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Inhibition of Endogenous Gastrin Activity by Antibodies to the Carboxyl-Terminal Tetrapeptide Amide of Gastrin
Vol. 58, No.2
GASTROENTEROLOGY
Copyright C 1970 by The Williams & Wilkins Co .
Printed in U.S .A.
INHIBITION OF ENDOGENOUS GASTRIN ACTIVITY BY ANTIBODIES TO THE CARBOXYL-TERMINAL TETRAPEPTIDE AMIDE OF GASTRIN BERNARD M. JAFFE, M.D., WILLIAM T. NEWTON, M.D., AND JAMES E. McGUIGAN, M.D . Departments of Surgery and Medicine, Washington University School of Medicine, and the John Cochran Veterans Administration Hospital, St. Louis, Missouri
Since incubation of gastrin with antibodies directed against its functional carboxyl-terminal tetrapeptide amide inhibits the gastric secretory effects of exogenous gastrin, this series of experiments was performed to determine whether the same antibody preparation also inhibits the physiological effects of endogenous gastrin. Endogenous gastrin release was stimulated in the perfused pylorus-ligated rat stomach by gastric irrigation with acetylcholine. The control rat experiments demonstrated that acid secretory responses to intragastric acetylcholine were reproducible and reliable; the second response in each rat varied from 10 % higher to 15 % lower than the first. In the experimental rats, passive administration of antibody-containing globulin during endogenous release inhibited the expected increase in acid secretion an average of 73 % (47 to 100 %). The experiments document that antibodies to the functional portion of the gastrin molecule inhibit the acid secretory effects of endogenous gastrin. The potent gastric acid stimulatory action of gastrin is dependent upon the hormone's carboxyl-terminal tetrapeptide sequence, tryptophyl- methionyl- aspartyl phenylalanine amide.! Antibodies have been produced in rabbits which are specific Received June 17, 1969. Accepted August 26, 1969. A preliminary report of this work was presented at the annual meeting of the American Gastroenterological Association held in Washington, D. C., in May 1969. Address requests for reprints to : Dr. James E. McGuigan, Department of Medicine, University of Florida College of Medicine, Gainesville, Florida 32601. This work was supported by Grant GM 00371 and Research Grant 1 ROl AM 10837 from the National Institutes of Health, United States Pub~ lic Health Service ; by Grant T -394 from The American Cancer Society; and by Grant 66-679 from The American Heart Association. Dr. McGuigan also received Research Career Development Award 1-K3-Al-19,499 from the National Institutes of Health .
for this tetrapeptide sequence. 2 Previous experiments have demonstrated that, by incubating antibody with gastrin, these antibodies inhibit the gastric acid secretory effects of exogenous gastrin.3 These inhibition experiments were performed using the perfused pylorus-ligated rat.4 Having demonstrated that antibody-gastrin complexes prepared in vitro were functionally inactive following intravenous administration, the presently reported experiments were performed in order to determine whether antibodies to the gastrin tetrapeptide also inhibit the gastric acid secretory effects of endogenous gastrin. For this series of experiments, a modification of the rat bioassay system was used in which acetylcholine irrigation of the stomach was employed to liberate endogenous gastrin. Experiments in dogs in which gastrin concentrations in portal and systemic veins were meaiS,ured directly utilizing a radioimmunoassay for gastrin 5 have demonstrated that acetylcholine irrigation of the antrum re151
Vol. 58, No.2
JAFFE ET AL.
152
suIts in the virtually immediate liberation of gastrin into the circulation. 6 The present work assumes that similar release of gastrin occurs in rats with acetylcholine irrigation. Furthermore, both by gastrin radioimmunoassay and by immunofluorescence (unpublished observations) gastrin has been localized in the rat antrum, as in the antra of other species so far studied. 7 Methods and Materials Production of antibody. Gastrin tetrapeptide (Cyclo Chemical Company, Los Angeles, Calif.) was conjugated to bovine y-globulin (Sigma Chemical Company, St. Louis, Mo.) using water soluble l-ethyl-3-dimethylaminopropyl) carbodiimide (Ott Chemical Company, Muskegon, Mich.) as the conjugating agent. The degree of substitution, an average of 8 to 12 gastrin tetrapeptide groups per globulin molecule, was determined by titration with N-bromosuccinimide.8 Ten rabbits were immunized with the conjugate emulsified in complete Freund's adjuvant at 0, 1, and 6 months. Ten days following the third immunization, the rabbits were bled by cardiac puncture and the sera were pooled. Antibody activity was characterized by quantitative immunochemical techniques including gel filtration on a Sephadex-G25 column," equilibrium dialysis; and charcoal radioimmunoassay· using tritiated acetyl tetrapeptide amide, as well as by a radioimmunoassay using an artificial radioiodinated tetrapeptide-containing test antigen (unpublished data). The antibodies bound gastrin tetrapeptide as well as the whole human gastrin molecule: For use in the inhibition experiments, antibodies in the pooled sera were precipitated by the drop-wise addition of cold saturated aqueous ammonium sulfate to a final concentration of 40% of saturation. After stirring for 16 hr at 4 C to allow for maximal precipitation, the material was centrifuged for 30 min at 2000 g and the supernatant fluid was discarded . The precipitate was dissolved in and dialyzed against 0.15 M NaCI-O.D1 M potassium phosphate, pH 7.4. The protein concentration was adjusted to 43 OD units per ml read at 278 mf-t on a Beckman DU spectrophotometer. Two-tenths milliliter of this globulin preparation was in antibody excess, relative to 100 ng of synthetic human gastrin I,· a dose sufficient to increase the rate of gastric acid secretion by the rat bioassay 2Y2-fold: The control globulin preparation was prepared identically from sera from rabbits immunized according to the same schedule only
with complete Freund's adjuvant alone and no tetrapeptide conjugate. Rat bioassay. The bioassay employed measured the rate of acid secretion by the perfused stomach. After being fed sugar cubes for 2 to 3 days, male Sprague-Dawley rats weighing between 217 and 283 g and averaging 251 g were anesthetized with 25% urethane (0.4 ml per 100 g) administered half intraperitoneally and half subcutaneously in two separate areas. Once a satisfactory anesthetic level was induced, tracheostomy was performed and the left jugular vein was cannulated with a PE 50 polyethylene cannula (fig. 1). A midline abdominal incision was made, the distal duodenum was tied off, and a no. 16 rubber catheter was inserted into the distal antrum through the duodenum and secured in place by a silk ligature. A no. 12 rubber catheter was inserted via the esophagus into the proximal cardia but, because it totally occluded the esophageal lumen, it was not necessary to tie it in place. The stomachs were irrigated clean and the preparation was allowed to equilibrate for 1 to 2 hr. Starting at the equilibrium phase and throughout the entire experi-
E"'":"l~Z!Z2IE1lZ-dP-S F,om Pump l V. Tubing ill
- S Jugum, VI ;"
FIG.
1. Schematic
bioassay.
representation of the rat
February 1970
153
INHIBITION OF GASTRIN ACTIVITY ENDOGENOUS GASTRIN INH I BITION EXPERIMENTS
4.4
Control Rat
3.6 COllf,ol Gl obuli ll
COII"ol Gl obu l i n
2.8
EJ(p~rim~nlal
~
Ral
eDIII,,,, Globulill
3.8 3.4
20
40
60
80
100
120
140
160
TIME (MINUTES)
FIG. 2. Plot of actual data from one control and one experimental rat experiment demonstrating the general format of the experiments . Acetylcholine was instilled in the stomach at the vertical arrows. The acid content of the interval represented by dashed lines is not recorded, as it reflects the acid content of the acetylcholine effluent.
ment, saline maintained at 35 C in a constant water bath was infused into the stomach at a constant rate of 0.75 ml per min by a roller pump (model RL175, Holter Company, Bridgeport, Pa.) . The gastric effluents, as lO-min collections, were titrated to pH 7.0 with 0.01 M sodium hydroxide. After a constant base line (differences between consecutive titrations showed only minimal random variations) of acid secretion was maintained for at least 30 min, the pump was turned off momentarily, 1.5 ml of 1% acetylcholine solution were instilled into the stomach via the esophageal catheter, and the pump was restarted. Since the volume of the bioassay irrigation system including the catheters was a.pproximately 3 ml, the acetyl-
choline was recovered in the first lO-min collection. In pilot experiments, 1.5-ml instillation of methylene blue solution into similar ra.t preparations essentially was cleared from the stomachs in 10 min . This was confirmed by the fact that the first titration required additional base equivalent to the 0.4 fLEq of base necessary to titrate the acetylcholine solution alone to pH 7.0. The peak of the gastric acid response occurred from the second to fourth lO-min intervals (most frequently the third lO-min interval) following gastric irrigation with acetylcholine and returned to constant values within approximately 1 hr. Individual rats varied in basal levels and responsiveness . Hence, it was necessary to express the gastrin-mediated, acetyl-
154
JAFFE ET AL. TABLE
1. Control rats
~
Rat no.
First stimulation
Second stimulation
Inhibition
Cl. .. . . . . . . . . . . . C2 ... . . .... .. .. . C3 ... . . . . . . . . . . . C4. . . . . .. . .. .. . C5 ... . . .. . C6 .. .. .. ... .. . . C7 .. ... . .. . .. . . .
1.67a
1.69 1.55 1. 78 3.00 2.06 1.77 1.73
-3 -10 -10 13 15 -5 9
1.94
1
••
,
Average ... ..
•
0
•
%
1.50 1.71 3.30 2.25 1.73 1.80 1.99
Ratio of peak stimulated acid secretion per lO-min interval basal acid secretion per 10-min interval a
choline-induced, gastric stimulatory responses as ratios of stimulated to basal acid secretory rates; i.e., Peak stimulated acid secretion (J.'Eq/lO min) Basal acid secretion (J.'Eq/lO min) There was no relationship between the magnitude of secretory responses and the basal levels. Simultaneous with the instillation of acetylcholine, the rats received 0.5 ml of globulin preparation (in experiments C, rats 1 and 2, and E, rats 1 and 2 the dose was 0.3 ml, see tables 1 and 2) slowly administered intravenously over from 3 to 5 min. The intravenous tubing was cleared of protein by the infusion of 0.2 ml of saline to ensure completeness of delivery. Experimental protocol. Each rat served as his own control in order to eliminate effects of ratto-rat variation in physiological responsiveness to acid stimulation. Once a constant base line was achieved, each rat received, as the initial sequence, intragastric acetylcholine and intravenous control globulin; then the acid response was characterized. The rats then were divided into two groups. The first group, consisting of 7 rats, received the identical sequence of acetylcholine and control globulin in order to demonstrate that the responses were reproducible and reliable. The second group, also consisting of 7 rats, received the identical gastric irrigation with acetylcholine, but this was accompanied by the intravenous infusion of a comparable amount of antibody-containing globulin. The sequence for the control and experimental rats is illustrated in figure 2, the graphs being the actual data from two individual rat experiments. The sequence of administration of the globulin
Vol. 58, No.2
preparations was not varied; had the order of administration of the globulin fractions been reversed, it might not have been possible to interpret control responses modified by antibody still circulating from the first injection. Thus, for each rat in both groups, the first response was considered the reference and the responses to the second stimulation were compared with these. The percentage inhibition by the second globulin preparation (control or antibody) was determined by the formula: Acid first response - acid second response . X 100 ACId first response This represents inhibition of expected increase in acid secretion due to liberation of endogenous gastrin compared with the reference first response.
Results
The base line acid secretory rates varied from 0.5 to 4.6 p.Eq of acid per 10 min and averaged 2.2 p.E'q per 10-min interval. These rates were almost identical with the average basal acid secretory rate reported previously.s In the absence of intravenous globulin, irrigation of the stomach with acetylcholine alone resulted in double the rate of acid secretion in nine experiments. When acetylcholine was given simultaneously with control globulin, the peak of secretion varied from 1.45 to 8.78 and averaged 4.35 p.Eq of acid per 10 min , almost exactly twice the average basal secretion. Control rats. The data for these seven experiments are tabulated in table 1. The average of responses to antral irrigation with acetylcholine accompanied by intraTABLE
2. Experimental rats First stimulation
Second stimulation
Inhibition
El. .. E2 .... .. . . . .. . . E3 .. . . .• 0· · · _. E4 .. . .. . . . . ..... E5. . . . .. . . E6 .. .. .. . . . . .. . . E7 ..... .. . . . . . .
2.10 2.90 2.05 1.45 1. 76 1.85 2.04
1.34 1.43 1.28 1.08 1.00 1.45 1.37
69 77 74 100 47 65
Average . . . ,.
2.00
1.37
73
Rat no.
% •
••
••••
,
•
•
•
0
•
•
••
0
83
February 1970
INHIBITION OF GASTRIN ACTIVITY
venous control globulin administration was approximately double the basal rate of acid secretion. There was little difference in the response to the two gastric irrigations; the second response varied from 15% lower to 10% higher than the first and the degree of inhibition as defined by the above formula was negligible. Experimental rats. The data for these experiments are presented in table 2. The average degree of response to the first stimulation was exactly that seen for the control group. However, in the experimental rats , the magnitude of response to the second stimulation (accompanied by antibody globulin) was, in each case, considerably less than the corresponding initial stimulation. The degree of antibody inhibition of the expected increase in gastric acid secretion varied from 47 to 100% and averaged 73% . There was no overlap in the degree of inhibition between any animals in the control and experimental groups. The differences between the first and second responses in the two groups were evaluated statistically using t-test analyses for paired data and the differences were found to be statistically significant with P value of 0.005 for the experimental group and not at all significant for the control group. Discussion
The ability of specific antihormone antibodies to neutralize particular biological effects of small «2000 mol wt) hormones has been described previously. Incubation of antibodies to melanocyte-stimulating hormone (conjugated to rabbit serum albumin) with melanocyte-stimulating hormone in vitro results in inhibition of the expected frog skin darkening effect of the peptide hormone.lO Inhibition of pressor activity followed in vitro incubation of angiotensin II with antibodies elicited by immunization of rabbits with angiotensin II absorbed to carbon particlesY Passive immunization of rats with antibodies to thyroxylthyroglobulin and thyroxyl serum globulin offered protection against the metabolic effects of both exogenous thyroglobulin and thyroxine. 12 Active immunization
155
a histamine-protein conjugate protected rabbits against anaphylactic responses to ovalbumin 13 and patients with diabetes insipidus have become vasopressin-resistant as the result of active immunization occurring during hormonal therapy.14 Initial experiments have confirmed the neutralizing effect of antibodies to gastrin tetrapeptide against the acid secretory effects of exogenous gastrin. 3 In the current series of experiments, the same antibody preparation inhibited the physiological effects of endogenous gastrin. Although the exact amino acid sequence of rat gastrin is not known , experiments utilizing antibodies to human gastrin 15. 7 demonstrate that rat gastrin is bound by antibodies to human gastrin (unpublished observations), suggesting that the two species-specific gastrin molecules are structurally similar. Since the gastrin molecules from all species thus far examined contain the functional carboxyl-terminal tetrapeptide sequence 15. 16 to which the antibodies used in these inhibition studies were directed, it is reasonable to expect that rat gastrin also contains the tetrapeptide sequence and that antibodies should bind both human and rat gastrins. In denervated canine pouches,17 as well as in modified rat bioassay preparations, antral irrigation with acetylcholine results in increased acid secretion only after a latent period, comparable with the latent period seen following intravenous administration of exogenous gastrin. 3 In canine experiments,6 measurement of gastrin directly by radioimmunoassay revealed that acetylcholine in contact with antral mucosa liberates gastrin almost immediately. Although the circulating half-life of gastrin,18 like glucagon,19 is short and the antibody globulin was infused into the rat preparations at the time when gastrin release was expected, these experiments do not determine the potential duration of inhibition nor do they delineate whether the locus of inhibition was at the site of gastrin release, within the circulation, or at the receptor cell. Inhibition of excessive gastrin-mediated acid secretion conceivably could have clin-
~ith
156
JAFFE ET AL .
ical application. It is possible that acid secretion rates and, thereby, disease processes associated with acid secretion may be modified by antibody bindings of endogenous gastrin molecules. The data from these experiments support the conclusion that antibodies to the functional carboxyl-terminal tetrapeptide amide inhibit the gastric secretory responses to endogenous (as well as exogenous) gastrin. REFERENCES 1. Tracy, H . J., and R. A. Gregory. 1964. Physiological properties of a series of synthetic peptides structurally related to gastrin 1. Nature (London) 204: 935. 2. McGuigan, J . E. 1967. Antibodies to the carboxyl-terminal tetrapeptide of gastrin. Gastroenterology 63: 697. 3. Jaffe, B. M., W. T. Newton, and J. E. McGuigan. 1969. Inhibition of gastrin activity with antibodies to the C-terminal tetrapeptide of gastrin . Surgery 65: 633. 4. Lai, S. K. 1964. Studies on gastrin . Gut 5: 327.
5. McGuigan, J . E. 1968. Immunochemical studies with synthetic gastrin. Gastroenterology 54: 1005. 6. McGuigan, J. E ., B. M. Jaffe, and W. T. Newton. 1969. Immunochemical measurement of endogenous release and circulation (abstr.> . Gastroenterology 56: 1181. 7. McGuigan, J . E. 1968. Gastric mucosal intracellular localization of gastrin by immunofluorescence. Gastroenterology 55 : 315. 8. Patehornik, A., W. B . Lawson, and B. Witkop. 1958. Selective cleavage of peptide bonds. II . The tryptophyl peptide bond and
Vol. 68, No.2
the cleavage of glucagon. J. Amer. Chem. Soc. 80: 4747. 9. McGuigan, J. E . 1968. Antibodies to the carboxyl-terminal tetrapeptide amide of gastrin in guinea pigs. J. Lab. Clin. Med. 71: 964.
10. McGuire, J., R. McGill, S. Leeman, and T . Goodfriend. 1965. The experimental generation of antibodies to a-Melanocyte stimulating hormone and adrenocorticotropic hormone. J. Clin. Invest. 44 : 1672. 11. Boyd, G. W., and W. S. Peart. 1968. The production of high-titre antibody against free angiotensin II. Lancet 2: 129. 12. Clutten, R. F., C. R. Harington, and M. E. Yuill. 1938. Studies in synthetic immunochemistry. III. Preparation and antigenic properties of thyroxyl derivatives of proteins, and physiological effects of their antisera. Biochem. J. 32: 1119. 13. Fell, N., G. Rodney, and D. E . Marshall. 1943. Histamine-protein complexes; synthesis and immunologic investigation ; histamine-azoprotein. J. Immun. 47: 237. 14. Roth, J ., S. M. Glick, L. A. Klein, and M. J. Petersen. 1966. Specific antibody to vasopressin in man. J. Clin. Endocr. 26: 671. 15. Gregory, R. A. 1966. The isolation and chemistry of gastrin. Gastroenterology 51: 953. 16. McGuigan, J. E. 1969. Studies of the immunochemical specificity of some antibodies to human gastrin. Gastroenterology 56: 429. 17. Cooke, A. R., and M. 1. Grossman. 1968. Comparison of stimulants of antral release of gastrin. Amer. J. Physiol. 215: 314. 18. Jaffe, B. M., and W. T. Newton. 1969. Distribution and localization of radio-iodinated gastrin. Surg. Forum. In press. 19. Unger, R. H., A. M. Eisentraut, M . S. McCall, and L. L. Madison . 1961. Glucagon antibodies and an immunoassay for glucagon. J. Clin. Invest. 40: 1280.
GASTROENTEROLOGY
Copyright C 1970 by The Williams & Wilkins Co .
Printed in U.S .A.
INHIBITION OF ENDOGENOUS GASTRIN ACTIVITY BY ANTIBODIES TO THE CARBOXYL-TERMINAL TETRAPEPTIDE AMIDE OF GASTRIN BERNARD M. JAFFE, M.D., WILLIAM T. NEWTON, M.D., AND JAMES E. McGUIGAN, M.D . Departments of Surgery and Medicine, Washington University School of Medicine, and the John Cochran Veterans Administration Hospital, St. Louis, Missouri
Since incubation of gastrin with antibodies directed against its functional carboxyl-terminal tetrapeptide amide inhibits the gastric secretory effects of exogenous gastrin, this series of experiments was performed to determine whether the same antibody preparation also inhibits the physiological effects of endogenous gastrin. Endogenous gastrin release was stimulated in the perfused pylorus-ligated rat stomach by gastric irrigation with acetylcholine. The control rat experiments demonstrated that acid secretory responses to intragastric acetylcholine were reproducible and reliable; the second response in each rat varied from 10 % higher to 15 % lower than the first. In the experimental rats, passive administration of antibody-containing globulin during endogenous release inhibited the expected increase in acid secretion an average of 73 % (47 to 100 %). The experiments document that antibodies to the functional portion of the gastrin molecule inhibit the acid secretory effects of endogenous gastrin. The potent gastric acid stimulatory action of gastrin is dependent upon the hormone's carboxyl-terminal tetrapeptide sequence, tryptophyl- methionyl- aspartyl phenylalanine amide.! Antibodies have been produced in rabbits which are specific Received June 17, 1969. Accepted August 26, 1969. A preliminary report of this work was presented at the annual meeting of the American Gastroenterological Association held in Washington, D. C., in May 1969. Address requests for reprints to : Dr. James E. McGuigan, Department of Medicine, University of Florida College of Medicine, Gainesville, Florida 32601. This work was supported by Grant GM 00371 and Research Grant 1 ROl AM 10837 from the National Institutes of Health, United States Pub~ lic Health Service ; by Grant T -394 from The American Cancer Society; and by Grant 66-679 from The American Heart Association. Dr. McGuigan also received Research Career Development Award 1-K3-Al-19,499 from the National Institutes of Health .
for this tetrapeptide sequence. 2 Previous experiments have demonstrated that, by incubating antibody with gastrin, these antibodies inhibit the gastric acid secretory effects of exogenous gastrin.3 These inhibition experiments were performed using the perfused pylorus-ligated rat.4 Having demonstrated that antibody-gastrin complexes prepared in vitro were functionally inactive following intravenous administration, the presently reported experiments were performed in order to determine whether antibodies to the gastrin tetrapeptide also inhibit the gastric acid secretory effects of endogenous gastrin. For this series of experiments, a modification of the rat bioassay system was used in which acetylcholine irrigation of the stomach was employed to liberate endogenous gastrin. Experiments in dogs in which gastrin concentrations in portal and systemic veins were meaiS,ured directly utilizing a radioimmunoassay for gastrin 5 have demonstrated that acetylcholine irrigation of the antrum re151
Vol. 58, No.2
JAFFE ET AL.
152
suIts in the virtually immediate liberation of gastrin into the circulation. 6 The present work assumes that similar release of gastrin occurs in rats with acetylcholine irrigation. Furthermore, both by gastrin radioimmunoassay and by immunofluorescence (unpublished observations) gastrin has been localized in the rat antrum, as in the antra of other species so far studied. 7 Methods and Materials Production of antibody. Gastrin tetrapeptide (Cyclo Chemical Company, Los Angeles, Calif.) was conjugated to bovine y-globulin (Sigma Chemical Company, St. Louis, Mo.) using water soluble l-ethyl-3-dimethylaminopropyl) carbodiimide (Ott Chemical Company, Muskegon, Mich.) as the conjugating agent. The degree of substitution, an average of 8 to 12 gastrin tetrapeptide groups per globulin molecule, was determined by titration with N-bromosuccinimide.8 Ten rabbits were immunized with the conjugate emulsified in complete Freund's adjuvant at 0, 1, and 6 months. Ten days following the third immunization, the rabbits were bled by cardiac puncture and the sera were pooled. Antibody activity was characterized by quantitative immunochemical techniques including gel filtration on a Sephadex-G25 column," equilibrium dialysis; and charcoal radioimmunoassay· using tritiated acetyl tetrapeptide amide, as well as by a radioimmunoassay using an artificial radioiodinated tetrapeptide-containing test antigen (unpublished data). The antibodies bound gastrin tetrapeptide as well as the whole human gastrin molecule: For use in the inhibition experiments, antibodies in the pooled sera were precipitated by the drop-wise addition of cold saturated aqueous ammonium sulfate to a final concentration of 40% of saturation. After stirring for 16 hr at 4 C to allow for maximal precipitation, the material was centrifuged for 30 min at 2000 g and the supernatant fluid was discarded . The precipitate was dissolved in and dialyzed against 0.15 M NaCI-O.D1 M potassium phosphate, pH 7.4. The protein concentration was adjusted to 43 OD units per ml read at 278 mf-t on a Beckman DU spectrophotometer. Two-tenths milliliter of this globulin preparation was in antibody excess, relative to 100 ng of synthetic human gastrin I,· a dose sufficient to increase the rate of gastric acid secretion by the rat bioassay 2Y2-fold: The control globulin preparation was prepared identically from sera from rabbits immunized according to the same schedule only
with complete Freund's adjuvant alone and no tetrapeptide conjugate. Rat bioassay. The bioassay employed measured the rate of acid secretion by the perfused stomach. After being fed sugar cubes for 2 to 3 days, male Sprague-Dawley rats weighing between 217 and 283 g and averaging 251 g were anesthetized with 25% urethane (0.4 ml per 100 g) administered half intraperitoneally and half subcutaneously in two separate areas. Once a satisfactory anesthetic level was induced, tracheostomy was performed and the left jugular vein was cannulated with a PE 50 polyethylene cannula (fig. 1). A midline abdominal incision was made, the distal duodenum was tied off, and a no. 16 rubber catheter was inserted into the distal antrum through the duodenum and secured in place by a silk ligature. A no. 12 rubber catheter was inserted via the esophagus into the proximal cardia but, because it totally occluded the esophageal lumen, it was not necessary to tie it in place. The stomachs were irrigated clean and the preparation was allowed to equilibrate for 1 to 2 hr. Starting at the equilibrium phase and throughout the entire experi-
E"'":"l~Z!Z2IE1lZ-dP-S F,om Pump l V. Tubing ill
- S Jugum, VI ;"
FIG.
1. Schematic
bioassay.
representation of the rat
February 1970
153
INHIBITION OF GASTRIN ACTIVITY ENDOGENOUS GASTRIN INH I BITION EXPERIMENTS
4.4
Control Rat
3.6 COllf,ol Gl obuli ll
COII"ol Gl obu l i n
2.8
EJ(p~rim~nlal
~
Ral
eDIII,,,, Globulill
3.8 3.4
20
40
60
80
100
120
140
160
TIME (MINUTES)
FIG. 2. Plot of actual data from one control and one experimental rat experiment demonstrating the general format of the experiments . Acetylcholine was instilled in the stomach at the vertical arrows. The acid content of the interval represented by dashed lines is not recorded, as it reflects the acid content of the acetylcholine effluent.
ment, saline maintained at 35 C in a constant water bath was infused into the stomach at a constant rate of 0.75 ml per min by a roller pump (model RL175, Holter Company, Bridgeport, Pa.) . The gastric effluents, as lO-min collections, were titrated to pH 7.0 with 0.01 M sodium hydroxide. After a constant base line (differences between consecutive titrations showed only minimal random variations) of acid secretion was maintained for at least 30 min, the pump was turned off momentarily, 1.5 ml of 1% acetylcholine solution were instilled into the stomach via the esophageal catheter, and the pump was restarted. Since the volume of the bioassay irrigation system including the catheters was a.pproximately 3 ml, the acetyl-
choline was recovered in the first lO-min collection. In pilot experiments, 1.5-ml instillation of methylene blue solution into similar ra.t preparations essentially was cleared from the stomachs in 10 min . This was confirmed by the fact that the first titration required additional base equivalent to the 0.4 fLEq of base necessary to titrate the acetylcholine solution alone to pH 7.0. The peak of the gastric acid response occurred from the second to fourth lO-min intervals (most frequently the third lO-min interval) following gastric irrigation with acetylcholine and returned to constant values within approximately 1 hr. Individual rats varied in basal levels and responsiveness . Hence, it was necessary to express the gastrin-mediated, acetyl-
154
JAFFE ET AL. TABLE
1. Control rats
~
Rat no.
First stimulation
Second stimulation
Inhibition
Cl. .. . . . . . . . . . . . C2 ... . . .... .. .. . C3 ... . . . . . . . . . . . C4. . . . . .. . .. .. . C5 ... . . .. . C6 .. .. .. ... .. . . C7 .. ... . .. . .. . . .
1.67a
1.69 1.55 1. 78 3.00 2.06 1.77 1.73
-3 -10 -10 13 15 -5 9
1.94
1
••
,
Average ... ..
•
0
•
%
1.50 1.71 3.30 2.25 1.73 1.80 1.99
Ratio of peak stimulated acid secretion per lO-min interval basal acid secretion per 10-min interval a
choline-induced, gastric stimulatory responses as ratios of stimulated to basal acid secretory rates; i.e., Peak stimulated acid secretion (J.'Eq/lO min) Basal acid secretion (J.'Eq/lO min) There was no relationship between the magnitude of secretory responses and the basal levels. Simultaneous with the instillation of acetylcholine, the rats received 0.5 ml of globulin preparation (in experiments C, rats 1 and 2, and E, rats 1 and 2 the dose was 0.3 ml, see tables 1 and 2) slowly administered intravenously over from 3 to 5 min. The intravenous tubing was cleared of protein by the infusion of 0.2 ml of saline to ensure completeness of delivery. Experimental protocol. Each rat served as his own control in order to eliminate effects of ratto-rat variation in physiological responsiveness to acid stimulation. Once a constant base line was achieved, each rat received, as the initial sequence, intragastric acetylcholine and intravenous control globulin; then the acid response was characterized. The rats then were divided into two groups. The first group, consisting of 7 rats, received the identical sequence of acetylcholine and control globulin in order to demonstrate that the responses were reproducible and reliable. The second group, also consisting of 7 rats, received the identical gastric irrigation with acetylcholine, but this was accompanied by the intravenous infusion of a comparable amount of antibody-containing globulin. The sequence for the control and experimental rats is illustrated in figure 2, the graphs being the actual data from two individual rat experiments. The sequence of administration of the globulin
Vol. 58, No.2
preparations was not varied; had the order of administration of the globulin fractions been reversed, it might not have been possible to interpret control responses modified by antibody still circulating from the first injection. Thus, for each rat in both groups, the first response was considered the reference and the responses to the second stimulation were compared with these. The percentage inhibition by the second globulin preparation (control or antibody) was determined by the formula: Acid first response - acid second response . X 100 ACId first response This represents inhibition of expected increase in acid secretion due to liberation of endogenous gastrin compared with the reference first response.
Results
The base line acid secretory rates varied from 0.5 to 4.6 p.Eq of acid per 10 min and averaged 2.2 p.E'q per 10-min interval. These rates were almost identical with the average basal acid secretory rate reported previously.s In the absence of intravenous globulin, irrigation of the stomach with acetylcholine alone resulted in double the rate of acid secretion in nine experiments. When acetylcholine was given simultaneously with control globulin, the peak of secretion varied from 1.45 to 8.78 and averaged 4.35 p.Eq of acid per 10 min , almost exactly twice the average basal secretion. Control rats. The data for these seven experiments are tabulated in table 1. The average of responses to antral irrigation with acetylcholine accompanied by intraTABLE
2. Experimental rats First stimulation
Second stimulation
Inhibition
El. .. E2 .... .. . . . .. . . E3 .. . . .• 0· · · _. E4 .. . .. . . . . ..... E5. . . . .. . . E6 .. .. .. . . . . .. . . E7 ..... .. . . . . . .
2.10 2.90 2.05 1.45 1. 76 1.85 2.04
1.34 1.43 1.28 1.08 1.00 1.45 1.37
69 77 74 100 47 65
Average . . . ,.
2.00
1.37
73
Rat no.
% •
••
••••
,
•
•
•
0
•
•
••
0
83
February 1970
INHIBITION OF GASTRIN ACTIVITY
venous control globulin administration was approximately double the basal rate of acid secretion. There was little difference in the response to the two gastric irrigations; the second response varied from 15% lower to 10% higher than the first and the degree of inhibition as defined by the above formula was negligible. Experimental rats. The data for these experiments are presented in table 2. The average degree of response to the first stimulation was exactly that seen for the control group. However, in the experimental rats , the magnitude of response to the second stimulation (accompanied by antibody globulin) was, in each case, considerably less than the corresponding initial stimulation. The degree of antibody inhibition of the expected increase in gastric acid secretion varied from 47 to 100% and averaged 73% . There was no overlap in the degree of inhibition between any animals in the control and experimental groups. The differences between the first and second responses in the two groups were evaluated statistically using t-test analyses for paired data and the differences were found to be statistically significant with P value of 0.005 for the experimental group and not at all significant for the control group. Discussion
The ability of specific antihormone antibodies to neutralize particular biological effects of small «2000 mol wt) hormones has been described previously. Incubation of antibodies to melanocyte-stimulating hormone (conjugated to rabbit serum albumin) with melanocyte-stimulating hormone in vitro results in inhibition of the expected frog skin darkening effect of the peptide hormone.lO Inhibition of pressor activity followed in vitro incubation of angiotensin II with antibodies elicited by immunization of rabbits with angiotensin II absorbed to carbon particlesY Passive immunization of rats with antibodies to thyroxylthyroglobulin and thyroxyl serum globulin offered protection against the metabolic effects of both exogenous thyroglobulin and thyroxine. 12 Active immunization
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a histamine-protein conjugate protected rabbits against anaphylactic responses to ovalbumin 13 and patients with diabetes insipidus have become vasopressin-resistant as the result of active immunization occurring during hormonal therapy.14 Initial experiments have confirmed the neutralizing effect of antibodies to gastrin tetrapeptide against the acid secretory effects of exogenous gastrin. 3 In the current series of experiments, the same antibody preparation inhibited the physiological effects of endogenous gastrin. Although the exact amino acid sequence of rat gastrin is not known , experiments utilizing antibodies to human gastrin 15. 7 demonstrate that rat gastrin is bound by antibodies to human gastrin (unpublished observations), suggesting that the two species-specific gastrin molecules are structurally similar. Since the gastrin molecules from all species thus far examined contain the functional carboxyl-terminal tetrapeptide sequence 15. 16 to which the antibodies used in these inhibition studies were directed, it is reasonable to expect that rat gastrin also contains the tetrapeptide sequence and that antibodies should bind both human and rat gastrins. In denervated canine pouches,17 as well as in modified rat bioassay preparations, antral irrigation with acetylcholine results in increased acid secretion only after a latent period, comparable with the latent period seen following intravenous administration of exogenous gastrin. 3 In canine experiments,6 measurement of gastrin directly by radioimmunoassay revealed that acetylcholine in contact with antral mucosa liberates gastrin almost immediately. Although the circulating half-life of gastrin,18 like glucagon,19 is short and the antibody globulin was infused into the rat preparations at the time when gastrin release was expected, these experiments do not determine the potential duration of inhibition nor do they delineate whether the locus of inhibition was at the site of gastrin release, within the circulation, or at the receptor cell. Inhibition of excessive gastrin-mediated acid secretion conceivably could have clin-
~ith
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JAFFE ET AL .
ical application. It is possible that acid secretion rates and, thereby, disease processes associated with acid secretion may be modified by antibody bindings of endogenous gastrin molecules. The data from these experiments support the conclusion that antibodies to the functional carboxyl-terminal tetrapeptide amide inhibit the gastric secretory responses to endogenous (as well as exogenous) gastrin. REFERENCES 1. Tracy, H . J., and R. A. Gregory. 1964. Physiological properties of a series of synthetic peptides structurally related to gastrin 1. Nature (London) 204: 935. 2. McGuigan, J . E. 1967. Antibodies to the carboxyl-terminal tetrapeptide of gastrin. Gastroenterology 63: 697. 3. Jaffe, B. M., W. T. Newton, and J. E. McGuigan. 1969. Inhibition of gastrin activity with antibodies to the C-terminal tetrapeptide of gastrin . Surgery 65: 633. 4. Lai, S. K. 1964. Studies on gastrin . Gut 5: 327.
5. McGuigan, J . E. 1968. Immunochemical studies with synthetic gastrin. Gastroenterology 54: 1005. 6. McGuigan, J. E ., B. M. Jaffe, and W. T. Newton. 1969. Immunochemical measurement of endogenous release and circulation (abstr.> . Gastroenterology 56: 1181. 7. McGuigan, J . E. 1968. Gastric mucosal intracellular localization of gastrin by immunofluorescence. Gastroenterology 55 : 315. 8. Patehornik, A., W. B . Lawson, and B. Witkop. 1958. Selective cleavage of peptide bonds. II . The tryptophyl peptide bond and
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the cleavage of glucagon. J. Amer. Chem. Soc. 80: 4747. 9. McGuigan, J. E . 1968. Antibodies to the carboxyl-terminal tetrapeptide amide of gastrin in guinea pigs. J. Lab. Clin. Med. 71: 964.
10. McGuire, J., R. McGill, S. Leeman, and T . Goodfriend. 1965. The experimental generation of antibodies to a-Melanocyte stimulating hormone and adrenocorticotropic hormone. J. Clin. Invest. 44 : 1672. 11. Boyd, G. W., and W. S. Peart. 1968. The production of high-titre antibody against free angiotensin II. Lancet 2: 129. 12. Clutten, R. F., C. R. Harington, and M. E. Yuill. 1938. Studies in synthetic immunochemistry. III. Preparation and antigenic properties of thyroxyl derivatives of proteins, and physiological effects of their antisera. Biochem. J. 32: 1119. 13. Fell, N., G. Rodney, and D. E . Marshall. 1943. Histamine-protein complexes; synthesis and immunologic investigation ; histamine-azoprotein. J. Immun. 47: 237. 14. Roth, J ., S. M. Glick, L. A. Klein, and M. J. Petersen. 1966. Specific antibody to vasopressin in man. J. Clin. Endocr. 26: 671. 15. Gregory, R. A. 1966. The isolation and chemistry of gastrin. Gastroenterology 51: 953. 16. McGuigan, J. E. 1969. Studies of the immunochemical specificity of some antibodies to human gastrin. Gastroenterology 56: 429. 17. Cooke, A. R., and M. 1. Grossman. 1968. Comparison of stimulants of antral release of gastrin. Amer. J. Physiol. 215: 314. 18. Jaffe, B. M., and W. T. Newton. 1969. Distribution and localization of radio-iodinated gastrin. Surg. Forum. In press. 19. Unger, R. H., A. M. Eisentraut, M . S. McCall, and L. L. Madison . 1961. Glucagon antibodies and an immunoassay for glucagon. J. Clin. Invest. 40: 1280.