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Effect of hepatocyte growth factor on experimental gastric ulcer healing in rats
A1004
AGA
ABSTRACTS
GASTROENTEROLOGY,
Vol.
108,
PAVfERN OF GASTRIC EMPTYING AND RELEASE OF INCRETINS AFTER GLUCOSE INGESTION. J. Schirra, M. I(utschinski, C. Weidmanu, T. Sch§fer, U. Wank, R. Arnold, 8. Gfkc. Dept. of Gastroenterology, University Hospital of Marburg, Germany. 1! has been claimed that glucosesolutions ranging from 0.2-1.0 kca]/ml empty from the stomach at a linear, energy-ennstautrate of about 2 kcal/min {Gastroenterology1983;85:78-82). By contrast, it is discussedif gastric emptying of caloric meals and subsequentrelease of incretin hormonesare influenced by the precedinginterdigestive phase. Therefore, we examinedthe emptying pattern of glucose solutions from the stomach, the resulting release of GIP, GLP-1 and insulin into the circulation, and the influence of the precedinginterdigestive phase. METHODS: 8 healthy male volunteers were studied on 4 separate days. In each subject, 50 g and 1O0 g of glucosedissolvedin 400 ml of water were orally administered in interdigestive phase I (P I} and phase II (P II). Antroduudenalmotility (2 autral, 4 duodenal ports)and gastric emptying {"double-markertechnique" according to Gastroenterology 1976;70:203-210) were simultaneously measuredfor 180 rain following ingestion of glucose.Rate constants (k) and slopes ([~)of the emptying curves of glucose were calculated using a power exponential fitting. Blood was regularly sampled for determination of glucose, IR-Insulin! IRGIP and IR-GLP-1 (antibody GA 1178). RESULTS: Mean_+SEM, *: p < 0.05 vs. P 1/50 g, #: p < 0.05 vs. P 1/100 g Gastricemptyingofglucose Parameter P 1/50 g P 11/50g P 1/100 g P t11100g k 0.020_+0.002 0.020-+0.001 0.014-+0.001"# 0.015-+0.00t*# 6 1.27-+0.08 1.20-+0.07 1.19-+0.09 1.20-+0.11 Lag phsse (rain) 12.8-+3.9 12.8-+4.5 23.3-+5.5 20.8-+7.5 50% retention (mini 41.4_-5.9 36.9_*3.0 54.7-+5.0*# 51.5_+5.5"# Time to phase Ill (mini 132-.19 128_+14 165-.12"# 168-_6"# % relentinn at phase U] 4.3-+2.3 6.4-+1.6 8.7-+2.3 5.4-+1.1 Deliveryof glucoseinto theduodenum[kcal/min) 0-30 min 2.7-+0.3 2.8-+0.2 4.0+_0.4*# 4.2-+0.5*# 60-90 rain 0.9-+0.1 0.9-+0.1 2.1_+0.2"# 2.1-+0.2"# 120-150 min 0.2-+0.1 0.2-+0.04 0.8_+0.1"# 0.8-+0.1"# Releaseof hormonesandplasmaglucoselevels(AUCo v e r basel.180m/n) IR-GIP (/zg/I) 3.4-+0.5 3,6-*0.4 4.6-+0.3*# 5.5-+0.7*# IR-GLP-1 (pmol/I) 12.2-+4.4 7.1 _+1.3 26.6-.5.3*# 24.7-+4.6*# IR-Inenlin (mU/I) 348.7-+82.3 392.3_+79.2 672.6_+101.2"# 642,8_+102.8"# Bleed glucose(mmnl/I) 8.8_+3.0 7.3-+2.6 12.7-.3.5"# 10.9_+3.7"# Duodenaldeliveryofglucosewhenincretinsmtomedto basalplasmalevels(kcal/min) 50 g glucose returnto basal 100 g glucose return to basal GLP-1 1.4_+0.1 60min 1.1-+0.1 120 min GIP 0.17-.0.07 150 min no return to basal CONCLUSIONS: Gastric emptying of glucosedisplays a power exponentialpattern with a short initial lag period. Correspondingly,the rate of glucosedelivery into the duodenumis not constant but exponentially declines over time. Increasing gastric glucose load raises rate of caloric delivery into the duodenum but nevertheless prolongs gastric emptying. In contrast to GIP a duodenal delivery of glucose exceeding 1 bcal/min is necessaryto release GLP-1. This would be compatible with duodenal nutrients activating an intestinal neuro-endecrineloop stimulating GLP-1 release from the ileum. In the dose range of this study, gastric emptying of glucose and hormone release are independentof the preceding interdigestive phase. Glucose loads of 200-400 kcal induce a fed motor pattern with re-appearance of phase III after approximately 95% of the meal have emptied.
• EFFECT OF H E P A T O C Y T E G R O W T H FACTOR ON E X P E R I M E N T A L GASTRIC ULCER H E A L I N G IN RATS. A. Schmassmann 1, Ch. Stettler 1, C. Hirschi 1, B. Flogerzi 1, L. Varga 1, B.M. Peskar 2, K. Matsumoto 3, T. Nakamnra 3, F. Halter 1. 1: Gastrointestinal Unit, Univ. Hospital, Inselspital, Bern, Switzerland. 2: Dep. of Exp. Clinical Medicine, Ruhr-University, Bochum, Germany. 3: Division of Biochemistry, Univ. School of Medicine, Osaka, Japan. Back,,round: Administration of hepatocyte growth factor (HGF) has been reported to increase tissue regeneration after experimental damage to liver and kidney. In gastric cell culture systems, HGF accelerates cell migration and proliferation. We report on the first in vivo study of the effect of HGF on gastric ulcer healing in rats. M e t h o d s : Serum levels of HGF were measured after i.v. and s.c. administration (Elisa). Cryoulcers were produced on gastric corpus in Wistar rats fitted with a gastric cannula. Rats were daily treated s. c. with either placebo, HGF (2 x 100 gg/kg), or omeprazole (1 x 40 gmol/kg). Ulcer healing rate was assessed by repeated quantitative video endoscopy. Prostaglandin synthesis in oxyntic mucosa, epithelial cell proliferation in ulcerated and non-ulcerated mucosa (injection of bromodeoxyuridine (BrdU) 1 hr before sacrifice), epithelial cell migration, angiogenesis, and maturation of granulation tissue were quantified on days 3, 8, and 15. Results: HGF was detected in serum during 5-20 min after injection. Compared with placebo (20 rats), HGF (20 rats) increased significantly 6keto-prostaglandin-Flc~ and E2 synthesis, respectively (day 3: +50% and +67%, P < 0.05; day 8: +23% and +37%, P < 0.05; day 15: +57% and +1%, n.s.). Omeprazole significantly increased ulcer healing rate, cell migration over the ulcer crater, and maturation of the granulation tissue by +16%, +20%, and +22%, respectively. Compared with placebo, HGF did not increase ulcer healing rate, epithelial cell migration, angiogenesis, and maturation of granulation tissue. The ratios BrdU (+) ceils in ulcer margin / non-ulcerated oxyntic mucosa were 4.3 (day 3), 3.9 (day 8), and 2.0 (day in placebo treated rats and 4.4 (day 3), 4.2 (day 8), and 3.6 (day 15) in HGF treated rats, respectively. The increase of +80% onday 15 in the HGF group was clearly significant (P < 0.001). While omeprazole significantly (P < 0.05) increased BrdU (+) cells in the non-ulcerated oxyntic mucosa on day 15 by +35%, HGF had no such effect. Conclusion: 1) HGF increased prostaglandin synthesis on days 3 and 8. 2) HGF increased epithelial cell proliferation in the ulcer margin in the late healing phase. 3) At the given dose, HGF did not accelerate cell migration and healing rate in the early phase of ulcer healing, but further studies on healing in the late remodeling phase look promising.
• NORTHERN BLOT ANALYSIS OF HISTAMINERGIC AND MUSCARINIC RECEPTORS ON RAT PARIETAL CELLS. J. Schmidtler, K. Dehne, V. Schusdziarra, M. Classen, *A. Polack, and W. Schepp. Dept. of Internal Medicine II, Technical Univ. of Munich, and *GSF-Institut fiir Klinische Molekularbiologie, Munich, Germany. Muscarinic agonists stimulate gastric exo- and endocrine cells, while histamine selectively stimulates parietal cells. However, the presence of appropriate receptors on gastric muocsal cells is controversial: While in situ hybridization failed to detect musearinic and histaminergic receptors in epithelial Cells of intact rat gastric mucosa (Science 1992;258:1662-5), PCR techniques revealed the mRNA of M3, but not of other muscarinic receptor subtypes in partially purified parietal cells and fundic glands of the rabbit gastric mucosa (Gastroenterology 1992; 103:870-5). In the present study we used highly purified fractions of isolated rat gastric mucosal cells to study receptor mRNA expression. Enzymatically isolated rat gastric mucosal cells were separated b.y counterfiow elutriation into 3 fractions (F3-F5) according to increasmg cell diameter and parietal cell content (4, 27, 80%). Density gradient centrifugation of F4 yielded highly enriched chief cells (90%; parietal cells < 5 %; F6) while density gradient centrifugation of F5 enriched parietal cells almost to purity ( > 9 5 % ; chief cells <5%; F7). Plasmid DNAs as probes for the muscarinic receptor subtypes M 1, M 2, M 3, and M 5 (rat) and M 2 (human) (T.I. Bonner, Bethesda) and plasmid DNA of the H 2 receptor (rat; L-C. Schwartz, Paris) were used to prepare [32p]labeled cDNAs as specific probes for Northern blot analysis of total cellular RNA of F3-F7. Signals were analysed by quantitative determination of radioactivity (BAS 1000, Fuji). We obtained signals not only for the M 3 (4.2 kB), but also for the M 1 (1.4 kB) receptor subtype. Expression of M 1 and M 3 slightly increased from F3 and F4 to F5 while it was weaker in F6 and maximal in F7 (relative mRNA expression levels in F3 : F4 : F5 : F6 : F7 = 0.69 : 0.79 : 0.89 : 0.47 : 1.00). No signal was obtained for the M2, M4, and M 5 subtypes. On the other hand, the H 2 receptor mRNA presented as a pronounced 5.6 kB band and a weaker 1.2 kB band. Corresponding to the contribution of parietal cells, the H 2 signal was detectable in F3 and F4, much more pronounced in F5, and maximal in F7 while it was totally absent from F6. We conclude that - unlike findings in rabbit gastric mucosal cells (Gastroenterology 1992; 103:8705) - in the rat gastric mucosa not only the M3, but also the M 1 receptor mRNA is expressed. Expression of both mnscarinic receptor messages is more pronounced in parietal cells (F7) than in chief (F6), mucus and endocrine cells (F3), while the H 2 receptor message is confined to parietal cells. Supported by DFG grant Sche 229/7-1.
• I D E N T I F I C A T I O N OF C H O L E C Y S T O K I N I N - B / G A S T R I N R E C E P T O R DOMAINS W H I C H CONFER H I G H AFFINITY FOR GASTRIN. F. Schmitz, M.-J. Wu, D. S. Pratt, M. Beinbom, and A. S. Kopin. Dept. of Medicine, New England Medical Center and Tufts University School of Medicine, Boston, MA.
•
No. 4
Pharmacologic comparison of the human CCK-B/gastrin receptor and the predominant CCK receptor from Xenopus laevis brain, CCK-X, reveals that both of these receptors share high affinity for CCK-8. A distinguishing feature of the human receptor is its >5000-fold higher affinity for gastrin. We have previously cloned both the human and Xenopus receptors; the deduced amino acid (aa) sequence identity approaches 51%. To map the receptor domains which confer high affinity for gastrin, we have constructed two series of chimeric receptors by sequential exchange of restriction fragments between the Xenopus and human cDNAs. In one group, increasing lengths of Xenopus aminoterminal sequence replaced the corresponding segment of the human receptor. The second series of chimeras had increasing amino-terminal contributions from human and a corresponding decrease in the Xenopus segment. All wild type and chimeric cDNAs were transiently expressed in COS-7 cells and affinities for CCK-8 and gastrin determined by 1251CCK8 competition experiments. CCK-8 affinities in the wild type range were considered an indication of conserved tertiary structure and were a requirement for further study. Exchange of the amino-terminal extracellular domain between Xenopus (66 aa) and human (52 aa) receptors resulted in little change in gastrin affinity. In contrast, introduction of the amino terminal third of the Xenopusreceptor, a segment including the N-terminus through the end of transmembrane domain (TMD) 3, into the human protein resulted in a 54:fold reduction in affinity for gastrin (1C50=54 riM). The converse receptor construct confirmed the importance of this domain; introduction of the amino terminal third of the human receptor into CCK-X increased gastrin affinity by >200-fold (IC50=25 nM vs. wild type CCKX, IC50 > 5000 riM). Introduction of a larger segment of the human receptor into CCK-X, including the amino terminus through transmembrane domain 5, further increased gastrin affinity to a value (IC50=2.3 nM) comparable to the CCK-B/gastrin receptor (IC50=1.0 riM). We conclude that gastrin affinity is conferred by an interaction of at least two receptor domains, the first in the region of TMDs 1-3 and the second mapping to the segment spanning TMDs 3-5. Furthermore, our data suggest that the amino terminus, TMDs 6, 7, the third extracellular loop, and the carboxy terminus are not important for gastrin selectivity.
AGA
ABSTRACTS
GASTROENTEROLOGY,
Vol.
108,
PAVfERN OF GASTRIC EMPTYING AND RELEASE OF INCRETINS AFTER GLUCOSE INGESTION. J. Schirra, M. I(utschinski, C. Weidmanu, T. Sch§fer, U. Wank, R. Arnold, 8. Gfkc. Dept. of Gastroenterology, University Hospital of Marburg, Germany. 1! has been claimed that glucosesolutions ranging from 0.2-1.0 kca]/ml empty from the stomach at a linear, energy-ennstautrate of about 2 kcal/min {Gastroenterology1983;85:78-82). By contrast, it is discussedif gastric emptying of caloric meals and subsequentrelease of incretin hormonesare influenced by the precedinginterdigestive phase. Therefore, we examinedthe emptying pattern of glucose solutions from the stomach, the resulting release of GIP, GLP-1 and insulin into the circulation, and the influence of the precedinginterdigestive phase. METHODS: 8 healthy male volunteers were studied on 4 separate days. In each subject, 50 g and 1O0 g of glucosedissolvedin 400 ml of water were orally administered in interdigestive phase I (P I} and phase II (P II). Antroduudenalmotility (2 autral, 4 duodenal ports)and gastric emptying {"double-markertechnique" according to Gastroenterology 1976;70:203-210) were simultaneously measuredfor 180 rain following ingestion of glucose.Rate constants (k) and slopes ([~)of the emptying curves of glucose were calculated using a power exponential fitting. Blood was regularly sampled for determination of glucose, IR-Insulin! IRGIP and IR-GLP-1 (antibody GA 1178). RESULTS: Mean_+SEM, *: p < 0.05 vs. P 1/50 g, #: p < 0.05 vs. P 1/100 g Gastricemptyingofglucose Parameter P 1/50 g P 11/50g P 1/100 g P t11100g k 0.020_+0.002 0.020-+0.001 0.014-+0.001"# 0.015-+0.00t*# 6 1.27-+0.08 1.20-+0.07 1.19-+0.09 1.20-+0.11 Lag phsse (rain) 12.8-+3.9 12.8-+4.5 23.3-+5.5 20.8-+7.5 50% retention (mini 41.4_-5.9 36.9_*3.0 54.7-+5.0*# 51.5_+5.5"# Time to phase Ill (mini 132-.19 128_+14 165-.12"# 168-_6"# % relentinn at phase U] 4.3-+2.3 6.4-+1.6 8.7-+2.3 5.4-+1.1 Deliveryof glucoseinto theduodenum[kcal/min) 0-30 min 2.7-+0.3 2.8-+0.2 4.0+_0.4*# 4.2-+0.5*# 60-90 rain 0.9-+0.1 0.9-+0.1 2.1_+0.2"# 2.1-+0.2"# 120-150 min 0.2-+0.1 0.2-+0.04 0.8_+0.1"# 0.8-+0.1"# Releaseof hormonesandplasmaglucoselevels(AUCo v e r basel.180m/n) IR-GIP (/zg/I) 3.4-+0.5 3,6-*0.4 4.6-+0.3*# 5.5-+0.7*# IR-GLP-1 (pmol/I) 12.2-+4.4 7.1 _+1.3 26.6-.5.3*# 24.7-+4.6*# IR-Inenlin (mU/I) 348.7-+82.3 392.3_+79.2 672.6_+101.2"# 642,8_+102.8"# Bleed glucose(mmnl/I) 8.8_+3.0 7.3-+2.6 12.7-.3.5"# 10.9_+3.7"# Duodenaldeliveryofglucosewhenincretinsmtomedto basalplasmalevels(kcal/min) 50 g glucose returnto basal 100 g glucose return to basal GLP-1 1.4_+0.1 60min 1.1-+0.1 120 min GIP 0.17-.0.07 150 min no return to basal CONCLUSIONS: Gastric emptying of glucosedisplays a power exponentialpattern with a short initial lag period. Correspondingly,the rate of glucosedelivery into the duodenumis not constant but exponentially declines over time. Increasing gastric glucose load raises rate of caloric delivery into the duodenum but nevertheless prolongs gastric emptying. In contrast to GIP a duodenal delivery of glucose exceeding 1 bcal/min is necessaryto release GLP-1. This would be compatible with duodenal nutrients activating an intestinal neuro-endecrineloop stimulating GLP-1 release from the ileum. In the dose range of this study, gastric emptying of glucose and hormone release are independentof the preceding interdigestive phase. Glucose loads of 200-400 kcal induce a fed motor pattern with re-appearance of phase III after approximately 95% of the meal have emptied.
• EFFECT OF H E P A T O C Y T E G R O W T H FACTOR ON E X P E R I M E N T A L GASTRIC ULCER H E A L I N G IN RATS. A. Schmassmann 1, Ch. Stettler 1, C. Hirschi 1, B. Flogerzi 1, L. Varga 1, B.M. Peskar 2, K. Matsumoto 3, T. Nakamnra 3, F. Halter 1. 1: Gastrointestinal Unit, Univ. Hospital, Inselspital, Bern, Switzerland. 2: Dep. of Exp. Clinical Medicine, Ruhr-University, Bochum, Germany. 3: Division of Biochemistry, Univ. School of Medicine, Osaka, Japan. Back,,round: Administration of hepatocyte growth factor (HGF) has been reported to increase tissue regeneration after experimental damage to liver and kidney. In gastric cell culture systems, HGF accelerates cell migration and proliferation. We report on the first in vivo study of the effect of HGF on gastric ulcer healing in rats. M e t h o d s : Serum levels of HGF were measured after i.v. and s.c. administration (Elisa). Cryoulcers were produced on gastric corpus in Wistar rats fitted with a gastric cannula. Rats were daily treated s. c. with either placebo, HGF (2 x 100 gg/kg), or omeprazole (1 x 40 gmol/kg). Ulcer healing rate was assessed by repeated quantitative video endoscopy. Prostaglandin synthesis in oxyntic mucosa, epithelial cell proliferation in ulcerated and non-ulcerated mucosa (injection of bromodeoxyuridine (BrdU) 1 hr before sacrifice), epithelial cell migration, angiogenesis, and maturation of granulation tissue were quantified on days 3, 8, and 15. Results: HGF was detected in serum during 5-20 min after injection. Compared with placebo (20 rats), HGF (20 rats) increased significantly 6keto-prostaglandin-Flc~ and E2 synthesis, respectively (day 3: +50% and +67%, P < 0.05; day 8: +23% and +37%, P < 0.05; day 15: +57% and +1%, n.s.). Omeprazole significantly increased ulcer healing rate, cell migration over the ulcer crater, and maturation of the granulation tissue by +16%, +20%, and +22%, respectively. Compared with placebo, HGF did not increase ulcer healing rate, epithelial cell migration, angiogenesis, and maturation of granulation tissue. The ratios BrdU (+) ceils in ulcer margin / non-ulcerated oxyntic mucosa were 4.3 (day 3), 3.9 (day 8), and 2.0 (day in placebo treated rats and 4.4 (day 3), 4.2 (day 8), and 3.6 (day 15) in HGF treated rats, respectively. The increase of +80% onday 15 in the HGF group was clearly significant (P < 0.001). While omeprazole significantly (P < 0.05) increased BrdU (+) cells in the non-ulcerated oxyntic mucosa on day 15 by +35%, HGF had no such effect. Conclusion: 1) HGF increased prostaglandin synthesis on days 3 and 8. 2) HGF increased epithelial cell proliferation in the ulcer margin in the late healing phase. 3) At the given dose, HGF did not accelerate cell migration and healing rate in the early phase of ulcer healing, but further studies on healing in the late remodeling phase look promising.
• NORTHERN BLOT ANALYSIS OF HISTAMINERGIC AND MUSCARINIC RECEPTORS ON RAT PARIETAL CELLS. J. Schmidtler, K. Dehne, V. Schusdziarra, M. Classen, *A. Polack, and W. Schepp. Dept. of Internal Medicine II, Technical Univ. of Munich, and *GSF-Institut fiir Klinische Molekularbiologie, Munich, Germany. Muscarinic agonists stimulate gastric exo- and endocrine cells, while histamine selectively stimulates parietal cells. However, the presence of appropriate receptors on gastric muocsal cells is controversial: While in situ hybridization failed to detect musearinic and histaminergic receptors in epithelial Cells of intact rat gastric mucosa (Science 1992;258:1662-5), PCR techniques revealed the mRNA of M3, but not of other muscarinic receptor subtypes in partially purified parietal cells and fundic glands of the rabbit gastric mucosa (Gastroenterology 1992; 103:870-5). In the present study we used highly purified fractions of isolated rat gastric mucosal cells to study receptor mRNA expression. Enzymatically isolated rat gastric mucosal cells were separated b.y counterfiow elutriation into 3 fractions (F3-F5) according to increasmg cell diameter and parietal cell content (4, 27, 80%). Density gradient centrifugation of F4 yielded highly enriched chief cells (90%; parietal cells < 5 %; F6) while density gradient centrifugation of F5 enriched parietal cells almost to purity ( > 9 5 % ; chief cells <5%; F7). Plasmid DNAs as probes for the muscarinic receptor subtypes M 1, M 2, M 3, and M 5 (rat) and M 2 (human) (T.I. Bonner, Bethesda) and plasmid DNA of the H 2 receptor (rat; L-C. Schwartz, Paris) were used to prepare [32p]labeled cDNAs as specific probes for Northern blot analysis of total cellular RNA of F3-F7. Signals were analysed by quantitative determination of radioactivity (BAS 1000, Fuji). We obtained signals not only for the M 3 (4.2 kB), but also for the M 1 (1.4 kB) receptor subtype. Expression of M 1 and M 3 slightly increased from F3 and F4 to F5 while it was weaker in F6 and maximal in F7 (relative mRNA expression levels in F3 : F4 : F5 : F6 : F7 = 0.69 : 0.79 : 0.89 : 0.47 : 1.00). No signal was obtained for the M2, M4, and M 5 subtypes. On the other hand, the H 2 receptor mRNA presented as a pronounced 5.6 kB band and a weaker 1.2 kB band. Corresponding to the contribution of parietal cells, the H 2 signal was detectable in F3 and F4, much more pronounced in F5, and maximal in F7 while it was totally absent from F6. We conclude that - unlike findings in rabbit gastric mucosal cells (Gastroenterology 1992; 103:8705) - in the rat gastric mucosa not only the M3, but also the M 1 receptor mRNA is expressed. Expression of both mnscarinic receptor messages is more pronounced in parietal cells (F7) than in chief (F6), mucus and endocrine cells (F3), while the H 2 receptor message is confined to parietal cells. Supported by DFG grant Sche 229/7-1.
• I D E N T I F I C A T I O N OF C H O L E C Y S T O K I N I N - B / G A S T R I N R E C E P T O R DOMAINS W H I C H CONFER H I G H AFFINITY FOR GASTRIN. F. Schmitz, M.-J. Wu, D. S. Pratt, M. Beinbom, and A. S. Kopin. Dept. of Medicine, New England Medical Center and Tufts University School of Medicine, Boston, MA.
•
No. 4
Pharmacologic comparison of the human CCK-B/gastrin receptor and the predominant CCK receptor from Xenopus laevis brain, CCK-X, reveals that both of these receptors share high affinity for CCK-8. A distinguishing feature of the human receptor is its >5000-fold higher affinity for gastrin. We have previously cloned both the human and Xenopus receptors; the deduced amino acid (aa) sequence identity approaches 51%. To map the receptor domains which confer high affinity for gastrin, we have constructed two series of chimeric receptors by sequential exchange of restriction fragments between the Xenopus and human cDNAs. In one group, increasing lengths of Xenopus aminoterminal sequence replaced the corresponding segment of the human receptor. The second series of chimeras had increasing amino-terminal contributions from human and a corresponding decrease in the Xenopus segment. All wild type and chimeric cDNAs were transiently expressed in COS-7 cells and affinities for CCK-8 and gastrin determined by 1251CCK8 competition experiments. CCK-8 affinities in the wild type range were considered an indication of conserved tertiary structure and were a requirement for further study. Exchange of the amino-terminal extracellular domain between Xenopus (66 aa) and human (52 aa) receptors resulted in little change in gastrin affinity. In contrast, introduction of the amino terminal third of the Xenopusreceptor, a segment including the N-terminus through the end of transmembrane domain (TMD) 3, into the human protein resulted in a 54:fold reduction in affinity for gastrin (1C50=54 riM). The converse receptor construct confirmed the importance of this domain; introduction of the amino terminal third of the human receptor into CCK-X increased gastrin affinity by >200-fold (IC50=25 nM vs. wild type CCKX, IC50 > 5000 riM). Introduction of a larger segment of the human receptor into CCK-X, including the amino terminus through transmembrane domain 5, further increased gastrin affinity to a value (IC50=2.3 nM) comparable to the CCK-B/gastrin receptor (IC50=1.0 riM). We conclude that gastrin affinity is conferred by an interaction of at least two receptor domains, the first in the region of TMDs 1-3 and the second mapping to the segment spanning TMDs 3-5. Furthermore, our data suggest that the amino terminus, TMDs 6, 7, the third extracellular loop, and the carboxy terminus are not important for gastrin selectivity.