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Identification and characterization of N-terminal and C-terminal flanking peptides of preprovasoactive intestinal peptide from human antral mucosa
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IDENTIFICATION AND CHARACTERIZATION OF N.-TERNIINAL AND C-TERMINAL FLANKING PEPTIDES OF PREPROVASOACTIVE INTESTINE PEPrIDE FROM HUMAN ANTRAL NIUCOSA. L. VOWLES, and R. DIMALINE.
Department of Physiology, University of Liverpool, Liverpool, U.K.
In addition to the biologically active peptides VIP and PHI, proVlP contains three further sequences of unknown function. These are, an N-terminal flanking peptide (NFP), a bridging peptide (BP) that separates PHI and VIP, and a C-terminal flanking peptide (CFP). We have raised antisera to the flanking peptides and used them in RIA to study biosynthetic processing pathways of preproVIP in human antral mucosa. Antisera specific for the Cterminus of NFP were raised in rabbits by immunising with the fragment YDVSRNA (preproVIP 73-79) conjugated to thyroglobulin through the tyros[no residue using glutaraldehyde. In a similar way antisera wore obtained to the C-terminus of CFP using the analogue YPEELE (Tyr-preproVIP i65-i69). The synthetic haptens were also used to produce radio]abel, and as RIA standards. Frozen human antral mucosa was chapped into small pieces, boiled in water for i0 min, homogenised and centrifuged. The supernatant was lyophilised, reconstituted in distilled water, ultracentrifuged and fractionated on 5ephadex GS0s (2.Sx140cm) equilibrated with 0.05M ammonium bicarbonate, at 4oc. A single major peak of C-terminal flanking peptide-like immunoreactivity (CFP-L[) was resolved at an elution volume of 40% (BSA=0%; i25I=i00%). Recovery of CFP-L] from the G50 eluates was l12±33pmol/g mucosa extracted (mean ~'SEM, n=6) which is similar to previously reported concentrations of VIP in this tissue. Immunoroactive eluates were pooled, the pH adjusted to 6.0, and loaded on to a Waters C18 radial compression cartridge. Elution of the sample with a gradient to 16% acetonitrile resolved a single major immunoreactive peak eluting at the gradient plateau, and a minor component eluting 10 rain. later. The major peak was purified to homogeneity on a Techsil C18 column and is undergoing structural character[sat[on. Concentrations of N-terminal flanking peptide-like immunoreactivity (NFP-LI) detected in the GS0 eluates were an order of magnitude lower than those of CFP-LI. Nevertheless, three distinct immunoreactive components were resolved that eiuted at 25%, 60%, and 83% of the elution volume, respectively. The earliest eluting material has been further purified by reversed phase HPLC. Conclusions 1. A peptide that cross reacts with antisera specific for the C-terminal flanking peptide of preproVIP has been isolated from human antra] mucosa; the peptide is present in concentrations similar to those of VIP. 2. Three components were resolved by gel filtration, that cross reacted with antisera specific for the N-terminal flanking peptide of preproVIP. However, concentrations of this material were an order of magnitude lower than those of VIP or CFP-LI.
IMMUNONEUTRALIZATION WITH A N T I G A S T R I N M O N O C L O N A L A N T I B O D Y R E V E A L S I M P O R T A N T GASTRIC ACID STIMULATORY ROLE FOR GASTRIN IN DOG DURING DISTENTION WITH GLUCOSE OR PEPTONE MEALS. J. H. WALSH and T. O. G. KOVACS, CURE/ UCLA/ Wadsworth VA Hospital, CA, 90073.
Los Angeles,
A monoclonal antibody was used to assess the role of circulating gastrin in r e g u l a t i o n of m e a l - s t i m u l a t e d g a s t r i c acid secretion in dogs. The antibody, ~l~ was specific for the C-terminal region of gastrin and CCE ( IC50 = 3-7)x M for all s u l f a t e d and n o n s u l f a t e d g a s t r i n s longer than 7 residues . This antibody bound labeled gastrin with similar high affinity at 37 C and in the presence of 40% dog serum. Acid secretion was measured in dogs with simple gastric fistulas by gravity drainage and titration or by automated intragastric titration. Stimulation was initiated by intravenous infusion of gastrin or histamine or by g a s t r i c d i s t e n t i o n with 300 ml 5.8% glucose or 8% peptone solutions, pH 5.5. Monoclonal antibody was given intrvenously over 15 min as 0.75 mg pure IgG in 10 ml 0.15 M NaCI containing 0.1% BSA. Meals were administered 15 min after antibody. Control studies included saline/BSA alone and 0.75 mg of another monoclonal antibody specific for glycine-extended gastrin. The gastrin antibody but not the gly-gastrin antibody comp~ete.~ inhibited acid responses to exogenou~ ga~trin-17 at a dose of 200 pmol.kg- .h- and inhibited a dose of 800 pmol.kg-X.h -~ by 70%. Monoclonal gastrin antibody had no effect on basal acid s e c r e t i o n but s i g n i f i c a n t l y inhibited the acid response to glucose distention from 8.4 mmol/h to 0.2 mmol/h; this antibody also inhibited the response to a subsequent peptone meal from 25 to 8 mmol/h, while gly-gastrin antibody had no effect. Maximal acid responses to histamine were similar to maximal responses to gastrin and were not inhibited by either gastrin or gly-gastrin antibody. These studies indicate that circulating gastrin can be antagonized by a monoclonal antibody to gastrin. Such antagonism almost completely eliminates the gastric acid response to gastric distention and markedly inhibits the response to a peptone meal. Thus circulating gastrin, acting alone or together with other agents such as h i s t a m i n e and a c e t y l c h o l i n e , has a major role in s t i m u l a t i o n of canine gastric acid secretion during the gastric phase of acid secretion.
IDENTIFICATION AND CHARACTERIZATION OF N.-TERNIINAL AND C-TERMINAL FLANKING PEPTIDES OF PREPROVASOACTIVE INTESTINE PEPrIDE FROM HUMAN ANTRAL NIUCOSA. L. VOWLES, and R. DIMALINE.
Department of Physiology, University of Liverpool, Liverpool, U.K.
In addition to the biologically active peptides VIP and PHI, proVlP contains three further sequences of unknown function. These are, an N-terminal flanking peptide (NFP), a bridging peptide (BP) that separates PHI and VIP, and a C-terminal flanking peptide (CFP). We have raised antisera to the flanking peptides and used them in RIA to study biosynthetic processing pathways of preproVIP in human antral mucosa. Antisera specific for the Cterminus of NFP were raised in rabbits by immunising with the fragment YDVSRNA (preproVIP 73-79) conjugated to thyroglobulin through the tyros[no residue using glutaraldehyde. In a similar way antisera wore obtained to the C-terminus of CFP using the analogue YPEELE (Tyr-preproVIP i65-i69). The synthetic haptens were also used to produce radio]abel, and as RIA standards. Frozen human antral mucosa was chapped into small pieces, boiled in water for i0 min, homogenised and centrifuged. The supernatant was lyophilised, reconstituted in distilled water, ultracentrifuged and fractionated on 5ephadex GS0s (2.Sx140cm) equilibrated with 0.05M ammonium bicarbonate, at 4oc. A single major peak of C-terminal flanking peptide-like immunoreactivity (CFP-L[) was resolved at an elution volume of 40% (BSA=0%; i25I=i00%). Recovery of CFP-L] from the G50 eluates was l12±33pmol/g mucosa extracted (mean ~'SEM, n=6) which is similar to previously reported concentrations of VIP in this tissue. Immunoroactive eluates were pooled, the pH adjusted to 6.0, and loaded on to a Waters C18 radial compression cartridge. Elution of the sample with a gradient to 16% acetonitrile resolved a single major immunoreactive peak eluting at the gradient plateau, and a minor component eluting 10 rain. later. The major peak was purified to homogeneity on a Techsil C18 column and is undergoing structural character[sat[on. Concentrations of N-terminal flanking peptide-like immunoreactivity (NFP-LI) detected in the GS0 eluates were an order of magnitude lower than those of CFP-LI. Nevertheless, three distinct immunoreactive components were resolved that eiuted at 25%, 60%, and 83% of the elution volume, respectively. The earliest eluting material has been further purified by reversed phase HPLC. Conclusions 1. A peptide that cross reacts with antisera specific for the C-terminal flanking peptide of preproVIP has been isolated from human antra] mucosa; the peptide is present in concentrations similar to those of VIP. 2. Three components were resolved by gel filtration, that cross reacted with antisera specific for the N-terminal flanking peptide of preproVIP. However, concentrations of this material were an order of magnitude lower than those of VIP or CFP-LI.
IMMUNONEUTRALIZATION WITH A N T I G A S T R I N M O N O C L O N A L A N T I B O D Y R E V E A L S I M P O R T A N T GASTRIC ACID STIMULATORY ROLE FOR GASTRIN IN DOG DURING DISTENTION WITH GLUCOSE OR PEPTONE MEALS. J. H. WALSH and T. O. G. KOVACS, CURE/ UCLA/ Wadsworth VA Hospital, CA, 90073.
Los Angeles,
A monoclonal antibody was used to assess the role of circulating gastrin in r e g u l a t i o n of m e a l - s t i m u l a t e d g a s t r i c acid secretion in dogs. The antibody, ~l~ was specific for the C-terminal region of gastrin and CCE ( IC50 = 3-7)x M for all s u l f a t e d and n o n s u l f a t e d g a s t r i n s longer than 7 residues . This antibody bound labeled gastrin with similar high affinity at 37 C and in the presence of 40% dog serum. Acid secretion was measured in dogs with simple gastric fistulas by gravity drainage and titration or by automated intragastric titration. Stimulation was initiated by intravenous infusion of gastrin or histamine or by g a s t r i c d i s t e n t i o n with 300 ml 5.8% glucose or 8% peptone solutions, pH 5.5. Monoclonal antibody was given intrvenously over 15 min as 0.75 mg pure IgG in 10 ml 0.15 M NaCI containing 0.1% BSA. Meals were administered 15 min after antibody. Control studies included saline/BSA alone and 0.75 mg of another monoclonal antibody specific for glycine-extended gastrin. The gastrin antibody but not the gly-gastrin antibody comp~ete.~ inhibited acid responses to exogenou~ ga~trin-17 at a dose of 200 pmol.kg- .h- and inhibited a dose of 800 pmol.kg-X.h -~ by 70%. Monoclonal gastrin antibody had no effect on basal acid s e c r e t i o n but s i g n i f i c a n t l y inhibited the acid response to glucose distention from 8.4 mmol/h to 0.2 mmol/h; this antibody also inhibited the response to a subsequent peptone meal from 25 to 8 mmol/h, while gly-gastrin antibody had no effect. Maximal acid responses to histamine were similar to maximal responses to gastrin and were not inhibited by either gastrin or gly-gastrin antibody. These studies indicate that circulating gastrin can be antagonized by a monoclonal antibody to gastrin. Such antagonism almost completely eliminates the gastric acid response to gastric distention and markedly inhibits the response to a peptone meal. Thus circulating gastrin, acting alone or together with other agents such as h i s t a m i n e and a c e t y l c h o l i n e , has a major role in s t i m u l a t i o n of canine gastric acid secretion during the gastric phase of acid secretion.