hormone, as first messenger, induces the formation of the second messenger, cyclic AMP, by activating the enzyme adenyl cyclase within the cell membrane.
Advances in Cyclic Nucleotide Research, Vol. I Raven Press, New York© 1972
Role of Cyclic AMP in the Regulation of Gastric Secretion in Dogs and Humans P.R. Bieck Division of Clinical Pharmacology, Vanderbilt University , School of Medicine, Nashville , Tennessee 37203
I. INTRODUCTION Since the discovery of cyclic adenosine 3',5'-monophosphate (cyclic AMP) in 1957, many hormones have been shown to influence their target tissue by increasing or decreasing the steady-state level of cyclic AMP in the cell. The hormone, as first messenger, induces the formation of the second messenger, cyclic AMP, by activating the enzyme adenyl cyclase within the cell membrane of the target cell (Robison, Butcher, and Sutherland, 1971). Alonso a nd Harris (1965) using isolated frog gastric mucosa demonstrated that gastric acid secretion and oxygen-consumption are stimulated equally by gastrin and by cyclic AMP (Harris and Alonso, 1965). Methylxanthines have the same effects (Harris, Nigon, and Alonso, 1969). The authors, therefore, suggested that cyclic AMP may be the intracellular mediator of gastric secretion in amphibians. Perfusion of the rat gastric mucosa! surface with cyclic AMP also stimula tes gastric acid secretion (Shaw and Ram well, 1968a,b ). Perrier and Laster (1970) described adenyl cyclase activity in stomach preparations of several mammalian species including man. Histamine and betazole, a histamine analogue, stimulated adenyl cyclase activity 3- to 4-fold in the parietal mucosa of the guinea pig, but gastrin was without effect. In contrast, Nakajima, Hi rschowitz, and Sachs, (197 1), in experiments with an amphibian adenyl cyclase preparation found that pentagastrin, histamine, and also fluoride, were all active stimulators of the enzyme. It has been suggested that a ll of the various actions of cyclic AMP are mediated by a cyclic AMP-dependent protein kinase, and such a protein has been demonstrated in bovine gastric mucosa (Kuo. Krueger, Sanes, and Greengard, 1970). 149
150
ROLE OF C YCLIC AMP IN GASTRIC SECRETION
There is therefore some evidence linking the actions of histamine and gastrin to cyclic AMP in the case of gastric acid secretion. But until now, direct measurements of the gastric mucosa cell content of cyclic AMP after hormonal stimulation have not been made in man or any other ma mmalian species. Obviously some of the cyclic nucleotide escapes from its cells of origin into the extracellular fluid : plasma, urine, and cerebrospinal fluid (Butcher and Sutherland, 1962 ; Broadus, K ami nsky, Northcutt, Hardman, Sutherland, and Liddle, 1970). This rate of extrusion of cyclic AMP into the extracellular space seems to be an important factor in the regulation of the intracellular levels of the nucleotide (Sutherland, 1970). Therefore, both cyclic AMP and HCl-secretion were measured in the fluid secreted by the gastric mucosa after hormonal stimulation (Bieck. Oates, Robison, and Adkins, 1971). In this way cyclic AMP secretion and the physiological response to stimulation could be examined at the same time. In another series of experiments in the dog the effects of histamine on acid-secretion were compared with the secretion rate of cyclic AMP into the gastric juice, with simultaneous measurements of the mucosa! cyclic AMP content being made. The data to be presented will deal further with the effects of theophylline, insulin, and prostaglandin E 2 on the HCI and cyclic AMP secretion. Cyclic ti MP was measured by the protein-binding assay of Gilman (1970) after purification of the samples on Dowex-50 columns and lyophilization and expressed as picomoles of cyclic AMP secreted per min. Total acid output was determined by titration with sodium hydroxide and calculated as µmoles HCI secreted per min.
II. DOG EXPERIMENTS Adult mongrel dogs weighing between 10 and 15 kg were prepared either with Heidenhain fundic pouches, which have an intact blood supply, but no vagal innervation, or with a simple gastric fistula. Groups of 2 to 3 dogs were used for the experiments. After fasting for 24 hr, the animals had their gastric juice collected at 5 to 10 min intervals; the juice from all the dogs was pooled and the volume recorded. Having established that cyclic AMP is secreted under basal conditions into the gastric juice at a rate of 3.1 ± 0.5 pmoles/min ( n = 18), we tested the effect of food (100 g of canned meat) and of half-maximal acid-stimulating doses (ED 50 ) of pentagastrin and hista mine on the cyclic AMP secretion. The ED 50 was calculated from a dose-response curve for each dog. All three of the stimuli caused highly significant increases in cyclic AMP secretion above the basal amount secreted by the same dog for one hour (Fig. 1).
ROLE OF CYCl
14
12
CAMP
10
PICOMOLES
MIN.
8 6
4 2 0
cc
FIG. I. Secretion of cyclic AMF ccllected during the first hour ol controls and : food p < 0.0025; pe
Increasing doses of bot rlependent elevation of cyclic Histamine differs from pent relatively greater increase ir HCl (Fig. 2). These experiments alon· between cyclic AMP output < 3S the second messenger in ga a time-relationship between t "lcid output. Indirect evidenc '.!Ild acid secretion is presente Fig. 3 shows a time-res pentagastrin. The HCl secre 35 min after the start of the A.M P had already reached its min later it had fallen to abou ihe pentagastrin infusion. Du and HCI output declined to c
RIC SECRETION ROLE OF CYCLIC
'1e actions of hist . :d . amme secretwn. But until ontent of cyclic AMP "a or ~ . any orher mamlllalk:, -apes_from its cells of ori ~ospmaJ fluid (Butcher -~ ardma = n, utherland ac.:: 1to t~e extracellular ;pz~ the in tracelluiar levels ,.
s
'.ion Were measured . o~aJ stimulation (Bi~ :lie AMP secret1on . a1H! . examined at the saml:' effects of histamine ~ >f eyel'ic AMP into £he~ •mucosa! cyclic AMP ·urther with the effects HCI and cyclic AMP
ig assay of Gilman Jmns and lyophiliza1 per min. Total acid •xide and calculated
AMP IN GASTRIC SECRETION
151 14 12
CAMP 10 n=60
PICOMOLES MIN. -
n=66
8 6 n =II
4
2
n=IB
o-;~::-:--~:-::-L....L--1-L__J'._ CONTROL
FOOD PENTAGASTRIN HISTAMINE
'
SAMPLES
'v------~' COLLECTED DURING
lat HOUR OF STIMULATION
FIG. I. Secretion of cyclic AMP into astri . . . . collected during the first hour of stimu~af c Juice of the He1denha10 dog. Samples were controls and · food p < 00025. . ion. 11 = number of samples. Difference between · · • pentagastnn P < 0.0005; histamine p < 0.0005.
Increasing d~ses of bo~h histamine and pentagastrin produced a dosedepend~nt el~vat1on of cyclic AMP and hydrochloric acid secretion (Fig. 2).
vere prepared either load supply, but no of 2 to 3 dogs were als had their gastric logs was pooled and er basal conditions = 18), we tested the 1al acid-stimulating jc AMP secretion. each dog. All three lie AMP secretion our (Fig. 1).
Histamme differs from pentagastrin in that at the higher doses there is a relatively greater increase in the output of cyclic AMP as compared with HCl (Fig. 2). These experiments alone do not establish a definite causal relationship between cyclic AMP output a nd HCI secretion. In order to accept cyclic AMP as the second messenger in gastric acid secretion it is necessary to demonstrate a time-relationship between the rise of cyclic AMP secretion and an increased acid output. Indirect evidence for a causal relationship between cyclic AMP and acid secretion is presented in Figs. 3, 4, and 5. Fig. 3 shows a time-response curve after stimulation with an ED 5 0 of pentagastrin. The HCl secretion almost reached the maximum level about 35 min after the start of the infusion. However, 15 min after the sta rt, cyclic AMP had already reached its maximal secretion rate of 25 pmoles per min ; 20 min later it had fallen to about 17 pmoles, and this was maintained to the end of the pentagastrin infusion. During the control-period (saline) both cyclic AMP and HCl output declined to control values wi thin 30 min.
152
ROLE OF C YCLJC AMP IN GASTRIC SECRETION
4
3 CAMP picomoles min.
2
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400
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300
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.... ...
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ROLE OF
It has been know: gastric acid secretion :- y stimulate HCI s1 , obertson, Rosiere, I
200 H_moles
30
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HISTAMINE (mg/hr. l.V. l CA M P pi comoles m in
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CAMP picomoles min.
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HCI }:!mole s min.
10
20 0
0 45
90
180
0
PENTAGASTRIN ( µg / hr. l.V. l FIG. 2. Dose-response curves to histamine and pentagastrin in 2 different Heidenhain dogs. O rdinate: pmoles/ min of cyclic AMP secreted into the gastric ju ice d uring I hr. Ordinate: 11moles/ min of HO secreted into the gastric juice during I hr. Abscissa: concentrations of hista mine or pentagastrin infused per hour. Each point represents the mean of 3 to 6 samples.
After histamine stimulation, a similar effect was seen (F ig. 4 and Fig. 5~ In the denervated Heidenhain-pouch dog (Fig. 4) and in the innervated gastric fistula dog (Fig. 5) histamine (ED 50 ) caused a rapid rise in cyclic AMP secretion_ reaching its maximum level between 10 and 20 min after starting the infusion. HCI secretion, in contrast, reached maximum values in both cases after 30 min. G astric mucosa! biopsies, taken from the fundus of the stomach at the same times as the gastric juice was collected, showed a more gradual increase in the amount of cyclic AMP than did the gastric juice samples (Fig. 5). But onoeagain it was noted that the maximal increase in cyclic AMP levels in the mucoS2 (30 min after start of the infusion) preceded the peak rate of HCl secretion. The change in the secretion rate of cyclic AMP into the gastric juice appears earlie7 and is much greater (about 5- to 10-fold) than the 2-fold increase in tiss~ levels which was observed.
• 3. Time-respo nse c1 P secretion (pmoles/ rr - · · n (ED 50 for 60 mi the mean of 4 s
~ts
T
.:.LE I. Effect of l.v. ad AMP st
:mtamine + thco)··· h :Seance
SECRETION
>O
153
It has been known for a long time that methylxanthines are stimulato rs of gastric acid secretion in man (Roth and Ivy, 1944). In the dog, these derivatives onl y stimulate HCI secreti on when given together with histamine or food (Robertson, Rosiere. Blickenstaff. and Grossman, 1950). The enzyme phos-
HCI 1
ROLE OF C YCLIC AMP IN GASTRIC SECRETION
~moles
min. CAMP
~
C AMP picomol e s
20
100
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(
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fa theophylline infusion gastric secretion of cyclic
30
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GASTRIC JUICE CAMP
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,
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Ordinates : 0-0 cyclic :cissa: histamine infusion single point represents the
tion of the values for uice on stimulation is :he maximum increase 0 min later than that .how the potentiating cyclic AMP secretion. e ED 50 of histamine 0.5 mg/kg/min. Histumoles HCJ and from ase of 40 µmoles HCl ;>eriod, when the dogs
30
60
MINUTES FIG. 5. Time-response curve to histamine in 3 total pouch dogs. Upper part : • - • cyclic AMP (pmoles/min) and 0-0 HCI (µmoles/ min) secreted into the gastric juice. Each single point represent~ the mean of 5- 7 samples. All values were significantly different (p < 0.05) from the controls. Lower part: bars represent the mean and the S.E.M. of the gastric mucosa) cyclic AMP content (5- 7 biopsies). Abscissa: histamine-infusion (ED 50) for 60 min.
were stimulated in the presence of theophylline, the effect of histamine was markedly enhanced, with a net increase of 195 µmoles HCI and 12.6 pmoles cyclic AMP per min. The potentiation by theophylline which produced an increase of 155 µmoles HCl and 6.4 pmoles cyclic AMP is significant for both parameters. In other experiments we found that stimulation with food, which is thought to exert its effect on gastric secretion in the Heidenhain dog via endogenously released gastrin, was also significantly potentiated by theophy1line. In 1968 Robert described the gastric antisecretory properties of several prostaglandins in the dog. The mechanism of this action is still largely unknown. Jacobsen (1970) demonstrated in dogs with a gastric fistula that the ratio between mucosa! blood flow and secretion rate fell with norepinephrine, but did
156
ROLE OF CYCLIC AMP IN GASTRIC SECRET/Ol'./
,,• - +
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THEOPHYLLINE
I \ I I I \ I \ NET INCR EASE CAMP
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% OF
CONTROL
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I
SECRETION
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HISTAMINE
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picomoles min .
ROLE
RATE
o I
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·---· I 0
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60
MINUTES F IG. 6. Effect or theophylline on histamine-stimulated cyclic AMP secretion into the gastric juice or one Hcidenhain dog. Ordinate: Net increase o r cyclic AMP secretion into the gastric juice. 0-0 histamine- controls: • - • histamine + theophylline (0.5 rng!kg·rnin) - theophyll ine (0.5 mg/kg/min) alone. Abscissa: histamine infusion IED 50) for 60 min. Each point represents the mean or 2 samples.
not change with prostaglandin E 1 (PGE 1 ). He concluded that the blood flow changes with prostaglandin were secondary to the inhibition of acid secretion. Studies by Shaw et al. ( 1968 a,b) and by Way and Durbin ( 1969) also indicate a direct action on the secretory mechanism. Way and Durbin found, in the isolated bullfrog mucosa, that PGE 1 inhibited the gastrin- and histamineinduced acid secretion, but did not block the stimulatory effect of cyclic AMP. This wou ld suggest that PG Et may act by preventing the formation of cyclic AMP, possibly by an inhibitory effect on adenyl cyclase. Shaw and Ramwell. in contrast, described an inhibitory effect of PGE 1 (in perfused rat stomach mucosa! s urface in vivo) on hydrogen-ion secretion caused by histamine, pentagastrin, cyclic AMP, and theophylline. This suggests that prostaglandin may also inhibit the action of 1he nucleotide. To make the situation even more complex, Perrier et al. (1970) found that PGE 1 stimulated guinea pig adenyl cyclase activity nearly as much as histamine did. In view of the demonstrated relationship between mucosa] and gastric juice cyclic AMP it appeared that it should be possible to detect any stimulatory effects of prostaglandiu on the cyclic AMP secretion rate in the experimental models used in this study. From Fig. 7 it can be seen that prostaglandin E 2
flG. 7. Effect or PGI Hadenhain dogs. Hist. 3h!es obtained I hou tarted was taken as I ~rval with histamine :-ate (ordinate). • - I ·~nts the mean ol bols = p < 0.05 It-
?GE.J, infused for < ......;sed a significant
=m compared to ion after an ini 3bout 40° 0 of th -- taglandin infusi< .:ereas the H Cl sec AMP levels ha< . These results ar· r - nting the form< with PGE 1 an ~lectrolyte
secret
'£CRETION
ROLE OF CYCLIC AMP IN GASTRIC SECRETION
150
THEOPHYLLINE
•
157
p < 0.05
o ns
% OF
HCI
I
CONTROL SECRETION
• I'
100
RATE
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7~
• - CAMP /
·-
__ . } PGE2
\ / ~---•/- HCI
50
)50
60
HISTAMINE EDso
0 0
30
60
90
120 150
MINUTES secretion into the gastric secretion into the gastric e (0.5 mg/kg/ min) - theo,J for 60 min. Each point
:d that the blood flow tion of acid secretion. in (1969) also indicate Durbin found, in the stria- and histamine. effect of cyclic AMP. ie formation of cyclic . Shaw and Ramwell, perfused rat stomach :aused by histamine, ts that prostaglandin ~ situation even more ed guinea pig adenyl
mucosaJ and gastric etect any stimulatory : in the experimental iat prostagJandin E 2
FIG. 7. EfTect of PGE 1 on histamine-stimulated gast ric acid a nd cyclic AMP secretion in 4 Heidenhain dogs. Histamine infusion (ED sol was continued for 4 hours. The mean of all acid values obtained I hour (-60 to 0 min) before the prostaglandin-infusion (I µg/kg/min) was started was taken as 1 00 ~ 0. 100 ~~ secretion rate of cyclic AMP was the value at each time interval with histamine stimulation alone. The changes were expressed as %of control secret io n rate (ordinate). • - • HCI (JJmoles•min). • - • cyclic AMP (pmoles/min). Each point represents the mean of 6-8 sam ples. Open symbols a re not significant from controls. filled symbols = p < 0.05 (t-test for paired values).
(PGE 2 ), infused for one hour at a rate of 1 JLg/kg/ min, together with histamine. caused a significant depression of the amount of cyclic AMP secreted per min when compared to the cyclic AMP levels after histamine alone. The HCl secretion after an initial (nonsignificant) increase was reduced at the same time to about 40" o of the control secretion rate. Thirty min after stopping the prostaglandin infusion, the cyclic AMP secretion rate had begun to increase. whereas the HCI secretion was still depressed. At the end of the experiment. cyclic AMP levels had reached initial values again, but the acid output remained low. These results are consistent with the hypothesis that PGE2 could act by preventing the formation of cyclic AMP by adenyl cyclase. Further experiments with PGEt and gastric biopsies during these experiments are currently under way. In 1966 Hirschowitz and Robbins described an inhibition of gastric acid and electrolyte secretion by insulin in the dog, which could not be prevented
158
ROLE OF CYCLIC AMP IN GASTRIC SECRETION
by correction or prevention of hypoglycemia. Infusion of potassium chloride completely reversed the insulin-inhibition (Hirschowitz and Sachs, 1966). This inhibitory effect of insulin on HCl secretion could also be demonstrated in isolated guinea-pig gastric mucosa, suggesting a direct effect of insulin on the electrolyte-secreting mechanisms of the stomach (Hirschowitz and Robbins, 1966). It has been demonstrated that insulin is able to produce a dramatic fall in the intracellular level of cyclic AMP when added to intact fat cells (Butcher et al., 1966). The data presently available are equally compatible with inhibition of adenyl cyclase, or activation of phosphodiesterase or stimulation of cyclic AMP effiux. It was therefore of interest to see what effect insulin would have on the cyclic AMP secretion into the gastric juice, when injected into a Heidenhain dog. Thirty min after a single intravenous injection of 0.5 U insulin per kg in dogs already stimulated with histamine, both cyclic AMP and HCI secretion were significantly depressed. As Fig. 8 shows, the acid output continued to fall for 120 min. At the end of the experiment both cyclic AMP and HCI secre-
100
--
/
~/
/
HCI
o---o-
ROLE OF CYC
Hon were still significantly does not act by promoting any lowering of the tissue cc should reveal this.
III. Gastric juice is relative: in it should permit better def secretion in the human. In two patients who ha· agus, thus ruling out saliva tStefanovich and Wells, 197; 101 pmoles per min were fo1
