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Effects of atropine and bethanechol on bombesin-stimulated release of pancreatic polypeptide and gastrin in dog
GASTROENTEROLOGY
77:714-718,1979
Effects of Atropine and Bethanechol on Bombesin-Stimulated Release of Pancreatic Polypeptide and Gastrin in Dog IAN L. TAYLOR, JOHN H. WALSH, DAVID JOHN WOOD, and MORTON I. GROSSMAN VA Wadsworth
Hospital
Center
and UCLA
School
In dogs with gastric fistulas, intravenous infusion of graded doses of bombesin gave graded increases in plasma concentrations of pancreatic polypeptide and of gastrin. The maximally effective dose, 500 ng kg-’ h-‘, gave a mean peak increase over basal of 240 pM for pancreatic polypeptide and 93 pM for gastrin. Atropine sulfate (0.1 mg kg-’ iv) inhibited the pancreatic polypeptide response to bombesin by 60 + 13% (P < 0.01) but had no significant effect on the gastrin response. Bethanechol (0.1 mg kg-’ h-‘) infused alone resulted in a small but statistically significant increment in plasma gastrin concentration (4.5 f 0.9 pM, P < 0.02) and a larger increment in plasma pancreatic polypeptide concentration (41.7 f 6.3 pM, P < 0.01). Bethanechoi (0.2 mg kg-’ h-‘) given together with bombesin (500 ng kg-’ he’) had no significant effect on the pancreatic polypeptide response but significantly (P < 0.02) inhibited the gastrin response by 50.1 f 8.9%. The finding that bethanechol inhibited bombesin-induced gastrin release complements earlier evidence for a cholinergic mechanism of inhibition of gastrin release. The findings of release of pancreatic polypeptide by bethanechol and suppression by atropine of bombesin-induced pancreatic polypeptide release add further evidence regarding cholinergic dependence of PP release. This study shows that bombesin, a known releaser of gastrin, Received Address
Fehruary 9, 1979. Accepted May 30,1979. requests for reprints to: Dr. M. 1. Grossman, VA Wadsworth Hospital Ccntcr, Building 115 Room 114. Los Angeles, California 90073. This work was supported by a Veterans Administration Senior Medical Investigatorship (MIG) and by Grants 08354 and 17328 from NIAMDD to CURE (Center for Ulcer Research and Education), I. L. Taylor was the recipient of a Nuffield Research Fellowship and D. C. Carter was the recipient of a Wellcome Research Fellowship. We thank Peter Chew for technical assistance, Ruth Abercromhio for preparing the illustrations, and Kuwa Chou for typing the manuscript. 0 1979 by the American Gastrocnterological Association 0016.5085/79/100714-05$02.00
of Medicine,
CARTER, Los Angeles,
California
also releases pancreatic polypeptide and 050 of bombesin for both of these actions 125 ng kg-’ h-‘.
that the is about
Bombesin, a peptide originally extracted from frog skin, has been shown to stimulate release of gastrin by an atropine resistant mechanism.‘,z In unpublished preliminary studies, we recently discovered that bombesin releases pancreatic polypeptide (PP) as well as gastrin. This prompted the present study which was directed toward comparing the dose response curves for release of gastrin and PP by bombesin and studying the effects of bethanechol and atropine on bombesin-induced gastrin and PP release. The effect of bethanechol alone on gastrin and PP release was also studied.
Materials
and Methods
The studies were done on four mongrel dogs (body weight 20.7-25.7 kg) with gastric fistulas. Synthetic bombesin was given by intravenous infusion for 2 h at one of 6 doses (31, 62, 125, 250, 500, and 1000 ng kg-’ h-‘). Each dose was given on a separate day and was preceded by a 30-min basal period. None of the doses produced any overt adverse effects in any of the animals tested. The effects of atropine and of bethanechol on the PP and gastrin responses to the 500 ng kg-’ h-’ dose of bombesin were studied. Bethanechol was given as an intravenous infusion in a dose of 0.1 mg kg-’ h-‘. The bethanechol and bombesin infusions were started simultaneously. Atropine sulfate (0.1 mg kg-‘) was given by rapid intravenous injection 10 min before starting the infusion of bombesin. Blood samples were taken into calcium oxalate tubes 30 min and immediately before the test drugs were given and then every 15 min during the 2-h period of study. Gastric juice was collected in 15 min samples. Acid concentration was measured by titration of 0.2 ml samples to pH 7.0 with 0.2 M NaOH on an automatic titrator (Radiometer, Copenhagen). The PP response to a meat meal (15 g kg-’ Friskies
October
1979
RELEASE
canned dog food) was measured and compared to the PP response to bombesin in the same animals. Lastly, the effect of bethanechol (0.1 mg kg-’ h-‘) on basal plasma PP and gastrin concentrations was studied in 11 other dogs (body weight 18.7-25.7 kg) with gas&c fistulas which were left open during the study. Blood samples were iaken 30 min and immediately before the start of the infusion and at 30 min intervals thereafter for a total of 2 h.
OF PANCREATIC
POLYPEPTIDE
BY BOMBESIN
715
500 i
22 f
--
40
\y
BOMBESIN. 500ng
kg-‘h-l
-
4
Radioimmunoassay The method used for radioimmunoassay of PP has been reported in detail elsewhere.” Briefly, antiserum against human PP (a generous gift of Dr. R. E. Chance, Lilly Laboratories) was used in a final dilution of l/ by a modification of the 1,~5O,ooO. Bovine PP was labeled chloramine T method,’ and the tracer was purified on a 1 x 100 cm Sephadex G50 &perfine.column. Activated charcoal was employed as the method of separation. Highly purified human PP was used as the stahdard. Porcine and human PP showed equal immunopotency in this assay system. As canine PP is identical in structure to porcine PP. one can assume that canine PP also shows equal immunopotency with our human standard. Plasma gastrin estimations were made according to described methods utilizing antibody 1296.” The specificity of this antibody is directed towards the carboxyl terminus of gastrin and measures all known biologically active gastrin components. Results are expressed in terms of a standard of pure natural human unsulfated little gastrin (hG17-I).
and Drugs
Peptides
Synthetic bombesin was a kind gift from Roberto de Castiglione of Farmitalia (Milan, Italy). Bethanechol
~-~60 -~ ----90
0-T
120
TIME, rnln Figure 2. Timecourse of PP and gastrin responses to the maximal cffectivc: dose of bombesin, 500 ng kg-’ h-‘.
was bought from Merck, Sharp and Dohme and atropine sulfate from Burroughs Wellcome.
Statistics response in the plasma PP The mean incremental and gastrin concentrations was calculated for each dog by subtracting the mean basal concentration from the mean concentration during the time period under study. These mean increments for individual dogs were ugd to calculate the mean increment for all the dogs studied, which, divided by its standard error, gave a t-value. To determine the statistical significance of differences between two treatments, the between-treatment difference in increment for each dog was calculated, and from these the mean difference for all dogs was calculated which, divided by its standard error, gave a t-value. Statistical analysis was performed on the raw data and after conversion to the percentage change to normalize the data. In all t-tests n is equal to the number of dogs tested. All values are expressed as mean plus or minus standard error.
Results $j
60
1,PP . GASTRIN
i!i $
40
i+ -$
20
5 Y
.
;.:::::I--;. 031
62
1%
250
500
1OOiJ
s!
P Figure
BOMBESIN
DOSE
ng kg-‘h-l
1 Dose response curves for increment above basal of PP ant1 gastrin in response to graded doses of bombesin. The responses are normalized to the maximal response to bombesin which is taken as 100. In this and subsequent figures, except Figure 6, results are for one test in each of four dogs.
Intravenous infusion of doubling doses of borrtbesin (31-1000 ng kg’ h-‘) produced dose-related increases in gastrin and PP concentrations (Figure 1). Maximal responses of both gastrin and PP occurred with the 500 ng kg-’ h-’ dose of bombesin, and the D50 was approximately 125 ng kg-’ h-’ for both peptides. The time-course of the response to bombesin was different for the two peptides; plasma PP concentrations increased rapidly after the infusion of bombesin, peaked early, and then decreased. In contrast, the gastrin response reached a peak at 45 min and remained relatively constant throughout the remaining period of bombcsin infusion (Figure 2). On a molar basis the peak increment in circulating PP was three- to fourfold greater than the peak
716
TAYLOR
ET AL.
GASTROENTEROLOGY
gastrin increment. At the maximal effective dose of bombesin, 500 ng kg-’ h-‘, the mean increment in serum PP (240 + 92 PM) was greater than the mean (142 + 24 PM) measured after ingestion of increment a meal in the same animals (Figure 3). However, the PP response to a meal was more sustained than the response to bombesin. Prior intravenous injection of atropine significantly (P < 0.01) inhibited the PP response to bombesin over the entire 2-h period of the study (Table 1, Figure 4). The mean percentage inhibition was 60 f 13%. The inhibition of PP release by atropine was highly significant (P < 0.01) whether the raw data or the percentage inhibition was analyzed. All the animals studied demonstrated inhibition of PP release after atropine. In contrast, the mean gastrin increment after atropine was not significantly different from that with bombesin alone (Table 1, Figure 4). Figure 5 shows the effect of a 2-h infusion of bethanechol (0.1 mg kg-’ hm’) on basal plasma PP and gastrin concentration in 11 dogs. Bethanechol caused a small (4.5 + 0.9 PM) but statistically significant (P < 0.01) increase in mean basal plasma gastrin concentration. The mean increment in plasma PP concen(41.7 * 6.3; P < trations in response to bethanechol 0.01) was almost lo-fold greater than the increment in plasma gastrin concentration. If the gastrin and PP responses to bethanechol are expressed as a percentage of the responses to bombesin, the responses are 5 and 170/o, respectively. Infusion of the same dose of bethanechol did not significantly alter the mean incremental PP response to bombesin (Figure 6). In contrast, bethanechol significantly inhibited (P
Vol. 77, No. 4, Part 1
PP
GASTRIN BOMBESIN ??BOMBESIN
ALONE + ATROPINE 2cm1
f 2
1501
z E 2
‘J’
100.
” /
i/‘:
50. r
i
O!io6
60 TIME
Figure
4. Effect
sponscs
01 -3Em
120
6b
lm,n)
TIME
120
lm,n)
of atropine (0.1 mg kg’ ) on the PP and gastrin to bombesin (500 ng kg’ h-l).
re-
< 0.01) the gastrin response to bombesin during all but the first 30 min of the test (Table 1, Figure 6). The mean incremental response was inhibited by 50.1 + 8.9%. This inhibition of gastrin release by bethanechol was highly significant (P < 0.01) whether the raw data or the percentage inhibition was analyzed. All the animals studied demonstrated inhibition of gastrin release by bethanechol. Figure 7 shows the rates of acid secretion observed under each experimental condition. The infusion of bethanechol with bombesin significantly (P < 0.05) enhanced the gastric acid response over that seen in response to bombesin alone. The injection of atropine virtually abolished the acid response to bombesin.
400.
z% la
300
_
200
. I 2 :
:
GASTRIN 25. ;L
100 _
0’
-30
Figure
0
30 TIME
60
90
0
30 TIME
120
(mln) 3. Comparison of the PP response to bombesin kg-‘) and to a meal in the same animals.
. -30
Figure
(500 ng
5. Effect
60
9’0
120
(min)
of an intravenous infusion of bethancchol kg-’ he’) on plasma gastrin and PP concentrations. test in each of 11 dogs.
(0.1 mg One
October
RELEASE
1979
Table
1. Mean +- SE Increment (PM) in Plasma Bombesin
and PP Concentrations
alone
Bombesin
of difference
from bombesin
Bombesin, a tetradecapeptide extracted from the skin of the European frog, Bombina bombina, is a potent stimulant of gastrin release in both man6 and dog.” Bombesin-like immunoreactivity has been demonstrated in stomach, intestine, and pancreas of the dog,’ and an increase in bombesin-like immunoreactivity has been described in the serum of man after the ingestion of food.” These findings suggest that a bombesin-like peptide may play a physiologic role in mammalian species. The present results demonstrate that bombesin is also a potent stimulant of pancreatic polypeptide release in the intact dog. However, bombesin does not stimulate PP release from the isolated dog pancreas.’ We have previously shown that the PP response to food can be virtually abolished by truncal vagotomy and atropine. In addition, the present study has shown that atropine significantly inhibits the PP response to bombesin. These findings suggest that a cholincrgic background is required for all stimulants of PP release to be effective. It is possible that this requirement for a cholinergic background is not met in the isolated organ preparation. The peak increment in plasma PP concentrations in response to the infusion of bombesin was three-to fourfold greater than the peak gastrin increment (Figure 2). This ratio of the circulating concentrations of the two peptides is similar to that seen after a meal and may well reflect a difference in the maximal secretory capacity of the PP and gastrin cells.
PP
717
with Various Treatments
+ atropine
Bombesin
+ bethanechol 45 -t gh 275 f 84
Accordingly, if the responses are expressed as a percentage of the maximal response, then the maximal effective doses and the D50s of bombesin for the release of both peptides are similar suggesting that bombesin is approximately equipotent in stimulating PP and gastrin release in the dog. Although bombesin is a potent stimulant of PP release in dog, preliminary studies suggest that it is a poor stimulant of PP release in man (Taylor and Lezoche, unpublished observations). This species difference is as yet without explanation. In dog, but not man, PP cells have been demonstrated by immunofluorescence in the antral mucosa.” However, the PP content of the antral mucosa is several hundred-fold less than that seen in the pancreas and total pancreatectomy in dog abolishes the PP response to food (Taylor and Modlin, unpublished observations). These findings suggest that pancreas is the only site of measurable release of PP into the circulation. Thus, it seems unlikely that bombesin would be selectively releasing PP from antral PP cells in the dog. Our present results suggest that cholinergic factors play contrasting roles in the release of PP and gastrin. Thus atropine inhibited PP release by bombesin, but had little effect on gastrin release. In contrast, a cholinomimetic, bethanechol, inhibited bombesin stimulated gastrin release, but had little effect on PP release. These findings support the previous suggestion that a cholinergic mechanism of inhibition of gastrin release exists.“’
20
GASTRIN BOMBESIN . BOMBESIN
1
BY BOMBESIN
alone: a P < 0.05; b P < 0.01.
Discussion
500
POLYPEPTIDE
109f26 78 f 36”
93 + 13 240 * 92
Gastrin increment PP increment Significance
Gastrin
OF PANCREATIC
ALONE + BETHANECHOL
R
BOMBESIN f s P
+ BETHANECHOL
10 1 1
::
01 BOMBESIN O_3rpr
sb TIME
Figure
----co Inun)
-30
0
60 TIME
-3’0
120
30 TIME
(m,n)
6. Effect of bethanechol (0.1 mg kg-’ h-l) on the PP and gastrin responses to bombesin (500 ng kg-’ h-l).
0
Figure
60
+ ATROPINE
p-1- 90
120
(mln)
7. Acid response to bombesin (500 ng kg-’ h-‘) alone with bethanechol (0.1 mg kg-’ h ‘).
and
718
GASTROENTEROLOGY
TAYLOR ET AL.
References 8. 1. Anastasi A, Erspamer V, Bucci M: Isolation and structure of bombesin and alytensin, two analogous active peptides from the skin of the Euorpean amphibians “Bombina” and “Alytes.” Experientia 27:166, 1971 2. Bertaccini G. Erspamer V, Melchiorri P, et al: Gastrin release by bombesin in the dog. Br J Pharmacol52:219-225,1974 3. Taylor IL, Impicciatore M, Carter DC, Walsh JH: Effect of atropine and vagotomy on the pancreatic polypeptide response to a meal in dogs. Am J Physio1235 (4):E443-E447,1978 4. Hunter WM, Greenwood FC: Preparation of iodine 131 labelled human growth hormone of high specific activity. Nature (Lond) 194:495-496,1962 5. Walsh JH, Trout HH, Debas HT: Immunochemical and biological properties of gastrins obtained from different species and of different molecular species of gastrins. In: Endocrinol-
7.
8.
9.
10.
Vol. 77, No. 4, Part 1
ogy of the Gut. Edited by WY Chey, F Brooks. Thorofare, NJ., Charles B. Slack, Inc., 1974, p 227-289 Erspamer V, Melchior P, Erspamer CF. et al: Polypeptides of the amphibian skin active on the gut and their mammalian counterparts. In: Gastrointestinal Hormones and Pathology of the Digestive System. Edited by M Grossman, V Speranza, N Basso, E Lezoche. New York, Plenum Press, 1978, p 51-64 Walsh JH, Wong HC: Bombesin-like peptides. In: Methods of Hormone Radioimmunoassay. 2nd edition. New York, N.Y., Academic Press, Inc., 1979, p 581-594 Adrian TE, Bloom SR, Hermanson K, Iversen J: Pancreatic polypeptide, glucagon and insulin secretion from the isolated perfused canine pancreas. Diabetologia 14:413-417,1978 Larsson L-I, Sundler F, Hakanson R: Pancreatic polypeptide. -A postulated new hormone: identification of its cellular storage site by light and electron microscopic immunocytochemistry. Diabetologia 12:211-228,1976 Walsh JH, Grossman MI: Gastrin. N Engl J Med 292:13241334,1377-1384,1975
77:714-718,1979
Effects of Atropine and Bethanechol on Bombesin-Stimulated Release of Pancreatic Polypeptide and Gastrin in Dog IAN L. TAYLOR, JOHN H. WALSH, DAVID JOHN WOOD, and MORTON I. GROSSMAN VA Wadsworth
Hospital
Center
and UCLA
School
In dogs with gastric fistulas, intravenous infusion of graded doses of bombesin gave graded increases in plasma concentrations of pancreatic polypeptide and of gastrin. The maximally effective dose, 500 ng kg-’ h-‘, gave a mean peak increase over basal of 240 pM for pancreatic polypeptide and 93 pM for gastrin. Atropine sulfate (0.1 mg kg-’ iv) inhibited the pancreatic polypeptide response to bombesin by 60 + 13% (P < 0.01) but had no significant effect on the gastrin response. Bethanechol (0.1 mg kg-’ h-‘) infused alone resulted in a small but statistically significant increment in plasma gastrin concentration (4.5 f 0.9 pM, P < 0.02) and a larger increment in plasma pancreatic polypeptide concentration (41.7 f 6.3 pM, P < 0.01). Bethanechoi (0.2 mg kg-’ h-‘) given together with bombesin (500 ng kg-’ he’) had no significant effect on the pancreatic polypeptide response but significantly (P < 0.02) inhibited the gastrin response by 50.1 f 8.9%. The finding that bethanechol inhibited bombesin-induced gastrin release complements earlier evidence for a cholinergic mechanism of inhibition of gastrin release. The findings of release of pancreatic polypeptide by bethanechol and suppression by atropine of bombesin-induced pancreatic polypeptide release add further evidence regarding cholinergic dependence of PP release. This study shows that bombesin, a known releaser of gastrin, Received Address
Fehruary 9, 1979. Accepted May 30,1979. requests for reprints to: Dr. M. 1. Grossman, VA Wadsworth Hospital Ccntcr, Building 115 Room 114. Los Angeles, California 90073. This work was supported by a Veterans Administration Senior Medical Investigatorship (MIG) and by Grants 08354 and 17328 from NIAMDD to CURE (Center for Ulcer Research and Education), I. L. Taylor was the recipient of a Nuffield Research Fellowship and D. C. Carter was the recipient of a Wellcome Research Fellowship. We thank Peter Chew for technical assistance, Ruth Abercromhio for preparing the illustrations, and Kuwa Chou for typing the manuscript. 0 1979 by the American Gastrocnterological Association 0016.5085/79/100714-05$02.00
of Medicine,
CARTER, Los Angeles,
California
also releases pancreatic polypeptide and 050 of bombesin for both of these actions 125 ng kg-’ h-‘.
that the is about
Bombesin, a peptide originally extracted from frog skin, has been shown to stimulate release of gastrin by an atropine resistant mechanism.‘,z In unpublished preliminary studies, we recently discovered that bombesin releases pancreatic polypeptide (PP) as well as gastrin. This prompted the present study which was directed toward comparing the dose response curves for release of gastrin and PP by bombesin and studying the effects of bethanechol and atropine on bombesin-induced gastrin and PP release. The effect of bethanechol alone on gastrin and PP release was also studied.
Materials
and Methods
The studies were done on four mongrel dogs (body weight 20.7-25.7 kg) with gastric fistulas. Synthetic bombesin was given by intravenous infusion for 2 h at one of 6 doses (31, 62, 125, 250, 500, and 1000 ng kg-’ h-‘). Each dose was given on a separate day and was preceded by a 30-min basal period. None of the doses produced any overt adverse effects in any of the animals tested. The effects of atropine and of bethanechol on the PP and gastrin responses to the 500 ng kg-’ h-’ dose of bombesin were studied. Bethanechol was given as an intravenous infusion in a dose of 0.1 mg kg-’ h-‘. The bethanechol and bombesin infusions were started simultaneously. Atropine sulfate (0.1 mg kg-‘) was given by rapid intravenous injection 10 min before starting the infusion of bombesin. Blood samples were taken into calcium oxalate tubes 30 min and immediately before the test drugs were given and then every 15 min during the 2-h period of study. Gastric juice was collected in 15 min samples. Acid concentration was measured by titration of 0.2 ml samples to pH 7.0 with 0.2 M NaOH on an automatic titrator (Radiometer, Copenhagen). The PP response to a meat meal (15 g kg-’ Friskies
October
1979
RELEASE
canned dog food) was measured and compared to the PP response to bombesin in the same animals. Lastly, the effect of bethanechol (0.1 mg kg-’ h-‘) on basal plasma PP and gastrin concentrations was studied in 11 other dogs (body weight 18.7-25.7 kg) with gas&c fistulas which were left open during the study. Blood samples were iaken 30 min and immediately before the start of the infusion and at 30 min intervals thereafter for a total of 2 h.
OF PANCREATIC
POLYPEPTIDE
BY BOMBESIN
715
500 i
22 f
--
40
\y
BOMBESIN. 500ng
kg-‘h-l
-
4
Radioimmunoassay The method used for radioimmunoassay of PP has been reported in detail elsewhere.” Briefly, antiserum against human PP (a generous gift of Dr. R. E. Chance, Lilly Laboratories) was used in a final dilution of l/ by a modification of the 1,~5O,ooO. Bovine PP was labeled chloramine T method,’ and the tracer was purified on a 1 x 100 cm Sephadex G50 &perfine.column. Activated charcoal was employed as the method of separation. Highly purified human PP was used as the stahdard. Porcine and human PP showed equal immunopotency in this assay system. As canine PP is identical in structure to porcine PP. one can assume that canine PP also shows equal immunopotency with our human standard. Plasma gastrin estimations were made according to described methods utilizing antibody 1296.” The specificity of this antibody is directed towards the carboxyl terminus of gastrin and measures all known biologically active gastrin components. Results are expressed in terms of a standard of pure natural human unsulfated little gastrin (hG17-I).
and Drugs
Peptides
Synthetic bombesin was a kind gift from Roberto de Castiglione of Farmitalia (Milan, Italy). Bethanechol
~-~60 -~ ----90
0-T
120
TIME, rnln Figure 2. Timecourse of PP and gastrin responses to the maximal cffectivc: dose of bombesin, 500 ng kg-’ h-‘.
was bought from Merck, Sharp and Dohme and atropine sulfate from Burroughs Wellcome.
Statistics response in the plasma PP The mean incremental and gastrin concentrations was calculated for each dog by subtracting the mean basal concentration from the mean concentration during the time period under study. These mean increments for individual dogs were ugd to calculate the mean increment for all the dogs studied, which, divided by its standard error, gave a t-value. To determine the statistical significance of differences between two treatments, the between-treatment difference in increment for each dog was calculated, and from these the mean difference for all dogs was calculated which, divided by its standard error, gave a t-value. Statistical analysis was performed on the raw data and after conversion to the percentage change to normalize the data. In all t-tests n is equal to the number of dogs tested. All values are expressed as mean plus or minus standard error.
Results $j
60
1,PP . GASTRIN
i!i $
40
i+ -$
20
5 Y
.
;.:::::I--;. 031
62
1%
250
500
1OOiJ
s!
P Figure
BOMBESIN
DOSE
ng kg-‘h-l
1 Dose response curves for increment above basal of PP ant1 gastrin in response to graded doses of bombesin. The responses are normalized to the maximal response to bombesin which is taken as 100. In this and subsequent figures, except Figure 6, results are for one test in each of four dogs.
Intravenous infusion of doubling doses of borrtbesin (31-1000 ng kg’ h-‘) produced dose-related increases in gastrin and PP concentrations (Figure 1). Maximal responses of both gastrin and PP occurred with the 500 ng kg-’ h-’ dose of bombesin, and the D50 was approximately 125 ng kg-’ h-’ for both peptides. The time-course of the response to bombesin was different for the two peptides; plasma PP concentrations increased rapidly after the infusion of bombesin, peaked early, and then decreased. In contrast, the gastrin response reached a peak at 45 min and remained relatively constant throughout the remaining period of bombcsin infusion (Figure 2). On a molar basis the peak increment in circulating PP was three- to fourfold greater than the peak
716
TAYLOR
ET AL.
GASTROENTEROLOGY
gastrin increment. At the maximal effective dose of bombesin, 500 ng kg-’ h-‘, the mean increment in serum PP (240 + 92 PM) was greater than the mean (142 + 24 PM) measured after ingestion of increment a meal in the same animals (Figure 3). However, the PP response to a meal was more sustained than the response to bombesin. Prior intravenous injection of atropine significantly (P < 0.01) inhibited the PP response to bombesin over the entire 2-h period of the study (Table 1, Figure 4). The mean percentage inhibition was 60 f 13%. The inhibition of PP release by atropine was highly significant (P < 0.01) whether the raw data or the percentage inhibition was analyzed. All the animals studied demonstrated inhibition of PP release after atropine. In contrast, the mean gastrin increment after atropine was not significantly different from that with bombesin alone (Table 1, Figure 4). Figure 5 shows the effect of a 2-h infusion of bethanechol (0.1 mg kg-’ hm’) on basal plasma PP and gastrin concentration in 11 dogs. Bethanechol caused a small (4.5 + 0.9 PM) but statistically significant (P < 0.01) increase in mean basal plasma gastrin concentration. The mean increment in plasma PP concen(41.7 * 6.3; P < trations in response to bethanechol 0.01) was almost lo-fold greater than the increment in plasma gastrin concentration. If the gastrin and PP responses to bethanechol are expressed as a percentage of the responses to bombesin, the responses are 5 and 170/o, respectively. Infusion of the same dose of bethanechol did not significantly alter the mean incremental PP response to bombesin (Figure 6). In contrast, bethanechol significantly inhibited (P
Vol. 77, No. 4, Part 1
PP
GASTRIN BOMBESIN ??BOMBESIN
ALONE + ATROPINE 2cm1
f 2
1501
z E 2
‘J’
100.
” /
i/‘:
50. r
i
O!io6
60 TIME
Figure
4. Effect
sponscs
01 -3Em
120
6b
lm,n)
TIME
120
lm,n)
of atropine (0.1 mg kg’ ) on the PP and gastrin to bombesin (500 ng kg’ h-l).
re-
< 0.01) the gastrin response to bombesin during all but the first 30 min of the test (Table 1, Figure 6). The mean incremental response was inhibited by 50.1 + 8.9%. This inhibition of gastrin release by bethanechol was highly significant (P < 0.01) whether the raw data or the percentage inhibition was analyzed. All the animals studied demonstrated inhibition of gastrin release by bethanechol. Figure 7 shows the rates of acid secretion observed under each experimental condition. The infusion of bethanechol with bombesin significantly (P < 0.05) enhanced the gastric acid response over that seen in response to bombesin alone. The injection of atropine virtually abolished the acid response to bombesin.
400.
z% la
300
_
200
. I 2 :
:
GASTRIN 25. ;L
100 _
0’
-30
Figure
0
30 TIME
60
90
0
30 TIME
120
(mln) 3. Comparison of the PP response to bombesin kg-‘) and to a meal in the same animals.
. -30
Figure
(500 ng
5. Effect
60
9’0
120
(min)
of an intravenous infusion of bethancchol kg-’ he’) on plasma gastrin and PP concentrations. test in each of 11 dogs.
(0.1 mg One
October
RELEASE
1979
Table
1. Mean +- SE Increment (PM) in Plasma Bombesin
and PP Concentrations
alone
Bombesin
of difference
from bombesin
Bombesin, a tetradecapeptide extracted from the skin of the European frog, Bombina bombina, is a potent stimulant of gastrin release in both man6 and dog.” Bombesin-like immunoreactivity has been demonstrated in stomach, intestine, and pancreas of the dog,’ and an increase in bombesin-like immunoreactivity has been described in the serum of man after the ingestion of food.” These findings suggest that a bombesin-like peptide may play a physiologic role in mammalian species. The present results demonstrate that bombesin is also a potent stimulant of pancreatic polypeptide release in the intact dog. However, bombesin does not stimulate PP release from the isolated dog pancreas.’ We have previously shown that the PP response to food can be virtually abolished by truncal vagotomy and atropine. In addition, the present study has shown that atropine significantly inhibits the PP response to bombesin. These findings suggest that a cholincrgic background is required for all stimulants of PP release to be effective. It is possible that this requirement for a cholinergic background is not met in the isolated organ preparation. The peak increment in plasma PP concentrations in response to the infusion of bombesin was three-to fourfold greater than the peak gastrin increment (Figure 2). This ratio of the circulating concentrations of the two peptides is similar to that seen after a meal and may well reflect a difference in the maximal secretory capacity of the PP and gastrin cells.
PP
717
with Various Treatments
+ atropine
Bombesin
+ bethanechol 45 -t gh 275 f 84
Accordingly, if the responses are expressed as a percentage of the maximal response, then the maximal effective doses and the D50s of bombesin for the release of both peptides are similar suggesting that bombesin is approximately equipotent in stimulating PP and gastrin release in the dog. Although bombesin is a potent stimulant of PP release in dog, preliminary studies suggest that it is a poor stimulant of PP release in man (Taylor and Lezoche, unpublished observations). This species difference is as yet without explanation. In dog, but not man, PP cells have been demonstrated by immunofluorescence in the antral mucosa.” However, the PP content of the antral mucosa is several hundred-fold less than that seen in the pancreas and total pancreatectomy in dog abolishes the PP response to food (Taylor and Modlin, unpublished observations). These findings suggest that pancreas is the only site of measurable release of PP into the circulation. Thus, it seems unlikely that bombesin would be selectively releasing PP from antral PP cells in the dog. Our present results suggest that cholinergic factors play contrasting roles in the release of PP and gastrin. Thus atropine inhibited PP release by bombesin, but had little effect on gastrin release. In contrast, a cholinomimetic, bethanechol, inhibited bombesin stimulated gastrin release, but had little effect on PP release. These findings support the previous suggestion that a cholinergic mechanism of inhibition of gastrin release exists.“’
20
GASTRIN BOMBESIN . BOMBESIN
1
BY BOMBESIN
alone: a P < 0.05; b P < 0.01.
Discussion
500
POLYPEPTIDE
109f26 78 f 36”
93 + 13 240 * 92
Gastrin increment PP increment Significance
Gastrin
OF PANCREATIC
ALONE + BETHANECHOL
R
BOMBESIN f s P
+ BETHANECHOL
10 1 1
::
01 BOMBESIN O_3rpr
sb TIME
Figure
----co Inun)
-30
0
60 TIME
-3’0
120
30 TIME
(m,n)
6. Effect of bethanechol (0.1 mg kg-’ h-l) on the PP and gastrin responses to bombesin (500 ng kg-’ h-l).
0
Figure
60
+ ATROPINE
p-1- 90
120
(mln)
7. Acid response to bombesin (500 ng kg-’ h-‘) alone with bethanechol (0.1 mg kg-’ h ‘).
and
718
GASTROENTEROLOGY
TAYLOR ET AL.
References 8. 1. Anastasi A, Erspamer V, Bucci M: Isolation and structure of bombesin and alytensin, two analogous active peptides from the skin of the Euorpean amphibians “Bombina” and “Alytes.” Experientia 27:166, 1971 2. Bertaccini G. Erspamer V, Melchiorri P, et al: Gastrin release by bombesin in the dog. Br J Pharmacol52:219-225,1974 3. Taylor IL, Impicciatore M, Carter DC, Walsh JH: Effect of atropine and vagotomy on the pancreatic polypeptide response to a meal in dogs. Am J Physio1235 (4):E443-E447,1978 4. Hunter WM, Greenwood FC: Preparation of iodine 131 labelled human growth hormone of high specific activity. Nature (Lond) 194:495-496,1962 5. Walsh JH, Trout HH, Debas HT: Immunochemical and biological properties of gastrins obtained from different species and of different molecular species of gastrins. In: Endocrinol-
7.
8.
9.
10.
Vol. 77, No. 4, Part 1
ogy of the Gut. Edited by WY Chey, F Brooks. Thorofare, NJ., Charles B. Slack, Inc., 1974, p 227-289 Erspamer V, Melchior P, Erspamer CF. et al: Polypeptides of the amphibian skin active on the gut and their mammalian counterparts. In: Gastrointestinal Hormones and Pathology of the Digestive System. Edited by M Grossman, V Speranza, N Basso, E Lezoche. New York, Plenum Press, 1978, p 51-64 Walsh JH, Wong HC: Bombesin-like peptides. In: Methods of Hormone Radioimmunoassay. 2nd edition. New York, N.Y., Academic Press, Inc., 1979, p 581-594 Adrian TE, Bloom SR, Hermanson K, Iversen J: Pancreatic polypeptide, glucagon and insulin secretion from the isolated perfused canine pancreas. Diabetologia 14:413-417,1978 Larsson L-I, Sundler F, Hakanson R: Pancreatic polypeptide. -A postulated new hormone: identification of its cellular storage site by light and electron microscopic immunocytochemistry. Diabetologia 12:211-228,1976 Walsh JH, Grossman MI: Gastrin. N Engl J Med 292:13241334,1377-1384,1975