Physiological and Immunological Studies with Desamidogastrin

Vol. 62, No.4 Printed in U.S.A.

GASTROENTEROLOGY

Copyright © 1972 by The Williams & Wilkins Co.

-PHYSIOLOGICAL AND IMMUNOLOGICAL STUDIES WITH DESAMIDOGASTRIN JAMES E. MCGUIGAN, M.D., AND HENRY F. THOMAS, M.D.

Division of Gastroenterology, Department of Medicine and the Department of Surgery, University of Florida College of Medicine, Gainesville, Florida

The carboxyl-terminal tetrapeptide amide of the gastrin molecule has been shown to be physiologically inactive when deprived of its terminal amide group. In this study, the physiological activity of nonamidated human gastrin I (desamidogastrin) was examined. Desamidogastrin, in doses of 0.1, 1.0, and 10 /-Lg per kg per hr, was administered by constant intravenous infusion to dogs prepared with Heidenhain pouches. In contrast to human gastrin I, desamidogastrin was found without measurable effect in stimulating acid output from the Heidenhain pouches. In addition, the immunological reactivity of desamidogastrin with antibodies to human gastrin I was assessed. Antibodies to human gastrin I utilized were those produced by repeated immunization of rabbrts with human gastrin I, residues 2 to 17, covalently conjugated to bovine serum albumin. In addition to the absence of demonstrable physiological effect in stimulating acid secretion, when the gastrin carboxyl-terminal amide group was removed, greater than 99% of the immunological reactivity of human gastrin I molecules with these antibodies to gastrin was lost. The gastrin heptadecapeptides, purified and structurally characterized by Gregory and Tracy 1 and Gregory et al. 2 are the most potent agents known in stimulating gastric hydrochloric acid secretion. The carboxyl-terminal tetrapeptide amide of gastrin possesses all of the physiological properties of the intact gastrin heptadecapeptides3, 4; therefore, the carboxylterminal tetrapeptide amide is, or contains, the active site of the gastrin molecule. Deamidation of the gastrin tetrapeptide amide has been shown to abolish its physiological activity.3, 4 Effects of

deamidation of the intact gastrin heptadecapeptide on its physiological activity or immunological reactivity have not been previously examined. In this study. nonamidated human gastrin I heptadecapeptide (desamidogastrin) was examined. The purposes of this study were 2-fold; first, to determine the effect of absence of the amide group on the capacity of human gastrin I to stimulate acid secretion, and, second, to assess the immunological reactivity of antibodies to human gastrin I with desamidogastrin.

Received November 30, 1970. Accepted Novem· ber 15, 1971. Address requests for reprints to: Dr. James E. McGuigan, Division of Gastroenterology, Department of Medicine. University of Florida College of Medicine, Gainesville. Florida 32601. This work was supported by Research Grants AM 13711 from the National Insitutes of Health and T -394 from The American Cancer Society.

Preparation of antibodies to gastrin. Antibodies to gastrin were as we have previously described and utilized 5-1 2 and their production will be only briefly summarized here. Randomly bred New Zealand white rabbits were repeatedly immunized with synthetic human gastrin I (residues 2 to 17) (SHG: 2 to 17), covalently coupled to bovine serum albumin. Conjugation was achieved by use of I-ethyl-3-

Materials and Methods

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(3-dimethylaminopropyl) carbodiimide as the peptide-protein linking agent. The human gastrin-bovine serum albumin conjugate was administered by footpad injection in complete Freund's adjuvant containing Mycobacterium butyrium. Antibodies to human gastrin I examined in this study were those contained in the serum of each of 3 rabbits obtained by cardiac puncture 2 weeks after booster injections which had followed initial immunization by 6, 12, and 16 months. Gastrin peptides. Synthetic human gastrin I (residues 2 to 17) was purchased from Imperial Chemicals Industries, Ltd. Synthetic human gastrin I (residues 1 to 17) was a kind gift from Dr. John Morley, Imperial Chemical Industries, Ltd. Synthetic nonamidated human gastrin I (residues 1 to 17) (desamidogastrin) was prepared and kindly supplied by Professor George Kenner, Department of Organic Chemistry, University of Liverpool. Characterization of antibody binding. Synthetic human gastrin I (residues 1 to 17) (SHG: 1 to 17) was radioiodinated using 1251 by a modification 7 of the method described by Hunter and Greenwood. 13 Antibodies to gastrin were diluted and incubated for 4 to 5 days in the presence of [ 1251]SHG: 1 to 17 (300 to 5000 counts per min). In addition, variable amounts of either unlabeled human gastrin I or unlabeled desamidogastrin were included in the immune incubation mixtures. The final volume of each reaction mixture was adjusted to 2.4 ml. All solutions were prepared and diluted in 0.02 M barbital solution containing 2 mg per ml of ovalbumin (Sigma Chemical Co.). Incubations were performed at 4 C in 7.5- by l00-mm disposable glass culture tubes. The final dilution of the antibody preparation was 1: 48,000. Following incubation of antibodies to gastrin with radiolabeled gastrin and varying amounts of the unlabeled gastrin heptadecapeptides, antibody-free [ 1251]SHG: 1 to 17 was separated from antibody-bound (1 251]SHG: 1 to 17 by means of the anion-binding resin, Rohm and Haas Amberlite CG-4B, 200 to 400 mesh. 14 Radioactivity was measured in the precipitates and supernatant solutions using a Nuclear-Chicago automatic 'Y spectrometer. Radioactivity contained in the precipitates, representing antibody-free radioactivity, was then compared with antibody-bound radioactivity contained in the supernatant solutions to determine individual bound to free ratios for [ 125 1]SHG: 1 to 17. Infusion of gastrin and desamidogastrin. Heidenhain (vagally denervated) pouches were constructed in 3 mongrel dogs, ranging from 13

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to 17 kg in weight. The pouches were drained by stainless steel cannulas. At least 6 weeks were permitted to elapse following construction of the pouches prior to initiation of the gastrin infusion experiments. The dogs were fasted for 24 hr prior to desamidogastrin infusions. A polyethylene catheter for intravenous infusion was inserted into a superficial vein. Pouch secretions were collected with the dogs in a modified Pavlov stand. From two to four 15-min samples of pouch secretion were collected prior to intravenous infusion in order to be certain that acid secretion was at or near zero. Intravenous infusion was conducted for 2 consecutive hr, Ringer's solution was infused by means of a peristaltic pump at a constant rate of 0.25 ml per min during the 2-hr period. During the 2nd hour an additional solution was administered via a Y-connector at a rate of 0.38 ml per min using a Harvard syringe constant infusion pump. The second solution contained either 154 mM NaCl alone or desamidogastrin in 154 nM NaCl: desamidogastrin was administered at rates of 0.1, 1.0, and 10Ilg per kg per hr. The order in which each dog received the various amounts of desamidogastrin was randomized. At least 48 hr were permitted to elapse between each infusion. Consecutive 15min collections of Heidenhain pouch secretion before and during saline or peptide in saline infusion were collected and acid output was measured by titration to the phenol red end point with 0.01 N NaOH. Acid output for periods of saline or peptide infusion was calculated and expressed as microequivalents per hour. Serum for gastrin radioimmunoassay was obtained from the right femoral vein at the initiation of the 2nd hr of infusions (immediately prior to gastrin peptide infusion), and at 30 and 60 min thereafter. Statistical comparisons were performed using t-test analyses for unpaired data. 15

Results Measurements of acid output. The mean rate of acid output for the 3 dogs for the 30-min control infusion period prior to infusion with saline or desamidogastrin was near zero, being 24 ± 4 (SEM) IlEq per hr. No increases in acid output from the Heidenhain pouches were seen with saline infusion or with desamidogastrin at any of the infusion concentrations. The mean acid output rates for infusion of saline and

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infusion of desamidogastrin in saline at 0.1 J,Lg per kg per hr were 19 ± 6 and 26 ± 11 J,LEq per hr respectively. Mean acid output for both 1 and 10 J,Lg per kg per hr desamidogastrin was < 10 J,LEq per hr. These results are in sharp contrast to those which we have obtained 16 with intravenous infusion of graded doses of intact human gastrin I, using 2 of the 3 dogs utilized in this study (table I). Marked increases in Heindenhain pouch acid output were found with all doses of intravenously infused human gastrin I which were examined (0.125 to 2.0 J,Lg per kg-hr). Even with the smallest administered amount of intact human gastrin I (0.125 J,Lg per kg-hr), an 8-fold increase in mean acid output was observed (table I). Immunological studies. In figure 1, data are presented from examination of antiserum from 1 rabbit obtained 2 weeks following injection 16 months after initial immunization: these data are representative of other antiserum preparations from this and the other 2 rabbits. Ratios of bound over free [125I]SHG: 1 to 17 were recorded in the presence of variable amounts of the gastrin heptadecapeptides. The effectiveness of human gastrin I and desamidogastrin in inhibiting the binding of [125I]SHG:l to 17 by antibodies directed to SHG: 2 to 17 are compared in figure 1. Displacement to the right of the curve for desamidogastrin when compared with human gastrin I indicated considerably less inhibition of the binding of [125I]SHG:l to 17 by antiTABLE

1. Acid output with constant intravenous infusion of intact human gastrin Ia

Rate of intravenous gastrin infusion ~g /k{!/hr

o (saline) 0.125 0.250 0.500

Heidenhain pouch acid output ~Eq/15

min

11 ± 4.9 b

88 ± 20.8 153 ± 30.9 476 ± 58.9 1.0 987 ± 28.9 2.0 1033 ± 76.5 a Plateau rates of acid output with constant infusion of graded doses of human gastrin I: results 'S included use of 2 of the 3 dogs utilized in this study. b

± SEM .

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FIG. 1. Radioimmunoassay calibration diagrams utilizing antibodies to synthetic human gastrin I, indicating the ratio of bound antibody over free ['2'I]SHG:1 to 17. Studies were conducted in the presence of variable amounts of synthetic human gastrin I (residues 1 to 17) (SHG( - NH,» or desamidogastrin (SHG( - OH)).

bodies to human gastrin I with desamidogastrin as compared with human gastrin I in its amidated form. The slopes of the two curves are not distinguishable. The relative inhibitory potency 14 of desamidogastrin in effecting 75% reduction of the bound over free (B: F) ratio when compared with synthetic human gastrin I was 0.0079. The relative inhibitory potency of desamidogastrin compared with human gastrin I resulting in 25% reduction of the B : F ratio was 0.0089. The mean relative inhibitory potencies for the nine antiserum preparations examined were 0.0081 ± 0.0004 (SEM) and 0.0092 ± 0.0006 (SEM) for 75% and 25% B: F reductions respectively. The data indicated in figure 1, and the calculated inhibitory potencies of desamidogastrin, when compared with human gastrin I, indicate that in excess of 100 desamidogastrin molecules are required to effect reduction in antibody binding of radiolabeled gastrin equivalent to that achieved by one molecule of human gastrin 1. As would be anticipated on the basis of the minimal immunoreactivity of desamidogastrin, there was no detectable increase in serum immunoassayable gastrin during infusion with any of the concentrations of desamidogastrin.

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Discussion The carboxyl-terminal tetrapeptide amide of gastrin (Try · Met· Asp· PheNH 2) possesses all of the physiological properties of intact gastrin heptadecapeptides. Morley 4 has extensively examined the structure-function relationships of the gastrin carboxyl-terminal tetrapeptide amide and many of its structural derivatives and analogues. The carboxyl-terminal amide group is of prime physiological importance inasmuch as the nonamidated gastrin tetrapeptide is completely devoid of activity. The results of this study demonstrate for the first time that, just as for the tetrapeptide of gastrin, the intact heptadecapeptide also loses its physiological activity when deprived of its carboxyl-terminal amide group. The precise mechanism by which gastrin molecules are inactivated in vivo has not been defined: it is possible that gastrin may be degradatively inactivated by structural modification of the carboxyl-terminal region of the molecule, e.g., with cleavage of the terminal phenylalanine amide or even by removal of its carboxyl-terminal amide group. In support of such a possibility, Glass and his co-workers l7 have identified carboxyamidopeptidase activity in homogenates from rat kidney. This enzyme demonstrates specificity for carboxyl-terminal amino acids with amidation of their terminal carboxyl groups. These investigators demonstrated that this enzyme cleaved the carboxyl-terminal amidated phenylalanyl residue from an analogue of the gastrin tetrapeptide amide, rendering it physiologically inactive. The product of the activity of this enzyme on the human gastrin I heptadecapeptide is desphenylalanyl gastrin. It is possible that a carboxyaminopeptidase system may be operational in the degradative deactivation of the carboxy-amidated gastrointestinal hormones, which include gastrin as well as the other gastrointestinal hormones, cholecystokinin-pancreozymin and secretin. 1-3. 18. 19 The observations of Glass and his colleagues 2o are in accord with studies in dogs indicating degradation of immunoreactive gastrin by the kid-

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ney. Laster and Walsh 2 1 have also studied the activity of rat tissue homogenates in modifying the carboxyl-terminal region of the gastrin tetrapeptide (t-Boc . Try · Met· Asp ·Phe-NH 2 ). In addition to recognition of carboxyamidopeptidase activity in renal tissue extracts, as noted by Glass and his colleagues, they also detected enzyme (amidase) activity in both liver and small intestinal mucosa. This enzyme catalyzed the removal of the terminal amide group, resulting in the production of the C-terminal desamidogastrin tetrapeptide. In this study detectable increases in Heidenhain pouch acid secretion were not obtained with intravenous infusion rates of 0.1, 1.0, and 10 J.l.g of desamidogastrin per kg per hr. In contrast, we have found 16 substantial increases in Heidenhain pouch acid output with infusions of the intact human gastrin I heptadecapeptide in doses from 0.125 to 2.0 J.l.g per kg per hr: 8-fold increases in acid output were produced with the lowest rate of human gastrin I heptadecapeptide infusion (0.125 J.l.g per kg per hr). Therefore, no stimulation of acid secretion was observed with desamidogastrin, even though the highest dose of desamidogastrin (10 J.l.g per kg-hr) exceeded the smallest dose of human gastrin I (0.125 J.l.g per kg-hr) which was physiologically active by almost 100-fold. In prior experiments lO we have examined gastrin release and circulation through the liver following acetylcholine stimulation of the canine stomach, measuring serum gastrin concentrations in portal, hepatic, and peripheral venous sera. At that time, we indicated that values measured and recorded were those of immunoreactive gastrin, which values mayor may not correspond closely with serum concentrations of physiologically active gastrin molecules. Recognizing the loss of physiological activity which results from deamidation of the carboxyl-terminal phenylalanine amide of the gastrin tetrapeptide, we cautioned that it was unknown to what extent deamidation of the gastrin molecule affected its immunoreactivity. The results of these studies indicate that with a slight, but strategically located, structural modification of the gastrin

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molecule i.e., deprivation of its carboxylterminal-amide group, physiological activity was greatly reduced, being undetectable under the conditions of these experiments, and greater than 99% of the immunological reactivity of the gastrin molecule was lost. If gastrin is structurally modified in its degradative deactivation by carboxyaminopeptidase activity, its immunological reactivity would be substantially reduced, as observed in these studies. In the application of a radioimmunoassay method, it is of prime importance to have a high degree of correlation between the immunoassayability of a substance and its physiological activity. When this is not the case, a substance, e.g., a polypeptide hormone, may be structurally modified in such a manner as to ablate physiological activity but preserve immunoreactivity. Such a phenomenon results in the undesired immunoassayability of a functionally inactivated substance. With antibodies used in these experiments there is sufficient antibody specificity for the physiologically active portion of gastrin, that inactivation of gastrin molecules, even by as structurally subtle a change as deamidation, would result in almost complete loss of immunoreactivity: such molecules would not be recorded by radioimmunoassay. The results of these experiments indicate no detectable increases in immunoassayable gastrin in canine serum with infusion of desamidogastrin at rates from 0.1 to 10 j.tg per kg per hr, even though the highest dose exceeded by lO-fold the dose of the intact gastrin heptadecapeptide required to evoke maximum acid secretion. 22 Conversely, increases in canine serum gastrin concentrations determined by radioimmunoassay can be readily demonstrated with infusion of the intact human gastrin I heptadecapeptide in doses as small as 0.125 j.tg per kg per hr.16 The results of these studies indicate the marked reductions in immunoreactivity which result from deamidation of the human gastrin I heptadecapeptide molecule. These investigations are in support of the view that with use of antibodies of

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this variety, human gastrin I molecules, which may be modified by deamidation, and be therefore physiologically inactive, would not contribute significantly to the immunological measurement of serum gastrin concentrations. REFERENCES 1. Gregory RA, Tracy HJ: The constitution and properties of two gastrins extracted from hog antral mucosa. I. The isolation of two gastrins from hog antral mucosa. II. The properties of two gastrins isolated from hog mucosa. Gut 5: 103-114, 1964 2. Gregory H, Hardy PM, Jones DS, et al: Structure of gastrin. Nature (Lond) 204:931-933, 1964 3. Morley JS, Tracy HJ, Gregory RA: Structurefunction relationships in the active C-terminal tetrapeptide sequence of gastrin. Nature (Lond) 207:1356, 1965 4. Morley JS: Structure-function relationships in gastrin-like peptides. Proc Roy Soc B 179:79111, 1968 5. McGuigan JE: Studies of the immunochemical I specificity of some antibodies to human gastrin. Gastroenterology 56:429- 438, 1969 6. McGuigan JE: Immunochemical studies with synthetic human gastrin. Gastroenterology 54: 1005-1011, 1968 7. McGuigan JE, Trudeau WL: Studies with antibodies to gastrin: radioimmunoassay in human serum and physiological studies. Gastroenterology 58:139-150, 1970 8. McGuigan JE, Trudeau WL: Immunochemical measurement of elevated levels of gastrin in the serum of patients with pancreatic tumors of the Zollinger-Ellison variety. N Engl J Med 278: 1308- 1313, 1968 9. McGuigan JE, Trudeau WL: Serum gastrin concentrations in pernicious anemia. N Engl J Med 282:358-361, 1970 10. McGuigan JE, Jaffe BM, Newton WT: Immunochemical measurements of endogenous gastrin release. Gastroenterology 59:499- 504, 1970 11. Trudeau WL, McGuigan JE: Serum gastrin levels in patients with peptic ulcer disease. Gastroenterology 59:6-12, 1970 12. Trudeau WL, McGuigan JE: Relations between serum gastrin levels and rates of gastric hydrochloric acid secretion. N Engl J Med 282:408412, 1971 13. Hunter WM, Greenwood FC: Preparation of iodine- 13 'labeled human growth hormone of high specific activity. Nature (Lond) 194:495-496, 1962 14. Yalow RS, Berson SA: 'Radioimmunoassay of gastrin. Gastroenterology 58: 1-14, 1970

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15. Snedecor GW: Statistical Methods. Fifth Edition. Ames, Iowa, Iowa State University Press, 1956, p 91 16. McGuigan JE, Isaza J, Landor JH: Relationships of gastrin dose, serum gastrin, and acid secretion. Gastroenterology 61:659-666, 1971 17. Glass JD, Schwartz IL, Walter R: Enzymatic inactivation of peptide hormones possessing a C-terminal amide group. Proc Natl Acad Sci USA 63:1426- 1430, 1969 18. Mutt V, Jorpes JE: Isolation of aspartyl-phenylalanine amide from cholecystokinin-pancreozymin . Biochem Biophys Res Commun 26:392- 397, 1967

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19. Mutt V, Jorpes JE: Contemporary developments in the biochemistry of the gastrointestinal hormones. Recent Progr Horm Res 23:483, 1967 20. Clendinnen BG, Davidson WD, Lemmi CAE, et al: Renal uptake and excretion of gastrin in the dog (abstr). Gastroenterology 58:935, 1970 21. Laster L, Walsh JH : Enzymatic degradation of C-terminal tetrapeptide amide of gastrin by mammalian tissue extracts. Fed Proc 27:13281330, 1968 22. Hirschowitz BI: Gastrin 1, pentagastrin and histamine in the fistula dog. Fed Proc 27:13181320, 1968