,260) receptor antagonists (20 lag). A quotient (Q), Q=5 min spike count after/before each treatment, was used t o represent the response magnitude. The time interval between the treatments was at least 30 min. Results: 1) Single unit-activities from 57 GVA terminals have been identified responding to CCK-8 ia (1-10 pmol) and all were activated also by local mechanical stimulus (camel's hair brush). 2) Consecutive ia injections of CCK-8 0.1, 1.0, 10, and 100 pmol increased the Qs of GVAs multi-unit discharge from 1.79 _+.0.77, to 2.58 _+0.89, 4.10 _+0.69 and 4.57 _+0.77, N=5, respectively. The response to CCK-8 (10 pmol) was suppressed by 20 lag devazepide pretreatment (4.10 -+ 0.67, N=5 vs 1.27 ± 0.87, N=3) but not by 20 lag L-365,260 (2.07_+0.22 vs 1.95_+0.22, N=8). 3) The terminals responsiveness to ia injection of 5 pmol CCK-8 were reduced from 1.74 _+0.24, to 1.48 _+0.21 and 1.13 -+ 0.04, when ambient CCK-8 level were increased from 0.1 to 1.0 and 10 pmol, respectively. These data indicted 1) in vitro, CCK induces dose-related, CCK-A receptor mediated activation of GVAs with polymodal endings; 2) CCK sensitivity is inversely related to the basal CCK levels; 3) the in vitro preparation is a suitable model to investigate the regulation of GVA terminals, Supported by NIH Grant DK48476.
Hormones, Transmitters, Growth Factors, and their Receptors Al189
promoter (-373/+26)-luciferase plasmid was treated with SssI methylase and transfected into a human hepatoma cell line, HepG2; luciferase activity was measured. RESULTS. (1) Five CpG sites are located in this region (-340 to +61) of the rat NT/N gene: -340, -190 (consensus AP-1 site), -147 (CRE halfsite), -31 and +61. The number and percentage of clones methylated at these sites are listed below: -340 -190 -147 14 d fetus: 7/14 (51%) 6/14 (43%) 6/14 (43%) 16 d fetus: 12/16 (73%) 9/16 (57%) 5/16 (31%) 60 d adult: 15/15 (100%) 15/15(100%) 15/15(00%) 120 d adult: 17/17(100%) 17/17(100%) 17/17(100%) ..........................................................................................................
-31 +61 4/14 (29%) 0/14 (0) 5/16 (31%) 0/16 (0) 5/15(33%) 2/15(13%) 8/17(47%) 4/17(24%)
CpG dinucleotides located at -340, -190 and -147 sites were 100% methylated in 60 and 120 d adult livers in which the NT/N gene is silenced. In contrast, fetal (14 and 16 d) livers, which express NT/N, demonstrate varying degrees of demethylation at these sites. (2) In addition, in vitro methylation of the NT/N-luciferase construct resulted in a 67-fold reduction in luciferase activity in HepG2 cells. CONCLUSIONS. Our results of NT/N gene methylation strongly correlate with developmental expression of NT/N in the liver with upstream sites methylated in the adult livers in which the NT/N gene is not expressed. Moreover, our data suggest that DNA methylation plays a key role in the silencing of the gut hormone NT/N in the adult liver; demethylation of these sites may explain the low level of NT/N expression noted in the fetal liver. We speculate that DNA methylation, in combination with binding of certain transcription factors, accounts for the developmental pattern of NT/N expression. • G4860
CYCLIC NUCLEOTIDE-GATED CATION CHANNELS IN RAT INTESTINE: DISTRIBUTION AND FUNCTIONAL REGULATION. .X.-T. Wang. W. Qiu, B. Lee and S. Guggino. Division of Gastroenterology, Johns Hopkins School of Medicine, Baltimore, MD. Previous studies (Am. J. Physiol. 238:F261-268, 1980) have shown the existence of sodium conductances which cannot be attributed to the amiloride sensitive sodium channel (ENaC). Nucleotide-gated channels are a small family of highly related nonselective cation channels which have a primary (CNC1, CNC2, CNC3) and secondary subunit (~CNCab). Our previous studies demonstrated that CNCI and CNC3 mRNA is expressed in all segments of intestine, while the CNC2 and [3CNCab mRNA is mainly restricted to the ileum and colon respectively. In rats fed a low-salt diet or injected with aldosterone, conditions which increase the message for the 13ENaC and are well-known to increase ENaC mediated net transepithelial sodium absorption (which restores body sodium homeostasis), the message for CNC1 is also increased in rat ileum and colon. We have now found that the changes at the message level reflect similar changes in CNC proteins. Using immunofluorescence and confocal microscopy, a polyclonal peptide antibody specific to rat CNC1 intensely labeled the rat retinal photoreceptor layer, which is the positive control tissue for CNCI. In the rat intestine, positive staining was seen mainly in the ascending colon, and the labeling localized to the apical membrane of the epithelial cells. After low-sait diet, the labeling was more intense. To further evaluate the function of these channels, sodium-mediated short circuit current (Isc) from rat colon was recorded. The basal sodium-mediated Isc was partially blocked by 10 laM amiloride. In the continuous presence of amiloride, 8-Br-cGMP increased the Isc, which was inhibited by apical addition of 1-cis-diltiazem and dichlorobenzamil, blockers of CNC. The above results provide critical data to support our hypothesis that cyclic nucleotide-gated cation channels represent another sodium transepithelial transport pathway which may regulate blood volume. • G4861
FUNCTIONAL
EXPRESSION
OF
THE
HUMAN
EPIDERMAL
G R O W T H FACTOR RECEPTOR IN ESCHERICHIA COLI: A NEW METHOD FOR ANALYZING THE BIOLOGICAL ACTIVITY OF EGF
ISOFORMS*. Yutian Wang, Jalu Hu, Suming Cheng. Xijing Hospital, Xi'an 710033, CHINA; Shengli Yang. Shanghai Bioengineering Investigation Center, Shanghai 200233 CHINA. The members of EGF family, which include epidermal growth factor(EGF), transforming growth factor-~(TGF-c0, amphiregulin(AR), heparin-binding EGF:like growth factor(HB-EGF), betacellulin(BTC), and epiregulin, share the same receptor, the EGF-R, and a spectrum of biological activities. In order to investigate the physiologic and pathologic role of the EGF family in digestive diseases and cancers, we try to establish a system that could quantitatively analyze the integral biological function of the EGF family. The main strategy of the study is to obtain an E.coli strain which expresses the extracellular domain of the human EGF-RIII (EGF-RIII), which mainly contributes to ligand-binding, in its membrane, by which we might create a method to measure the biological activity of EGF family according to the theory that multi-ligands bind competitively to the same receptor. It contained three steps: firstly, to clone the DNA of EGF-RIII; secondly, to construct the expressing vector pLERIII in which the coding region of EGF-RIII was fused in phase to the 5' part of the Lamb gene(coding for the first 279 amino-acids of the LamB protein) under ptacl2 promoter control; thirdly, to obtain the stable expressing clone. The results of the study showed that after induction
with isopmpyl-13-D-galactopyranoside(IPTG), the strain of E.coli JM109 transformed with the plasmid pLERIII could express specific and saturable binding sites for 125I-EGF, which were discovered mainly on its membrane by immunoelectmnmicroscopy. By Scatchard analysis, the results suggested that the number of the sites on the stable expressing strain was about 738 sites per cell, and its dissociation constant was about 3.0x10 -11 tool/1. Basing on the stable expressing strain, competition experiments showed all the members of EGF family could bind competitively to these sites. The study indicated that system might become a useful tool for analyzing the biological activity of EGF family. * Funded by National Nature Science Research Foundation of China. G4862
REGULATION OF CHOLECYSTOKININ SECRETION IN STC-1 CELLS THROUGH pH-SENSITIVE POTASSIUM CHANNELS. Y. Wang, V. Prpic, J. G. Fitz, and R. A. Liddle. Departments of Medicine, Durham VA and Duke University Medical Centers, Durham, NC and University of Colorado, Denver, CO. Cholecystokinin (CCK) is secreted into the blood from cells of the upper small intestine upon ingestion of food. Previous studies have shown that the resting membrane potential is determined in large part by K + channel activity and that membrane depolarization produced by K÷ channel blockade by barium, disopyramide and other maneuvers is a stimulant of CCK secretion. Moreover, inhibition of Na+/H÷ exchange by amiloride analogs which would tend to acidify the intracellular compartment, has also been reported to stimulate CCK secretion. Since certain K÷ channels are pH-sensitive, the purpose of these studies was to determine whether alterations in intracellular pH (pHi) in STC-1 cells directly affect K + channel activity and CCK secretion. The effects of extraceUular pH (pile) and pHi on K+ channel activity and CCK secretion was examined in STC-1 cells. Basal secretion of CCK over 15 minutes at pH 7.4 and 37°C averaged 6.3 fmol/well (-106 cells). Substitution of buffer at pH 7.0 significantly stimulated CCK release by 232%. To examine whether pH directly affects membrane K+ permeability, outward currents carried by K+ were measured using whole cell patch clamp techniques. Under basal conditions (pHe 7.4, pHi 7.2), potassium current (IK) measured at 0 mV averaged 902 -+ 118 (mean -+ SE) pA. I K was significantly inhibited by lowering pHe to 7.0 (I K = 325 _+ 132 pA) or 6.5 OK = 180 _+ 152 pA). These effects appear to be mediated through changes in pH i since intracellular dialysis with acidic solutions (pHe 7.4, pH i 7.0) nearly eliminated current activity [6-+ 13 pA (p<0.002)]. These findings indicate that the K+ permeability of STC-1 cells is tightly regulated by changes in pHi. Consequently, inhibition of K+ efflux by effects of phi on K+ channels may serve as a potent stimulus for hormone secretion, linking cell metabolism and secretory functions. Q G4863 CItOLECYSTOKININ-RESPONSIVE GASTRIC VAGAL AFFERENTS IN VITRO IN RATS. Yu Hua Wang, Yvette Tach6 and Jen Yu Wek Dept. of
Med., CURE Digest. Dis. Res. Ctr. and BRI, UCLA Sch. of Med., Los Angeles, CA 90095. Back~,round: Although cholecystokinin (CCK)-responsive gastric vagal afferents (GVAs) have been studied in vivo for more than a decade, the terminal locations and properties remain the subject of investigation. To reduce the sample bias and the interference from different levels of reflexes, and to improve the accessibility of the receptive fields, CCK-responsive GVAs were further investigated on an in vitro isolated stomach. Purpos~ in vitro 1) to determine whether there are CCK-responsive GVAs and whether the terminal is polymodal ending; 2) to investigate if the response is doserelated and the action is CCK receptor mediated; and 3) to study whether the terminal's CCK sensitivity is effected by ambient CCK level. Methods~ Experiments were conducted on the rat in vitro gastric vagus-stomach preparation. The single and multi-unit activities of GVAs were recorded and analyzed from the ventral gastric branch of the vagus nerve strands. The left gastric artery was cannulated for intra-arterial injections (ia) of vehicle (0.1 ml), CCK-8 (0.1 to 100 pmol), CCK-A (devazepide) and CCK-B (L365,260) receptor antagonists (20 lag). A quotient (Q), Q=5 min spike count after/before each treatment, was used t o represent the response magnitude. The time interval between the treatments was at least 30 min. Results: 1) Single unit-activities from 57 GVA terminals have been identified responding to CCK-8 ia (1-10 pmol) and all were activated also by local mechanical stimulus (camel's hair brush). 2) Consecutive ia injections of CCK-8 0.1, 1.0, 10, and 100 pmol increased the Qs of GVAs multi-unit discharge from 1.79 _+.0.77, to 2.58 _+0.89, 4.10 _+0.69 and 4.57 _+0.77, N=5, respectively. The response to CCK-8 (10 pmol) was suppressed by 20 lag devazepide pretreatment (4.10 -+ 0.67, N=5 vs 1.27 ± 0.87, N=3) but not by 20 lag L-365,260 (2.07_+0.22 vs 1.95_+0.22, N=8). 3) The terminals responsiveness to ia injection of 5 pmol CCK-8 were reduced from 1.74 _+0.24, to 1.48 _+0.21 and 1.13 -+ 0.04, when ambient CCK-8 level were increased from 0.1 to 1.0 and 10 pmol, respectively. These data indicted 1) in vitro, CCK induces dose-related, CCK-A receptor mediated activation of GVAs with polymodal endings; 2) CCK sensitivity is inversely related to the basal CCK levels; 3) the in vitro preparation is a suitable model to investigate the regulation of GVA terminals, Supported by NIH Grant DK48476.