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Effects of the histamine H2-receptor antagonist T-593 on histamine H2-receptor: Two enantiomers and analogues
April 1995
• ALTERATION OF THE GASTRIN/CCK-B RECEPTOR BY ENDOGENOUS HYPERGASTRINEMIA IN TRANSFORMED ECL CELLS. LH Tanm GP Lawton, K Miu, IM Modlin Yale GI Surgery and West Haven VAMC ,CT. The enterochromaffin-like (ECL) cell of the gastric fundus is profoundly affected by gastrin. Gastrin stimulation of the ECL cell results in histamine secretion, and DNA synthesis through a CCK-B/gastrin receptor, which can be blocked by the specific receptor antagonist L365,260. A principal role of the ECL cell is the regulation of parietal cell acid secretion. In addition, ECL cell proliferation is gastrin driven. The effects of elevated gastrin levels on the ECL cell CCK-B receptor are unknown. Homospecific upregulation of receptor population has been reported in many endocrine cell systems. The purpose of this study was to determine the effect of endogenous hypergastrinemia on ECL cell gastrin receptors and evaluate the functional effects on DNA synthesis and histamine secretion. Hypergastrinemia was generated in the mastomys rodent by the histamine-2 receptor antagonist loxtidine (lox)(llmg/kg/day). Two groups (n=27 each) of young (3-6 moo mastomys were studied. Gastrin levels were determined by RIA. In the untreated, 8 wk and 16 wk lox groups, gastrin levels were 46 ± 5, 196 ± 35 and 204 ± 45 pmol/L (p<0.05), respectively. Sections of fundic mucosa (n=3) in each group were evaluated utilizing polyclonal gastrin receptor antibody (1:200). Immunocytuchemical staining was significantly increased after both 8 wks and 16 wks of lox. Fundic mucosal homogenates were then prepared (n=3, each group) and Western blotting performed using anti-gastrin receptor Ab (l: 1000). Membrane bound Ab were detected using a luminol-chemihiminesccncesystem, and densitometer quantified. There was a significant increase in a major protein band (Mr ±70 kDa) consistent with the gastrin receptor in the 8 wk and 16 wk groups. To evaluate the functional significance of these alterations, we isolated pure ECL cells, and quantified histamine secretion (RIA), and DNA synthesis by measuring the incorporation of 5-bromo-2'-deoxyuridine (BrdU)(ELISA) in primary culture. ECL cells were isolated (n=4 preps/group) by pronase digestion and EDTA exposure, followed by particle size and density separation using counterflow elutriation and Nycodenz gradient centrifugation. Cells were obtained with a 200-fold enrichment and a purity of 90-95% as determined by total histamine content and chromogranin immunofluorescence. Trypan blue exclusion demonstrated >95% viability. The ECS0 was 2x 10-l 1M and 2x 10-10M for gastrin-stimulated BrdU uptake in the untreated group and the 8 wk group respectively. The EC50 for gasttinstimulated histamine secretion was increased from 3 x 10-10M to 10-9M and 5x 10-gM, in the untreated, 8wk and 16 wk groups, respectively. Homospecific upregulation of the gastrin receptor is evident in ECL cells after prolonged periods of endogenous hypergastrinemia. The proliferative and secretory responses to gastrin, however, are attenuated. Thus, in the transformation from normal to neoplasia, ECL cell regulation becomes less gastrin responsive. The identification of the specific ECL cell proliferative regulatory mechanism in neoplasia will be of considerable pathological relevance since neoplasia progresses despite loxtidine cessation and the advent of normal gastrin levels.
EFFECTS OF THE HISTAMINE H2-RECEPTOR ANTAGONIST T - 5 9 3 ON HISTAMINE H2-RECEPTOR: TWO ENANTIOMERS AND ANALOGUES. T. Tashiro. T. W a t a n a b e , K. Mild, M. Ichinose, N. Kakei, M. M a t s u s h i m a , N. Yahagi, M. Fddo, M. Oka, S. I s h i h a m a , Y. M a t s u b a r a , T. Okuda, K. Kurokawa. 1st Dept. Int. Med., U n i v e r s i t y of T o k y o Medical School, Tokyo, JAPAN H i s t a m i n e p l a y s a n i m p o r t a n t role in r e g u l a t i n g g a s t r i c acid s e c r e t i o n . C a n i n e H2 h i s t a m i n e r e c e p t o r has b e e n c l o n e d a n d t h r e e p a r t i c u l a r a m i n o acid r e s i d u e s h a v e b e e n r e p o r t e d to be i m p o r t a n t for specific b i n d i n g of h i s t a m i n e to H2 h i s t a m i n e r e c e p t o r . T e r t i a r y s t r u c t u r e of H2 h i s t a m i n e r e c e p t o r is n o t k n o w n i n detail. T-593, ( E ) - ( ± ) - l - [ 2 - h y d r o x y - 2 - ( 4 hydroxyphenyl) ethyl]-3-[2-[[[5-(methylarnino) methyl-2furyl] m e t h y l ] thio] e t h y l ] - 2 - ( m e t h y l s u l f o n y l ) g u a n i d i n e , is a n e w l y s y n t h e s i z e d s e c o n d g e n e r a t i o n H2 a n t a g o n i s t a n d a r a c e m i c c o m p o u n d c o m p o s e d of two k i n d s of e n a n t i o m e r s : R(+)T-593 a n d S(-)-T-593. We r e p o r t d i f f e r e n c e s of i n h i b i t o r y effects of two e n a n t i o m e r s a n d a n a l o g u e s o n H2 h i s t a m i n e r e c e p t o r . It is a n e f f o r t to e v a l u a t e t e r t i a r y s t r u c t u r e of H2 h i s t a m i n e r e c e p t o r . METHODS: HEPA cells t r a n s f e c t e d to e x p r e s s the c a n i n e H2 h i s t a m i n e r e c e p t o r g e n e w e r e i n c u b a t e d i n EBSS c o n t a i n i n g 0.1% BSA a n d v a r y i n g c o n c e n t r a t i o n s of h i s t a m i n e a n d T-593 or its a n a l o g u e s for 60 re.in at 37C. cAMP c o n t e n t was m e a s u r e d b y RIA a s s a y k i t (Yamasa, JAPAN). RESULTS: (1) S(-)-T-593 s h o w e d a m o r e p o t e n t h i s t a m i n e H2a n t a g o n i s m t h a n r a c e m i c T-593. (IC50=1x10-6) (2) R(+)-T-593 s h o w e d n o specific a n t a g o n i s m to h i s t a m i n e H2-receptor. (3) T6 4 9 ( D e s b e n z y l T-593) a n d T - 6 5 7 ( D e s m e t h y l a m i n o m e t h y l T593) s h o w e d n o specific a n t a g o n i s m to h i s t a m i n e H 2 - r e c e p t o r . O u r r e s u l t s s h o w i n g the d i f f e r e n t i n h i b i t o r y a c t i o n of e a c h enantiomer indicate that hydroxy moiety on asymmetric c a r b o n of T-593 m a y i n t e r a c t w i t h one of the b i n d i n g sites w h e r e h i s t a m i n e a c t i v a t e s H 2 - r e c e p t o r . Differences of t h e t e r t i a r y s t r u c t u r e of e a c h e n a n t i o m e r are i m p o r t a n t to d i s t i n g u i s h the a n t a g o n i s m , a n d t e r t i a r y s t r u c t u r e of T-593, e s p e c i a l l y the p o s i t i o n of c h a r g e d o r p o l a r a t o m s , is critical for the a n t a g o n i s m .
Hormones and Receptors
A1011
E N I ) O C T r o s I s OF GASTRIN IN GASTRIN RF_~EP'FOR EXPRESSING CELLS. N. Tarasova, S. A. Wank*, E. Hudson, G. Czerwinski, V. l.Romanov,
J. Resau, C J. Miehejda. ABL-Basic Research Program, National Cancer Institute, Frederick Cancer Research and Development Center, P.O. Box B, Frederick, MD, 21702. * Digestive Disease Branch, National Institute of Diabetes and Digestive And Kidney Diseases, National Institute of Health, Bethesda, MD. The growth promoting effects ofgastrin on many GI cell lines are well known, but the mechanism of that effect is poorly understood. As a first step in the study of the hormone interaction with gastrin receptor(GR) expressing cells, three fluorescent derivatives of heptagastrin were synthesized, characterized and tested with confocal laser scanning microscopy (CLSM) on a number of GR expressing cell lines. Cyanine dye, Cy3.29 and borfluoropyrroraethene (BODIPY) derivatives of the hormone were tound to be absorbed into the cells and concentrated in perinuclear organelles by a passive mechanism. The BODIPY derivative turned out to be chemically unstable and was bleached by the laser beam very rapidly. Rhodamine Greenheptagastrin, however turned out to be suitable for the studies of GR interaction with the ligand. Rapid clustering (within 4-7 rain) and internalization of the compound upon binding at room temperature and at 370 was observed in rat pancreatic acinar cells AR42J, human gastric adenocarcinomas AGS-P and SIIA, human colon adenocarcinoma HCTI 16 and NIH 3T3 ceils, transfected with human GR cDNA. At 37°C partial colocalization with the lysosome marker neutral red was detected by CLSM, suggesting that internalized gastrin ends up in lysosomes. Lowering the temperature to 20°C inhibited translocation of the ligand to lysosomes. Decreasing the temperature to 4°C completely abolished clustering and internalization of the labeled hormone. Immuno-electron microscopy studies with the antibodies to gastrin revealed the presence of internalized hormone in multivesieular particles and endosomes. Almost no gastrin was detected in lysosomes with the antibodies, probably due to the rapid degradation of the peptide. A negligible amount of the hormone was detected in the nucleus by CLSM/EM. Continuous accumulation of fluorescent label in the cells was observed by CLSM in the presence of protein synthesis inhibitor cycloheximide suggesting that gastrin recycled back to. the cell membrane after delivering the hormone into intracellular compartments. (Research sponsored in part by the National Cancer Institute, DHHS, under contract No. NO1-CO-46000 with ABL)
• GASTRIN AND GLYCINE-EXTENDED PROGASTR1N PROCESSING INTERMEDIATES INDUCE DIPI~ERENT PROGRAMS OF EARLY GENE ACTIVATION. A. Todisco, Y. Takeuchi, C.J. Dickinson, T. Yamada. Depts. Int. IVied. & Pods., U. Mich. Med. Ctr., Ann Arbor, MI. Progastrin is post-translationally processed to gastrin (G-NH2) via glycine-extended intermediates (G-Gly) which, initially thought to be biologically inactive, have recently been shown to have actions of their own as stimulants of cellular growth. Since G-NHz and G-Gly have distinct receptors, we sought to investigate whether they exert their trophic actions through separate mechanisms. We first examined whether the growth stimulatory effects of G-NH2 and G-Gly on the rat pancreatic acinar cell line, AR4-ZI, i s associated with induction of the expression of the early response genes c-fos and c-jun. G17-NH2(10-10 M-10-SM) increased dosedependently c-fos and c-jun specific m-RNAs (determined by Northern blots) in serum starved AR4-ZI cells but G17-Gly did not. As growth factors activate c-fos gene expression through a serum response element (SRE), we examined the effects of G17-NH 2 (10-SM) or G17-GIy (10-SM) on AR4-2J cells transfected with an SRE-luciferase reporter gene plasmid. After 6h of incubation G17-NH 2 induced luciferase activity seven-fold but G17-GIy had no effect. Growth factor activation of the c-fos SRE is known to involve the activation of the small GTP binding protein RAS, thus we examined whether inhibition of RAS expression with a dominantly expressed mutant (inactive) RAS gene transfected into AR4-2J cells could inhibit the effects of Gl7-NH z on c-fos gene transcription. In the presence of dominant negative RAS, G 17-NH2 induction of SRE-luciferase activity was reduced by 30%. Since c-jun is known to be activated via aminoterminal phosphorylation by JUN-kinase, we examined the effect of G 17NH2 (10-8M) or G17-Gly (10-SM) on AR4-2J cell JUN-kinase activity by co-transfecting the chimeric GAL4-c-jun expression plasmid and the 5XGAL-luciferase reporter plasmid. In this system GAL4-c-jun can transactivate and stimulate luciferase activity only if tbe c-jun aminoterminus is phosphorylated. In contrast to our previous observations, G17Gly induced 5XGAL-luciferase activity by three-fold, indicating JUNkinase activation, while G17-NH2 had no effect. Our results lead us to conclude that G-NH2 and G-Gly both stimulate cell proliferation through different programs of early gene activation. While G-NH 2 stimulates c-los and c-jun gene expression via a RAS dependent pathway, G-Gly regulates early gene activation through post-translational modification. Our studies present a novel model in which both the precursor and the product of a key processing reaction, peptide ~-amidation, stimulate cellular proliferation via distinct receptors linked to different signal transduction pathways.
• ALTERATION OF THE GASTRIN/CCK-B RECEPTOR BY ENDOGENOUS HYPERGASTRINEMIA IN TRANSFORMED ECL CELLS. LH Tanm GP Lawton, K Miu, IM Modlin Yale GI Surgery and West Haven VAMC ,CT. The enterochromaffin-like (ECL) cell of the gastric fundus is profoundly affected by gastrin. Gastrin stimulation of the ECL cell results in histamine secretion, and DNA synthesis through a CCK-B/gastrin receptor, which can be blocked by the specific receptor antagonist L365,260. A principal role of the ECL cell is the regulation of parietal cell acid secretion. In addition, ECL cell proliferation is gastrin driven. The effects of elevated gastrin levels on the ECL cell CCK-B receptor are unknown. Homospecific upregulation of receptor population has been reported in many endocrine cell systems. The purpose of this study was to determine the effect of endogenous hypergastrinemia on ECL cell gastrin receptors and evaluate the functional effects on DNA synthesis and histamine secretion. Hypergastrinemia was generated in the mastomys rodent by the histamine-2 receptor antagonist loxtidine (lox)(llmg/kg/day). Two groups (n=27 each) of young (3-6 moo mastomys were studied. Gastrin levels were determined by RIA. In the untreated, 8 wk and 16 wk lox groups, gastrin levels were 46 ± 5, 196 ± 35 and 204 ± 45 pmol/L (p<0.05), respectively. Sections of fundic mucosa (n=3) in each group were evaluated utilizing polyclonal gastrin receptor antibody (1:200). Immunocytuchemical staining was significantly increased after both 8 wks and 16 wks of lox. Fundic mucosal homogenates were then prepared (n=3, each group) and Western blotting performed using anti-gastrin receptor Ab (l: 1000). Membrane bound Ab were detected using a luminol-chemihiminesccncesystem, and densitometer quantified. There was a significant increase in a major protein band (Mr ±70 kDa) consistent with the gastrin receptor in the 8 wk and 16 wk groups. To evaluate the functional significance of these alterations, we isolated pure ECL cells, and quantified histamine secretion (RIA), and DNA synthesis by measuring the incorporation of 5-bromo-2'-deoxyuridine (BrdU)(ELISA) in primary culture. ECL cells were isolated (n=4 preps/group) by pronase digestion and EDTA exposure, followed by particle size and density separation using counterflow elutriation and Nycodenz gradient centrifugation. Cells were obtained with a 200-fold enrichment and a purity of 90-95% as determined by total histamine content and chromogranin immunofluorescence. Trypan blue exclusion demonstrated >95% viability. The ECS0 was 2x 10-l 1M and 2x 10-10M for gastrin-stimulated BrdU uptake in the untreated group and the 8 wk group respectively. The EC50 for gasttinstimulated histamine secretion was increased from 3 x 10-10M to 10-9M and 5x 10-gM, in the untreated, 8wk and 16 wk groups, respectively. Homospecific upregulation of the gastrin receptor is evident in ECL cells after prolonged periods of endogenous hypergastrinemia. The proliferative and secretory responses to gastrin, however, are attenuated. Thus, in the transformation from normal to neoplasia, ECL cell regulation becomes less gastrin responsive. The identification of the specific ECL cell proliferative regulatory mechanism in neoplasia will be of considerable pathological relevance since neoplasia progresses despite loxtidine cessation and the advent of normal gastrin levels.
EFFECTS OF THE HISTAMINE H2-RECEPTOR ANTAGONIST T - 5 9 3 ON HISTAMINE H2-RECEPTOR: TWO ENANTIOMERS AND ANALOGUES. T. Tashiro. T. W a t a n a b e , K. Mild, M. Ichinose, N. Kakei, M. M a t s u s h i m a , N. Yahagi, M. Fddo, M. Oka, S. I s h i h a m a , Y. M a t s u b a r a , T. Okuda, K. Kurokawa. 1st Dept. Int. Med., U n i v e r s i t y of T o k y o Medical School, Tokyo, JAPAN H i s t a m i n e p l a y s a n i m p o r t a n t role in r e g u l a t i n g g a s t r i c acid s e c r e t i o n . C a n i n e H2 h i s t a m i n e r e c e p t o r has b e e n c l o n e d a n d t h r e e p a r t i c u l a r a m i n o acid r e s i d u e s h a v e b e e n r e p o r t e d to be i m p o r t a n t for specific b i n d i n g of h i s t a m i n e to H2 h i s t a m i n e r e c e p t o r . T e r t i a r y s t r u c t u r e of H2 h i s t a m i n e r e c e p t o r is n o t k n o w n i n detail. T-593, ( E ) - ( ± ) - l - [ 2 - h y d r o x y - 2 - ( 4 hydroxyphenyl) ethyl]-3-[2-[[[5-(methylarnino) methyl-2furyl] m e t h y l ] thio] e t h y l ] - 2 - ( m e t h y l s u l f o n y l ) g u a n i d i n e , is a n e w l y s y n t h e s i z e d s e c o n d g e n e r a t i o n H2 a n t a g o n i s t a n d a r a c e m i c c o m p o u n d c o m p o s e d of two k i n d s of e n a n t i o m e r s : R(+)T-593 a n d S(-)-T-593. We r e p o r t d i f f e r e n c e s of i n h i b i t o r y effects of two e n a n t i o m e r s a n d a n a l o g u e s o n H2 h i s t a m i n e r e c e p t o r . It is a n e f f o r t to e v a l u a t e t e r t i a r y s t r u c t u r e of H2 h i s t a m i n e r e c e p t o r . METHODS: HEPA cells t r a n s f e c t e d to e x p r e s s the c a n i n e H2 h i s t a m i n e r e c e p t o r g e n e w e r e i n c u b a t e d i n EBSS c o n t a i n i n g 0.1% BSA a n d v a r y i n g c o n c e n t r a t i o n s of h i s t a m i n e a n d T-593 or its a n a l o g u e s for 60 re.in at 37C. cAMP c o n t e n t was m e a s u r e d b y RIA a s s a y k i t (Yamasa, JAPAN). RESULTS: (1) S(-)-T-593 s h o w e d a m o r e p o t e n t h i s t a m i n e H2a n t a g o n i s m t h a n r a c e m i c T-593. (IC50=1x10-6) (2) R(+)-T-593 s h o w e d n o specific a n t a g o n i s m to h i s t a m i n e H2-receptor. (3) T6 4 9 ( D e s b e n z y l T-593) a n d T - 6 5 7 ( D e s m e t h y l a m i n o m e t h y l T593) s h o w e d n o specific a n t a g o n i s m to h i s t a m i n e H 2 - r e c e p t o r . O u r r e s u l t s s h o w i n g the d i f f e r e n t i n h i b i t o r y a c t i o n of e a c h enantiomer indicate that hydroxy moiety on asymmetric c a r b o n of T-593 m a y i n t e r a c t w i t h one of the b i n d i n g sites w h e r e h i s t a m i n e a c t i v a t e s H 2 - r e c e p t o r . Differences of t h e t e r t i a r y s t r u c t u r e of e a c h e n a n t i o m e r are i m p o r t a n t to d i s t i n g u i s h the a n t a g o n i s m , a n d t e r t i a r y s t r u c t u r e of T-593, e s p e c i a l l y the p o s i t i o n of c h a r g e d o r p o l a r a t o m s , is critical for the a n t a g o n i s m .
Hormones and Receptors
A1011
E N I ) O C T r o s I s OF GASTRIN IN GASTRIN RF_~EP'FOR EXPRESSING CELLS. N. Tarasova, S. A. Wank*, E. Hudson, G. Czerwinski, V. l.Romanov,
J. Resau, C J. Miehejda. ABL-Basic Research Program, National Cancer Institute, Frederick Cancer Research and Development Center, P.O. Box B, Frederick, MD, 21702. * Digestive Disease Branch, National Institute of Diabetes and Digestive And Kidney Diseases, National Institute of Health, Bethesda, MD. The growth promoting effects ofgastrin on many GI cell lines are well known, but the mechanism of that effect is poorly understood. As a first step in the study of the hormone interaction with gastrin receptor(GR) expressing cells, three fluorescent derivatives of heptagastrin were synthesized, characterized and tested with confocal laser scanning microscopy (CLSM) on a number of GR expressing cell lines. Cyanine dye, Cy3.29 and borfluoropyrroraethene (BODIPY) derivatives of the hormone were tound to be absorbed into the cells and concentrated in perinuclear organelles by a passive mechanism. The BODIPY derivative turned out to be chemically unstable and was bleached by the laser beam very rapidly. Rhodamine Greenheptagastrin, however turned out to be suitable for the studies of GR interaction with the ligand. Rapid clustering (within 4-7 rain) and internalization of the compound upon binding at room temperature and at 370 was observed in rat pancreatic acinar cells AR42J, human gastric adenocarcinomas AGS-P and SIIA, human colon adenocarcinoma HCTI 16 and NIH 3T3 ceils, transfected with human GR cDNA. At 37°C partial colocalization with the lysosome marker neutral red was detected by CLSM, suggesting that internalized gastrin ends up in lysosomes. Lowering the temperature to 20°C inhibited translocation of the ligand to lysosomes. Decreasing the temperature to 4°C completely abolished clustering and internalization of the labeled hormone. Immuno-electron microscopy studies with the antibodies to gastrin revealed the presence of internalized hormone in multivesieular particles and endosomes. Almost no gastrin was detected in lysosomes with the antibodies, probably due to the rapid degradation of the peptide. A negligible amount of the hormone was detected in the nucleus by CLSM/EM. Continuous accumulation of fluorescent label in the cells was observed by CLSM in the presence of protein synthesis inhibitor cycloheximide suggesting that gastrin recycled back to. the cell membrane after delivering the hormone into intracellular compartments. (Research sponsored in part by the National Cancer Institute, DHHS, under contract No. NO1-CO-46000 with ABL)
• GASTRIN AND GLYCINE-EXTENDED PROGASTR1N PROCESSING INTERMEDIATES INDUCE DIPI~ERENT PROGRAMS OF EARLY GENE ACTIVATION. A. Todisco, Y. Takeuchi, C.J. Dickinson, T. Yamada. Depts. Int. IVied. & Pods., U. Mich. Med. Ctr., Ann Arbor, MI. Progastrin is post-translationally processed to gastrin (G-NH2) via glycine-extended intermediates (G-Gly) which, initially thought to be biologically inactive, have recently been shown to have actions of their own as stimulants of cellular growth. Since G-NHz and G-Gly have distinct receptors, we sought to investigate whether they exert their trophic actions through separate mechanisms. We first examined whether the growth stimulatory effects of G-NH2 and G-Gly on the rat pancreatic acinar cell line, AR4-ZI, i s associated with induction of the expression of the early response genes c-fos and c-jun. G17-NH2(10-10 M-10-SM) increased dosedependently c-fos and c-jun specific m-RNAs (determined by Northern blots) in serum starved AR4-ZI cells but G17-Gly did not. As growth factors activate c-fos gene expression through a serum response element (SRE), we examined the effects of G17-NH 2 (10-SM) or G17-GIy (10-SM) on AR4-2J cells transfected with an SRE-luciferase reporter gene plasmid. After 6h of incubation G17-NH 2 induced luciferase activity seven-fold but G17-GIy had no effect. Growth factor activation of the c-fos SRE is known to involve the activation of the small GTP binding protein RAS, thus we examined whether inhibition of RAS expression with a dominantly expressed mutant (inactive) RAS gene transfected into AR4-2J cells could inhibit the effects of Gl7-NH z on c-fos gene transcription. In the presence of dominant negative RAS, G 17-NH2 induction of SRE-luciferase activity was reduced by 30%. Since c-jun is known to be activated via aminoterminal phosphorylation by JUN-kinase, we examined the effect of G 17NH2 (10-8M) or G17-Gly (10-SM) on AR4-2J cell JUN-kinase activity by co-transfecting the chimeric GAL4-c-jun expression plasmid and the 5XGAL-luciferase reporter plasmid. In this system GAL4-c-jun can transactivate and stimulate luciferase activity only if tbe c-jun aminoterminus is phosphorylated. In contrast to our previous observations, G17Gly induced 5XGAL-luciferase activity by three-fold, indicating JUNkinase activation, while G17-NH2 had no effect. Our results lead us to conclude that G-NH2 and G-Gly both stimulate cell proliferation through different programs of early gene activation. While G-NH 2 stimulates c-los and c-jun gene expression via a RAS dependent pathway, G-Gly regulates early gene activation through post-translational modification. Our studies present a novel model in which both the precursor and the product of a key processing reaction, peptide ~-amidation, stimulate cellular proliferation via distinct receptors linked to different signal transduction pathways.