Control Factors in the Release of Gastrin by Direct Electrical Stimulation of the Vagus C.L. Smith, BSc, MB, ChB, J. Kewenter, MD, PhD, A.M. Connell, MD, J. Ardill, PhD, R. Hayes, BSc, MD, and K. Buchanan, MD The release of gastrin by direct electrical stimulation of the vagus was studied together with the relative effects on the response of antral and duodenal acidification. As expected, gastrin levels increased to three times the normal simulated response following antral neutralization. In contrast, duodenal acidification failed to influence the vagal release of gastrin when the antrum was neutralized although it had a minor effect when the antrum was acidified. Thus the antral pH dominates over duodenal pH as a factor in controlling gastrin release. Surprisingly, atropine in doses which blocked acid release and produced marked cardiac effects failed to inhibit the release of gastrin from the antrum on vagal stimulation. This suggests that, using this model, vagal release of gastrin, if cholinergic, is highly resistant to atropine.
During the last decade, attention has been focused on the interaction between the vagus nerves (neural) and antral gastrin (hormonal) mechanisms in the control of gastric acid secretion, leading to the assumption that both the cephalic and gastric phases of acid secretion involve cholinergic neural mechanisms. Thus gastric acid secretion was thought to result not only from a direct effect of vagal stimulation on the parietal cells but also through cholinergic release of gastrin from the antrum (1). However, although there are a few studies using direct vagal stimulation, much information has been derived indirectly. Uvnas (2) first suggested vagal release of antral gastrin in response to electrical stimulation by showing decreased acid secretion if the antrum were denervated, resected or From the Division of Digestive Diseases, University of Cincinnati Medical Center and Jewish Hospital, Cincinnati, Ohio, and Queen's University of Belfast, Department of Medicine, Belfast, Northern Ireland. This research was supported by a grant from MerrellNational Laboratories. Address for reprint requests: Dr. A.M. Connell, Division of Digestive Diseases, University of Cincinnati Medical Center, Cincinnati, Ohio 45229.
Digestive Diseases, Vol. 20, No. 1 (January 1975)
cocainized. W i t h the advent of radioimmunoassay (3), a more direct examination of the relationship between vagus and gastrin release has been possible. Gastrin measured by radioimmunoassay is released promptly by vagal stimulation (4, 5), though Becker et al (5) used supraphysiological frequencies. Lanciault et al (4) concluded that while the neurotransmitter is acetylcholine, the results suggested a possible direct innervation of the antral cells. Thus while vagal stimulation results in gastrin release its contribution to the overall control of gastrin is not clear. Certainly antral p H has a major influence on gastrin release (6) and vagotomy in no way diminishes the response to feeding (7). It is not clear if vagal release of gastrin is wholly cholinergic. Nilsson et al (8) found a reduction of gastrin level after atropinization in sham-fed dogs, but others (9, 10), found that atropine did not inhibit gastrin release in response to a test meal. This study investigates the release of gastrin by direct electrical stimulation of the vagus and the relative role of antral and duodenal p H and the effect of atropinization on gastrin release.
13
SMITH ET AL
MATERIALS AND METHODS Eighteen mongrel female dogs weighing between 14 and 20 kg were used for the studies. After an overnight fast of at least 16 hours, the dogs were anesthetized using intravenous nembutal. Anesthesia was maintained as required by intravenous doses of nembutal. The vagus nerves were identified in the neck, sectioned and the distal portion attached through circular electrodes. A 0.5-cm cannula was inserted into the stomach at the junction of body and antrum and a further 0.5-cm cannula inserted in the body to allow free drainage during gastric irrigation. A further 0.5-cm cannula was inserted in the duodenum to facilitate pH determination. During experiments where duodenal irrigation was performed a 4-ram-diameter polyethylene tube was inserted through the third part of the duodenum so that the openings lay in the first and second parts of the duodenum. Antral irrigation was carried out in a similar manner with a 4-ram-diameter tube inserted through the body of the stomach so that the tip came to be near the pylorus. The pylorus was ligated in all experiments to prevent contamination of the duodenum with gastric contents or the antrum with duodenal contents. Antral and duodenal pH were measured by an electrode through the appropriate eannula and displayed on a digital p H meter (Radiometer, Copenhagen). Pulse rate was determined by digital palpation of the right femoral artery while the blood pressure was measured using a mercury manometer and an indwelling catheter in the left femoral artery. Pulse rate, blood pressure and pH were recorded at 5-minute intervals throughout. A normal gastrin response of the dogs was confirmed by a rise in peripheral serum gastrin levels following an 8-ounce meal given prior to operation.
Antral Alkalinization and Duodenal Acidification Phosphate buffer (pH 7.5) was used for antral irrigation. Dilute HCI acid (pH 1.5) was used for duodenal acidification. The rate of irrigation was that necessary to maintain the antrum above pH 6 and duodenum below pH 3. Free drainage of the antrum and duodenum were allowed through their respective cannulae.
ment was made with 5% dextrose in 0.9% saline by intravenous infusion into the left femoral vein.
Radioimmunoassay for Gastrin The assay was performed as detailed by Ardill (11). Antibodies were raised in New Zealand white rabbits to synthetic human gastrin I (2-17) conjugated to or-albumin or rabbit albumin using glutaraldehyde (12). The antibody used recognized two dog gastrins comparable to human gastrins, the heptadecapeptide (13), and "big gastrin" (14) and exhibits minimal cross reactivity with C C K / P Z . Gastrin was iodinated by a modification of the chloramine-T method (15) to give a specific activity of 700-900 mCi/mg. Standard gastrin was obtained from Medical Research Council, London (Gastrin H u m a n Type Synthetic 68/ 4399) and was made up in dog plasma which had been rendered hormone-free by treatment with charcoal. The assay was performed in 33% dog plasma using a final antibody dilution of 1/6000 with the addition of labelled gastrin equivalent to approximately 20,000 counts/ 100 seconds ( ~ 10 pg/tube). Separation of antibody bound from free hormone was achieved using dextrancoated charcoal (0.5%), I ml being added to each tube (16). Sensitivity of the assay was 0.5 pg absolute or 5 pg/ml. Each sample was assayed in triplicate.
Vagal Stimulation Vagat electrical stimulation was achieved directly using a Grass $4 stimulator through electrodes applied to the distal portion of the vagus exposed and divided in the neck. Stimulation was by supramaximal square wave pulses (2 msec and 8v) at frequencies between 2 cycles/second and 16 cycles/second.
RESULTS Steady Vagal Stimulation Results Raised Antral Mean Gastrin Levels Vagal stimulation maintained
in
at 4 c y c l e s / s e c
o v e r a p e r i o d of 3 0 m i n u t e s p r o d u c e d a s i g n i f i cant
r i s e in s e r u m
gastrin
levels in s a m p l e s
Blood Sampling
d r a w n f r o m t h e a n t r a l v e i n in 6 d o g s ( F i g u r e 1).
In all experiments, splenectomy was performed to facilitate nonocclusive cannulation of the antral vein via the splenic vein. In a similar way, fine polyethylene cannulae were inserted into the pancreaticoduodenal vein and the right external jugular vein. The blood was allowed to clot, centrifuged and the serum stored at -20 ~ C until assayed. To compensate for blood loss due to sampling, replace-
The
mean prestimulation
and
0 minutes
14
31 -4- 10 p g / m l ,
were
l e v e l s at 2 0 m i n u t e s 41 •
19 p g / m l
and
respectively. After stimulation
a n t r a l v e i n g a s t r i n levels r o s e to 2 1 2 4- 4 3 p g / m l at 10 m i n u t e s
a n d 2 7 7 • 69 p g / m l
at 20
minutes and remained elevated throughout
the
p e r i o d of s t i m u l a t i o n . O n s t o p p i n g s t i m u l a t i o n
Digestive Diseases, Vol. 20, No. 1 (January 1975)
CONTROL OF VAGAL GASTRIN RELEASE
8 ~
~
4
~
~ DUODENUM
~
'~ ANTRUM
VAGAL STIMULATION 175 I50 125 1.
_ :
IO0
300 T
200
~ [00
Fig 1. Serum gastrin levels in antral and peripheral veins in response to vagal stimulation (mean • SE).
I '~. . . . . .
I
-20
levels fell to normal within 15 minutes (Figure 1). In samples drawn from the jugular vein prestimulation serum gastrin levels at 20 and 0 minutes were 20 4- 7.4 and 14 4- 6.2 pg/ml rising after stimulation to 57 4- 17.5 pg/ml at 10 minutes, 67 4- 14.8 pg/ml at 20 minutes, and 75 4- 16 pg/ml at 30 minutes. On stopping stimulation there was a return to baseline levels (Figure 1). Antral pH fell gradually over the period of stimulation from approximately 5.0 m 1.0 to pH 1.6 4- 0.2 (Figure 1). Duodenal pH remained unchanged at 7.1 throughout the experiment. The pulse rate fell as expected during vagal stimulation. Digestive Diseases, Vol. 20, No. 1 (January 1975)
\ I
-I0
PERIPHERAL
:t" I
O
/0
20 TIME
30
40
50
60
(MIN)
With Antral Acidification, Vagal Stimulation Has Optimal Effects on Serum Gastrin at 8 Cycles/Sec
The effect of graded increases in vagal stimulation was examined in 4 dogs (Figure 2). Stimulation was applied at frequencies of 2, 4, 8, 12, and 16 cycles/second for 15 minutes each. Basal antral gastrin levels were 64 • 28 pg/ml and 51 4- 17 p g / m l rising on stimulation to 286 4 - 1 5 4 p g / m l with 2 cycles/second, 301 4- 125 pg/ml with 4 cycles/second, and a maximum of 330 4- 247 pg/ml with 8 cycles/ second but declining with higher frequencies of stimulation. Serum gastrin levels in blood from the duode15
SMITH ET AL
DUODENUM ANTRUM
16
VAGAL STIMULATION
4 0 600
500
. 400 % 300
200
I00
j-
~/,/
...
-..
/ -20
0
ANT.AL
~ ~.OUODENAL 20
40
60 TIME
80
I00
120
(MIN)
nal vein rose less than in the antral vein on stimulation, the highest levels being 191 -4- 90 pg/ml with the 8 cycles/second stimulus. Both antral and duodenal venous gastrin levels returned to baseline 50 minutes after cessation of stimulation. Antral pH dropped to 2.2 during the stimulation period at 4 cycles/second and remained at this level throughout the study. Even so the 8 cycle/second stimulus was the only one which resulted in a further increase in serum gastrin. D u o d e n a l A c i d i f i c a t i o n has O n l y M i n o r Effects on V a g a l l y - l n d u c e d Gastrin Release
The same dogs were subjected to increasing 16
140
Fig 2. Serum gastrin levels in antral and duodenal veins in response to graded increases in vagal stimulation (mean :E SE).
vagal stimulation but with the duodenal pH maintained at 2.0. The results are shown in Figure 3. There was a rise in serum gastrin levels in samples taken from the antral vein from basal levels of 30 -4- 9.4 pg/ml and 13.7 -4- 5.2 pg/ml to 215 -4- 29 pg/ml at stimulation frequencies of 2 cycles/second. During the ensuing 15-minute periods there was a progressive fall in antral serum gastrin levels to 81.7 -4- 24.5 pg/ml at 12 cycles/second. The fall in antral gastrin levels during continuing and increasing vagal stimulation appears to occur earlier than that noted in the same dogs without duodenal acidification. The pH of the duodenum continued at 2.0 -4- 0.4 during the period of stimulation and Digestive Diseases, Vol. 20, No. 1 (January 1975)
CONTROL OF VAGAL GASTRIN RELEASE 8
DUODENUM "~P~'-----"~ANTRUM
'!I
"~vY//////////~
. . . . . . . .
VAGAL STIMULATION
g~//////////~E"AL ~C~O,F,CAr,O.'//?////////////A
300
T T -~
200
%
ANTRAL
Z_ i
Fig 3, Effect of graded vagal stimulation on gastrin release during duodenal acidification (mean + SE)-
antral pH fell to 2.0 • 0.2 at 4 cycles/second, at which level it remained during stimulation. There was the expected fall in pulse rate with vagal stimulation. Determination of the serum gastrin level in samples from the pancreaticoduodenal vein revealed less dramatic changes than in antral blood. Basal levels were 9 :~ 4 pg/ml and 6.5 • 3 pg/ml rising to 34 ~ 9.5 pg/ml on stimulation. There was a subsequent gradual fall in duodenal serum gastrin levels.
Duodenal Acidification Fails to Inhibit Gastrin Levels Stimulated by Antral Neutralization During Vagal Stimulation The vagus was stimulated in 6 dogs while the pH of the antrum was maintained at 6.5. During antral neutralization, antral serum gastrin levels rose from control values of 78 4- 30 pg/ ml to the high level of 602 • pg/ml 15 minutes after starting stimulation and were Digestive Diseases, Voi. 20, No. 1 (January 1975)
~ _ ~
DUODENAL
I
I
T
r
I
i
- 20
- I0
0
I0
20
30
TIME
f
I
r
40
50
60
(rain)
maintained at this level during the period of antral neutralization (Figure 4). Contrary to expectation, changing duodenal pH did not affect the level of antrat serum gastrin which continued at 577 ~ 157 and 757 + 337 pg/ml in the next two 15-minute periods. On cessation of stimulation there was a fall in antral serum gastrin level to 469 -4- 233 pg/ml at 15 minutes after stimulation. The levels of gastrin achieved in this experiment are approximately double those reached in any of the other studies in this series. During the period of duodenal acidification only one dog showed a fall in serum gastrin but equally another dog had a rise to 2250 pg/ml.
Atropine Fails to Inhibit Vagally Released Gastrin Atropine administered in doses of either 0.1 or 0.2 mg/kg body weight to two groups of 3 dogs produced the expected blocking of vagally induced acid secretion and fall in pulse rate 17
SMITH
ET AL
8 6
v
"~
v
ANTRAL DUODENAL
4 2 175 150 125 IOO 75 ANTRALNEUTRALISATION
I ~OOE~'L'C'O'~'CAT,ON
ioo !
BOO
600 c~
400
200
I -20
I
I
20 40 TIME (rain.)
I 60
80
Fig 4. T h e s e r u m g a s t r i n r e s p o n s e t o v a g a l s t i m u l a t i o n d u r i n g a n t r a l n e u t r a l i z a t i o n a n d d u o d e n a l a c i d i f i c a t i o n ( m e a n ~ SE ).
18
Digestive Diseases, VoL 20, No. 1 (January 1975)
CONTROL OF VAGAL GASTRIN RELEASE
175 150 125 8 VAGAL STIMULATION
8oo I
600 !
%
40O -
ATROPINE o ~mg/kg BODY WEIGHT/
Fig 5. The effect of atropine on gastrin release in response to vagal stimulation (mean ~:SE,).
(Figure 5). In contrast to these findings, atropine did not block vagally induced gastrin release. Atropine at doses of 0.1 mg/kg and 0.2 mg/ kg body weight did not affect basal antral gastrin levels (Figure 5). On stimulation of the vagus, however, there was a rise to 245 4- 101 pg/ml in 10 minutes, which was sustained during the period of stimulation. With a dose of atropine of 0.2 mg/kg body weight there was a greater rise in antral gastrin levels over basal, but this group of dogs started with higher basal levels. Even so, there was no effect of atropine on basal levels. The gastrin levels attained with this dose of atropine were similar to those reached with vagal stimulation during antral neutralization. Digestive Diseases, Vol. 20, No. 1 (January 1975)
- 20
0
20
40
60
80
TIME (min)
Therefore, under these conditions while atropine blocked the other cholinergic mechanisms of heart rate and acid release; it had no effect on vagally induced gastrin release.
DISCUSSION
It is well known that indirect vagal stimulation using insulin hypoglycemia (17) or sham feeding (18) results in gastrin release and that these effects can be abolished by antral denervation (19). Lanciault et al (4) have described the kinetics of gastrin release in response to direct vagal stimulation at 10-15 cycles/second. Becker et al (5) showed a sustained release of gastrin from the neutralized antrum using supraphysiological frequencies of 20 cycles/sec19
SMITH ET AL
ond applied to the antral nerves only. They recorded a fall in vagally stimulated gastrin release on antral acidification. Our study amplifies this finding by showing that acidification of the antrum overcomes vagal stimulation even at physiological frequencies. We do not feel that the decreased gastrin output represents fatigue of G cells in view of the known sustained release of gastrin which occurs at 20 cycles/second (5). Duodenal acidification under certain circumstances decreases serum gastrin levels possibly by liberation of secretin (20-22). The present study has determined the relative dominance of antral and duodenal acidification in inhibition of vagally stimulated gastrin release. It seems clear that the duodenal effect is the minor one since when the antrum was maintained at neutrality duodenal acidification was ineffective in altering gastrin release. This might appear to conflict with the observations of Becker et al (21). They stimulated gastrin release by perfusion of the antrum with acetylcholine at pH 7 and under these circumstances perfusion of the duodenum with acid at pH 1 resulted in reduction in gastrin output. The experimental models, however, are different. In our study we used direct vagal stimulation whereas Becker et al (21) stimulated the mucosa with acetylcholine which does not necessarily mimic the mechanism of action of the vagus. The levels of gastrin in the pancreaticoduodenal vein were lower than in the antral vein. Dilution in the systemic circulation could account for most of this difference but it is consistent with the suggestion that gastrin is removed from the circulation in the small bowel (25). Possibly the most interesting result of this study is that atropine fails to block vagally induced gastrin release, which has previously been assumed to be cholinergic, especially since direct application of acetylcholine to the antral mucosa releases gastrin (24, 25). Some studies, however, have previously suggested a noncholinergic mechanism (8). Thus while atropinization reduced the gastrin response to the indirect stimuli of sham 20
feeding (7) and insulin (17) in dogs, it fails to block gastrin release in response to a test meal in dogs and man (7-9). Species differences occur in the response to atropinization. Atropine does not block the release of acid to infused gastrin in cats (26) but does in dogs (27). It is possible that the use of anesthesia may affect the responses (26), but no conclusive evidence of this exists. Indeed previous studies (7-9) have been carried out in the unanesthetized and in these atropine failed to block the gastrin response. In this present study we now have direct evidence that under the conditions of the experiment atropine in the effective dose of 0.1 mg/kg and 0.2 mg/kg fails to block gastrin release to direct vagal stimulation in dogs. At these doses, pulse rate and gastric acid response were blocked. Csendes et al (17) using similar dose levels of atropine blocked gastrin release by insulin only with a dose of 0.2 mg/kg but blocked acid release throughout the range. That vagal gastric innervation may not be wholly cholinergic is supported by the finding that a number of positive Hollander tests could be converted to negative by ~-adrenergic blockade (28). It has also been shown (29, 30) that catecholamines will stimulate gastrin release, and this release can be blocked by adrenergic blocking agents (29). Further evidence of complex vagal activity is that it has been suggested that receptive relaxation of the stomach induced by vagal stimulation uses a noncholinergi c nonadrenergic mechanism (31). In conclusion, this study suggests that the mechanism of vagal release of gastrin is noncholinergic, or if cholinergic, is highly resistant to atropine. ACKNOWLEDGMENT
We would like to thank Michael MacLcan, of the Jewish Hospital, Cincinnati, for his technical assistance. REFERENCES
1. Grossman MI: Neural and hormonal stimulation of gastric secretion of acid. Handbook of Physiology--Alimentary Canal, Vol. 2. CF Digestive Diseases, Vol. 20, No. 1 (January 1975)
CONTROL OF VAGAL GASTRIN RELEASE
Code (ed). Washington DC, American Physiological Society, 1967, pp 835-864 2. Uvnas B: The part played by the pyloric region in the cephalic phase of gastric secretion. Acta Physiol Scand 4: Suppl. XIII, 1942 3. McGuigan JE: Immunochemical studies with synthetic human gastrin. Gastroenterology 54:1005-1011, 1968 4. Lanciauh G, Bonoma C, Kaneman G, Brooks FB: Kinetics of gastrin release and degradation in response to electrical vagal stimulation in the dog. Proc Soc Exp Biol Med 142:740-743, 1973 5. Becker HD, Reeder DD, Thompson JC: Direct measurement of vagal release of gastrin. Surgery 75:101-106, 1974 6. Hansky J, Korman MG, Soveny C, St John DJB: Radioimmunoassay of gastrin: Studies in pernicious anaemia. Gut 12:97-101, 1971 7. Korman MG, Soveny C, Hansky J~ Effect of food on serum gastrin evaluated by radioimmunoassay. Gut 12:619-624, 1971 8. Nilsson G, Simon J, Yalow RS, Berson SA: Plasma gastrin and gastric acid responses to sham feeding and feeding in dogs. Gastroenterology 63:51-59, 1972 9. Walsh JH, Yalow RS, Berson SA: The effect of atropine on plasma gastrin response to feeding. Gastroenterology 60:16-21, 1971 10. Korman MG, Soveny C, Hansky J: Serum gastrin in duodenal ulcer. I. Basal levels and effect of food and atropine. Gut 12:899-902, 1971 1 I. Ardill J: The measurement of gastrin by radioimmuno~assay. PhD Thesis, Queen's University of Belfast, 1973 12. Sachs PH, Winn H J: The use of glutaraldehyde as a coupling agent for ribonuclease and bovine serum albumin. Immunochemistry 7:581-585, 1970 13. Gregory RA, Tracy H J, Grossman MI: Isolation of two gastrins from human antral mucosa. Nature 209:583, 1966 14. Yalow RS, Berson SA: Size and charge distinctions between endogenous human plasma gastrin in peripheral blood and hepatadecapeptide gastrins. Gastroenterology 58:609-615, 1970 15. Hunter WM, Greenwood FC: Preparation of iodine-131-1abelled growth hormone of high specific activity. Nature 194:495-496, 1962 16. Herbert V, Lau KS, Gottlieb CW, Bleicher SJ:
Digestive Diseases, VoL 20, No. 1 (January 1975)
Coated charcoal immunoassay of insulin. J Clin Endoer Metab 25 : 1375-1384, 1965 17. Hansky J, Soveny C, Korman MG: Role of the vagus in insulin-mediated gastrin release. Gastroenterology 63:387-391, 1972 18. Csendes A, Walsh JH, Grossman MI: Effect of atropine and of antral acidification on gastrin release and acid secretion in response to insulin and feeding in dogs. Gastroenterology 63:257263, 1972 19. Tepperman BL, Walsh JH, Preshaw RM: Effect of antral denervation on gastrin release by sham feeding and insulin hypoglycemia in dogs. Gastroenterology 63:973-980, 1972 20. Brooks AM, Stening GF, Grossman MI: Effect of gastric vagal denervation on inhibition of acid secretion by secretin. Am J Dig Dis 16:193-202, 1971 21. Becker HD, Evans JC, Reeder DD, Thompson JC: Duodenal acidification and antral release of gastrin. Arch Surg 180:201-207, 1974 22. Hansky J, Soveny C, Korman MG: Effect of secretin on serum gastrin as measured by immunoassay. Gastroenterology 61:62-68, 1971 23. Becker HD, Reeder DD, Thompson JC: Extraction of circulating endogenous gastrin by the small bowel. Gastroenterology 65:903-906, 1973 24. Robertson GR, Langlois K, Martin CG, Stezak G, Grossman MI: Release of gastrin in response to battering the pyloric mucosa with acetylcholine. Am J Physiol 163:27-33, 1950 25. Jackson BM, Reeder DD, Thompson JC: Dynamic characteristics of gastrin release. Am J Surg 123:137-142, 1972 26. Blair EL, Harper AA, Lake H J, Reed JD: The effect of atropine upon gastrin-stimulated acid gastric secretion. J Physiol 159:72-73, 1961 27. Gregory RA, Tracy H J: The preparation and properties of gastrin. J Physiol 156:523-543, 196l 28. Read RC, Thompson BW, Hall WH: Conversion of Hollander tests in man from positive to negative--~ adrenergic blockade with propanolol. Arch Surg 104:573-578, 1972 29. Hayes JR, Ardill J, Kennedy TL, Shanks RG, Buchanan KD: Stimulation of gastrin release by catecholamines. Lancet 1:819-821, 1972 30. Stadil F, Rehfeld JF: Release of gastrin by epi-
21
SMITH ET AL
nephrine in man. Gastroenterology 65:210-215, 1973 31. Martinson J: Vagal relaxation of the stomach--
22
Experimental reinvestigation of the concept of the transmission mechanism. Acta Physiol Stand 64:453-462, 1965
Digestive Oiseases, Voi. 20, No. 1 (January 1975)
During the last decade, attention has been focused on the interaction between the vagus nerves (neural) and antral gastrin (hormonal) mechanisms in the control of gastric acid secretion, leading to the assumption that both the cephalic and gastric phases of acid secretion involve cholinergic neural mechanisms. Thus gastric acid secretion was thought to result not only from a direct effect of vagal stimulation on the parietal cells but also through cholinergic release of gastrin from the antrum (1). However, although there are a few studies using direct vagal stimulation, much information has been derived indirectly. Uvnas (2) first suggested vagal release of antral gastrin in response to electrical stimulation by showing decreased acid secretion if the antrum were denervated, resected or From the Division of Digestive Diseases, University of Cincinnati Medical Center and Jewish Hospital, Cincinnati, Ohio, and Queen's University of Belfast, Department of Medicine, Belfast, Northern Ireland. This research was supported by a grant from MerrellNational Laboratories. Address for reprint requests: Dr. A.M. Connell, Division of Digestive Diseases, University of Cincinnati Medical Center, Cincinnati, Ohio 45229.
Digestive Diseases, Vol. 20, No. 1 (January 1975)
cocainized. W i t h the advent of radioimmunoassay (3), a more direct examination of the relationship between vagus and gastrin release has been possible. Gastrin measured by radioimmunoassay is released promptly by vagal stimulation (4, 5), though Becker et al (5) used supraphysiological frequencies. Lanciault et al (4) concluded that while the neurotransmitter is acetylcholine, the results suggested a possible direct innervation of the antral cells. Thus while vagal stimulation results in gastrin release its contribution to the overall control of gastrin is not clear. Certainly antral p H has a major influence on gastrin release (6) and vagotomy in no way diminishes the response to feeding (7). It is not clear if vagal release of gastrin is wholly cholinergic. Nilsson et al (8) found a reduction of gastrin level after atropinization in sham-fed dogs, but others (9, 10), found that atropine did not inhibit gastrin release in response to a test meal. This study investigates the release of gastrin by direct electrical stimulation of the vagus and the relative role of antral and duodenal p H and the effect of atropinization on gastrin release.
13
SMITH ET AL
MATERIALS AND METHODS Eighteen mongrel female dogs weighing between 14 and 20 kg were used for the studies. After an overnight fast of at least 16 hours, the dogs were anesthetized using intravenous nembutal. Anesthesia was maintained as required by intravenous doses of nembutal. The vagus nerves were identified in the neck, sectioned and the distal portion attached through circular electrodes. A 0.5-cm cannula was inserted into the stomach at the junction of body and antrum and a further 0.5-cm cannula inserted in the body to allow free drainage during gastric irrigation. A further 0.5-cm cannula was inserted in the duodenum to facilitate pH determination. During experiments where duodenal irrigation was performed a 4-ram-diameter polyethylene tube was inserted through the third part of the duodenum so that the openings lay in the first and second parts of the duodenum. Antral irrigation was carried out in a similar manner with a 4-ram-diameter tube inserted through the body of the stomach so that the tip came to be near the pylorus. The pylorus was ligated in all experiments to prevent contamination of the duodenum with gastric contents or the antrum with duodenal contents. Antral and duodenal pH were measured by an electrode through the appropriate eannula and displayed on a digital p H meter (Radiometer, Copenhagen). Pulse rate was determined by digital palpation of the right femoral artery while the blood pressure was measured using a mercury manometer and an indwelling catheter in the left femoral artery. Pulse rate, blood pressure and pH were recorded at 5-minute intervals throughout. A normal gastrin response of the dogs was confirmed by a rise in peripheral serum gastrin levels following an 8-ounce meal given prior to operation.
Antral Alkalinization and Duodenal Acidification Phosphate buffer (pH 7.5) was used for antral irrigation. Dilute HCI acid (pH 1.5) was used for duodenal acidification. The rate of irrigation was that necessary to maintain the antrum above pH 6 and duodenum below pH 3. Free drainage of the antrum and duodenum were allowed through their respective cannulae.
ment was made with 5% dextrose in 0.9% saline by intravenous infusion into the left femoral vein.
Radioimmunoassay for Gastrin The assay was performed as detailed by Ardill (11). Antibodies were raised in New Zealand white rabbits to synthetic human gastrin I (2-17) conjugated to or-albumin or rabbit albumin using glutaraldehyde (12). The antibody used recognized two dog gastrins comparable to human gastrins, the heptadecapeptide (13), and "big gastrin" (14) and exhibits minimal cross reactivity with C C K / P Z . Gastrin was iodinated by a modification of the chloramine-T method (15) to give a specific activity of 700-900 mCi/mg. Standard gastrin was obtained from Medical Research Council, London (Gastrin H u m a n Type Synthetic 68/ 4399) and was made up in dog plasma which had been rendered hormone-free by treatment with charcoal. The assay was performed in 33% dog plasma using a final antibody dilution of 1/6000 with the addition of labelled gastrin equivalent to approximately 20,000 counts/ 100 seconds ( ~ 10 pg/tube). Separation of antibody bound from free hormone was achieved using dextrancoated charcoal (0.5%), I ml being added to each tube (16). Sensitivity of the assay was 0.5 pg absolute or 5 pg/ml. Each sample was assayed in triplicate.
Vagal Stimulation Vagat electrical stimulation was achieved directly using a Grass $4 stimulator through electrodes applied to the distal portion of the vagus exposed and divided in the neck. Stimulation was by supramaximal square wave pulses (2 msec and 8v) at frequencies between 2 cycles/second and 16 cycles/second.
RESULTS Steady Vagal Stimulation Results Raised Antral Mean Gastrin Levels Vagal stimulation maintained
in
at 4 c y c l e s / s e c
o v e r a p e r i o d of 3 0 m i n u t e s p r o d u c e d a s i g n i f i cant
r i s e in s e r u m
gastrin
levels in s a m p l e s
Blood Sampling
d r a w n f r o m t h e a n t r a l v e i n in 6 d o g s ( F i g u r e 1).
In all experiments, splenectomy was performed to facilitate nonocclusive cannulation of the antral vein via the splenic vein. In a similar way, fine polyethylene cannulae were inserted into the pancreaticoduodenal vein and the right external jugular vein. The blood was allowed to clot, centrifuged and the serum stored at -20 ~ C until assayed. To compensate for blood loss due to sampling, replace-
The
mean prestimulation
and
0 minutes
14
31 -4- 10 p g / m l ,
were
l e v e l s at 2 0 m i n u t e s 41 •
19 p g / m l
and
respectively. After stimulation
a n t r a l v e i n g a s t r i n levels r o s e to 2 1 2 4- 4 3 p g / m l at 10 m i n u t e s
a n d 2 7 7 • 69 p g / m l
at 20
minutes and remained elevated throughout
the
p e r i o d of s t i m u l a t i o n . O n s t o p p i n g s t i m u l a t i o n
Digestive Diseases, Vol. 20, No. 1 (January 1975)
CONTROL OF VAGAL GASTRIN RELEASE
8 ~
~
4
~
~ DUODENUM
~
'~ ANTRUM
VAGAL STIMULATION 175 I50 125 1.
_ :
IO0
300 T
200
~ [00
Fig 1. Serum gastrin levels in antral and peripheral veins in response to vagal stimulation (mean • SE).
I '~. . . . . .
I
-20
levels fell to normal within 15 minutes (Figure 1). In samples drawn from the jugular vein prestimulation serum gastrin levels at 20 and 0 minutes were 20 4- 7.4 and 14 4- 6.2 pg/ml rising after stimulation to 57 4- 17.5 pg/ml at 10 minutes, 67 4- 14.8 pg/ml at 20 minutes, and 75 4- 16 pg/ml at 30 minutes. On stopping stimulation there was a return to baseline levels (Figure 1). Antral pH fell gradually over the period of stimulation from approximately 5.0 m 1.0 to pH 1.6 4- 0.2 (Figure 1). Duodenal pH remained unchanged at 7.1 throughout the experiment. The pulse rate fell as expected during vagal stimulation. Digestive Diseases, Vol. 20, No. 1 (January 1975)
\ I
-I0
PERIPHERAL
:t" I
O
/0
20 TIME
30
40
50
60
(MIN)
With Antral Acidification, Vagal Stimulation Has Optimal Effects on Serum Gastrin at 8 Cycles/Sec
The effect of graded increases in vagal stimulation was examined in 4 dogs (Figure 2). Stimulation was applied at frequencies of 2, 4, 8, 12, and 16 cycles/second for 15 minutes each. Basal antral gastrin levels were 64 • 28 pg/ml and 51 4- 17 p g / m l rising on stimulation to 286 4 - 1 5 4 p g / m l with 2 cycles/second, 301 4- 125 pg/ml with 4 cycles/second, and a maximum of 330 4- 247 pg/ml with 8 cycles/ second but declining with higher frequencies of stimulation. Serum gastrin levels in blood from the duode15
SMITH ET AL
DUODENUM ANTRUM
16
VAGAL STIMULATION
4 0 600
500
. 400 % 300
200
I00
j-
~/,/
...
-..
/ -20
0
ANT.AL
~ ~.OUODENAL 20
40
60 TIME
80
I00
120
(MIN)
nal vein rose less than in the antral vein on stimulation, the highest levels being 191 -4- 90 pg/ml with the 8 cycles/second stimulus. Both antral and duodenal venous gastrin levels returned to baseline 50 minutes after cessation of stimulation. Antral pH dropped to 2.2 during the stimulation period at 4 cycles/second and remained at this level throughout the study. Even so the 8 cycle/second stimulus was the only one which resulted in a further increase in serum gastrin. D u o d e n a l A c i d i f i c a t i o n has O n l y M i n o r Effects on V a g a l l y - l n d u c e d Gastrin Release
The same dogs were subjected to increasing 16
140
Fig 2. Serum gastrin levels in antral and duodenal veins in response to graded increases in vagal stimulation (mean :E SE).
vagal stimulation but with the duodenal pH maintained at 2.0. The results are shown in Figure 3. There was a rise in serum gastrin levels in samples taken from the antral vein from basal levels of 30 -4- 9.4 pg/ml and 13.7 -4- 5.2 pg/ml to 215 -4- 29 pg/ml at stimulation frequencies of 2 cycles/second. During the ensuing 15-minute periods there was a progressive fall in antral serum gastrin levels to 81.7 -4- 24.5 pg/ml at 12 cycles/second. The fall in antral gastrin levels during continuing and increasing vagal stimulation appears to occur earlier than that noted in the same dogs without duodenal acidification. The pH of the duodenum continued at 2.0 -4- 0.4 during the period of stimulation and Digestive Diseases, Vol. 20, No. 1 (January 1975)
CONTROL OF VAGAL GASTRIN RELEASE 8
DUODENUM "~P~'-----"~ANTRUM
'!I
"~vY//////////~
. . . . . . . .
VAGAL STIMULATION
g~//////////~E"AL ~C~O,F,CAr,O.'//?////////////A
300
T T -~
200
%
ANTRAL
Z_ i
Fig 3, Effect of graded vagal stimulation on gastrin release during duodenal acidification (mean + SE)-
antral pH fell to 2.0 • 0.2 at 4 cycles/second, at which level it remained during stimulation. There was the expected fall in pulse rate with vagal stimulation. Determination of the serum gastrin level in samples from the pancreaticoduodenal vein revealed less dramatic changes than in antral blood. Basal levels were 9 :~ 4 pg/ml and 6.5 • 3 pg/ml rising to 34 ~ 9.5 pg/ml on stimulation. There was a subsequent gradual fall in duodenal serum gastrin levels.
Duodenal Acidification Fails to Inhibit Gastrin Levels Stimulated by Antral Neutralization During Vagal Stimulation The vagus was stimulated in 6 dogs while the pH of the antrum was maintained at 6.5. During antral neutralization, antral serum gastrin levels rose from control values of 78 4- 30 pg/ ml to the high level of 602 • pg/ml 15 minutes after starting stimulation and were Digestive Diseases, Voi. 20, No. 1 (January 1975)
~ _ ~
DUODENAL
I
I
T
r
I
i
- 20
- I0
0
I0
20
30
TIME
f
I
r
40
50
60
(rain)
maintained at this level during the period of antral neutralization (Figure 4). Contrary to expectation, changing duodenal pH did not affect the level of antrat serum gastrin which continued at 577 ~ 157 and 757 + 337 pg/ml in the next two 15-minute periods. On cessation of stimulation there was a fall in antral serum gastrin level to 469 -4- 233 pg/ml at 15 minutes after stimulation. The levels of gastrin achieved in this experiment are approximately double those reached in any of the other studies in this series. During the period of duodenal acidification only one dog showed a fall in serum gastrin but equally another dog had a rise to 2250 pg/ml.
Atropine Fails to Inhibit Vagally Released Gastrin Atropine administered in doses of either 0.1 or 0.2 mg/kg body weight to two groups of 3 dogs produced the expected blocking of vagally induced acid secretion and fall in pulse rate 17
SMITH
ET AL
8 6
v
"~
v
ANTRAL DUODENAL
4 2 175 150 125 IOO 75 ANTRALNEUTRALISATION
I ~OOE~'L'C'O'~'CAT,ON
ioo !
BOO
600 c~
400
200
I -20
I
I
20 40 TIME (rain.)
I 60
80
Fig 4. T h e s e r u m g a s t r i n r e s p o n s e t o v a g a l s t i m u l a t i o n d u r i n g a n t r a l n e u t r a l i z a t i o n a n d d u o d e n a l a c i d i f i c a t i o n ( m e a n ~ SE ).
18
Digestive Diseases, VoL 20, No. 1 (January 1975)
CONTROL OF VAGAL GASTRIN RELEASE
175 150 125 8 VAGAL STIMULATION
8oo I
600 !
%
40O -
ATROPINE o ~mg/kg BODY WEIGHT/
Fig 5. The effect of atropine on gastrin release in response to vagal stimulation (mean ~:SE,).
(Figure 5). In contrast to these findings, atropine did not block vagally induced gastrin release. Atropine at doses of 0.1 mg/kg and 0.2 mg/ kg body weight did not affect basal antral gastrin levels (Figure 5). On stimulation of the vagus, however, there was a rise to 245 4- 101 pg/ml in 10 minutes, which was sustained during the period of stimulation. With a dose of atropine of 0.2 mg/kg body weight there was a greater rise in antral gastrin levels over basal, but this group of dogs started with higher basal levels. Even so, there was no effect of atropine on basal levels. The gastrin levels attained with this dose of atropine were similar to those reached with vagal stimulation during antral neutralization. Digestive Diseases, Vol. 20, No. 1 (January 1975)
- 20
0
20
40
60
80
TIME (min)
Therefore, under these conditions while atropine blocked the other cholinergic mechanisms of heart rate and acid release; it had no effect on vagally induced gastrin release.
DISCUSSION
It is well known that indirect vagal stimulation using insulin hypoglycemia (17) or sham feeding (18) results in gastrin release and that these effects can be abolished by antral denervation (19). Lanciault et al (4) have described the kinetics of gastrin release in response to direct vagal stimulation at 10-15 cycles/second. Becker et al (5) showed a sustained release of gastrin from the neutralized antrum using supraphysiological frequencies of 20 cycles/sec19
SMITH ET AL
ond applied to the antral nerves only. They recorded a fall in vagally stimulated gastrin release on antral acidification. Our study amplifies this finding by showing that acidification of the antrum overcomes vagal stimulation even at physiological frequencies. We do not feel that the decreased gastrin output represents fatigue of G cells in view of the known sustained release of gastrin which occurs at 20 cycles/second (5). Duodenal acidification under certain circumstances decreases serum gastrin levels possibly by liberation of secretin (20-22). The present study has determined the relative dominance of antral and duodenal acidification in inhibition of vagally stimulated gastrin release. It seems clear that the duodenal effect is the minor one since when the antrum was maintained at neutrality duodenal acidification was ineffective in altering gastrin release. This might appear to conflict with the observations of Becker et al (21). They stimulated gastrin release by perfusion of the antrum with acetylcholine at pH 7 and under these circumstances perfusion of the duodenum with acid at pH 1 resulted in reduction in gastrin output. The experimental models, however, are different. In our study we used direct vagal stimulation whereas Becker et al (21) stimulated the mucosa with acetylcholine which does not necessarily mimic the mechanism of action of the vagus. The levels of gastrin in the pancreaticoduodenal vein were lower than in the antral vein. Dilution in the systemic circulation could account for most of this difference but it is consistent with the suggestion that gastrin is removed from the circulation in the small bowel (25). Possibly the most interesting result of this study is that atropine fails to block vagally induced gastrin release, which has previously been assumed to be cholinergic, especially since direct application of acetylcholine to the antral mucosa releases gastrin (24, 25). Some studies, however, have previously suggested a noncholinergic mechanism (8). Thus while atropinization reduced the gastrin response to the indirect stimuli of sham 20
feeding (7) and insulin (17) in dogs, it fails to block gastrin release in response to a test meal in dogs and man (7-9). Species differences occur in the response to atropinization. Atropine does not block the release of acid to infused gastrin in cats (26) but does in dogs (27). It is possible that the use of anesthesia may affect the responses (26), but no conclusive evidence of this exists. Indeed previous studies (7-9) have been carried out in the unanesthetized and in these atropine failed to block the gastrin response. In this present study we now have direct evidence that under the conditions of the experiment atropine in the effective dose of 0.1 mg/kg and 0.2 mg/kg fails to block gastrin release to direct vagal stimulation in dogs. At these doses, pulse rate and gastric acid response were blocked. Csendes et al (17) using similar dose levels of atropine blocked gastrin release by insulin only with a dose of 0.2 mg/kg but blocked acid release throughout the range. That vagal gastric innervation may not be wholly cholinergic is supported by the finding that a number of positive Hollander tests could be converted to negative by ~-adrenergic blockade (28). It has also been shown (29, 30) that catecholamines will stimulate gastrin release, and this release can be blocked by adrenergic blocking agents (29). Further evidence of complex vagal activity is that it has been suggested that receptive relaxation of the stomach induced by vagal stimulation uses a noncholinergi c nonadrenergic mechanism (31). In conclusion, this study suggests that the mechanism of vagal release of gastrin is noncholinergic, or if cholinergic, is highly resistant to atropine. ACKNOWLEDGMENT
We would like to thank Michael MacLcan, of the Jewish Hospital, Cincinnati, for his technical assistance. REFERENCES
1. Grossman MI: Neural and hormonal stimulation of gastric secretion of acid. Handbook of Physiology--Alimentary Canal, Vol. 2. CF Digestive Diseases, Vol. 20, No. 1 (January 1975)
CONTROL OF VAGAL GASTRIN RELEASE
Code (ed). Washington DC, American Physiological Society, 1967, pp 835-864 2. Uvnas B: The part played by the pyloric region in the cephalic phase of gastric secretion. Acta Physiol Scand 4: Suppl. XIII, 1942 3. McGuigan JE: Immunochemical studies with synthetic human gastrin. Gastroenterology 54:1005-1011, 1968 4. Lanciauh G, Bonoma C, Kaneman G, Brooks FB: Kinetics of gastrin release and degradation in response to electrical vagal stimulation in the dog. Proc Soc Exp Biol Med 142:740-743, 1973 5. Becker HD, Reeder DD, Thompson JC: Direct measurement of vagal release of gastrin. Surgery 75:101-106, 1974 6. Hansky J, Korman MG, Soveny C, St John DJB: Radioimmunoassay of gastrin: Studies in pernicious anaemia. Gut 12:97-101, 1971 7. Korman MG, Soveny C, Hansky J~ Effect of food on serum gastrin evaluated by radioimmunoassay. Gut 12:619-624, 1971 8. Nilsson G, Simon J, Yalow RS, Berson SA: Plasma gastrin and gastric acid responses to sham feeding and feeding in dogs. Gastroenterology 63:51-59, 1972 9. Walsh JH, Yalow RS, Berson SA: The effect of atropine on plasma gastrin response to feeding. Gastroenterology 60:16-21, 1971 10. Korman MG, Soveny C, Hansky J: Serum gastrin in duodenal ulcer. I. Basal levels and effect of food and atropine. Gut 12:899-902, 1971 1 I. Ardill J: The measurement of gastrin by radioimmuno~assay. PhD Thesis, Queen's University of Belfast, 1973 12. Sachs PH, Winn H J: The use of glutaraldehyde as a coupling agent for ribonuclease and bovine serum albumin. Immunochemistry 7:581-585, 1970 13. Gregory RA, Tracy H J, Grossman MI: Isolation of two gastrins from human antral mucosa. Nature 209:583, 1966 14. Yalow RS, Berson SA: Size and charge distinctions between endogenous human plasma gastrin in peripheral blood and hepatadecapeptide gastrins. Gastroenterology 58:609-615, 1970 15. Hunter WM, Greenwood FC: Preparation of iodine-131-1abelled growth hormone of high specific activity. Nature 194:495-496, 1962 16. Herbert V, Lau KS, Gottlieb CW, Bleicher SJ:
Digestive Diseases, VoL 20, No. 1 (January 1975)
Coated charcoal immunoassay of insulin. J Clin Endoer Metab 25 : 1375-1384, 1965 17. Hansky J, Soveny C, Korman MG: Role of the vagus in insulin-mediated gastrin release. Gastroenterology 63:387-391, 1972 18. Csendes A, Walsh JH, Grossman MI: Effect of atropine and of antral acidification on gastrin release and acid secretion in response to insulin and feeding in dogs. Gastroenterology 63:257263, 1972 19. Tepperman BL, Walsh JH, Preshaw RM: Effect of antral denervation on gastrin release by sham feeding and insulin hypoglycemia in dogs. Gastroenterology 63:973-980, 1972 20. Brooks AM, Stening GF, Grossman MI: Effect of gastric vagal denervation on inhibition of acid secretion by secretin. Am J Dig Dis 16:193-202, 1971 21. Becker HD, Evans JC, Reeder DD, Thompson JC: Duodenal acidification and antral release of gastrin. Arch Surg 180:201-207, 1974 22. Hansky J, Soveny C, Korman MG: Effect of secretin on serum gastrin as measured by immunoassay. Gastroenterology 61:62-68, 1971 23. Becker HD, Reeder DD, Thompson JC: Extraction of circulating endogenous gastrin by the small bowel. Gastroenterology 65:903-906, 1973 24. Robertson GR, Langlois K, Martin CG, Stezak G, Grossman MI: Release of gastrin in response to battering the pyloric mucosa with acetylcholine. Am J Physiol 163:27-33, 1950 25. Jackson BM, Reeder DD, Thompson JC: Dynamic characteristics of gastrin release. Am J Surg 123:137-142, 1972 26. Blair EL, Harper AA, Lake H J, Reed JD: The effect of atropine upon gastrin-stimulated acid gastric secretion. J Physiol 159:72-73, 1961 27. Gregory RA, Tracy H J: The preparation and properties of gastrin. J Physiol 156:523-543, 196l 28. Read RC, Thompson BW, Hall WH: Conversion of Hollander tests in man from positive to negative--~ adrenergic blockade with propanolol. Arch Surg 104:573-578, 1972 29. Hayes JR, Ardill J, Kennedy TL, Shanks RG, Buchanan KD: Stimulation of gastrin release by catecholamines. Lancet 1:819-821, 1972 30. Stadil F, Rehfeld JF: Release of gastrin by epi-
21
SMITH ET AL
nephrine in man. Gastroenterology 65:210-215, 1973 31. Martinson J: Vagal relaxation of the stomach--
22
Experimental reinvestigation of the concept of the transmission mechanism. Acta Physiol Stand 64:453-462, 1965
Digestive Oiseases, Voi. 20, No. 1 (January 1975)
