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Effects of cimetidine, atropine and pirenzepine on basal and stimulated gastric acid secretion in the rat
European Journal of Pharmacology, 138 (1987) 225-232
225
Elsewer EJP 00787
Effects of cimetidine, atropine and pirenzepine on basal and stimulated gastric acid secretion in the rat M. Ekelund 1, R. H~kanson 2,, and S. Vallgren 1 Departments of ] Surgery and "-Pharmacology, Unwerstty of Lund, Lund, Sweden Recexved 11 September 1986, revised MS received 16 December 1986, accepted 24 March 1987
The gastric ant~-secretagogue effects of clmetxdme (a histamine H2-receptor antagomst) and of atropine (a non-selecUve muscarlnic receptor antagomst) and p~renzepme (a selective muscarlmc Mx-receptor antagomst) were examined in conscious gastric fistula rats both under basal condiuons and after stimulauon with maxamal doses of pentagastrin and histamine. Cimeudme blocked basal as well as sUmulated acid secretion. The c~metldine dose-response curves and the calculated EDso values were s~malar in the different experimental SltUaUons. Atropine blocked equally effecuvely the basal and the stimulated acid secreuon. The antisecretagogue and pupal ddatmg effects were compared. The EDs0 values for the anU-secretagogue effect and for the pupal dilating effect were m the same range though not ldenucal. P~renzepme blocked acid secretion, whether basal or sUmulated, with s~milar potency. It was much more potent to block acid secretion than to cause pupd dilatation The greater potency of p~renzeplne to block acid secretion than to cause pupil ddatat~on suggests that the chohnerg~c pathway of acid secretion revolves neuronal muscarimc M~-receptors within the intramural gangha of the stomach wall. In conclusion, clmetldme, atropine and pirenzepme effectwely blocked basal as well as pentagastrin- and tustamine-sumulated acid secretxon, indicating that both bastamine and acetylcholine are ~mportant m the control of the parietal cell. Histamine has been cla~med to be the final common chermcal mediator of acid secretion Thts wew is at odds w~th the fact that muscannic blockang agents also inhabit basal and stimulated acid secreUon. H2-receptor antagomsts; Atropine; Plrenzepine; AnUchohnerg~c drugs, Gastric acid secretion
1. Introduction Histamine H2-receptor blocking agents antagonize the effects of histamine on gastric acid secretion in a competitive manner (Black et al., 1972). However, histamine Hz-receptor antagonists inhibit not only the histamine-stimulated acid secretion but also the basal, gastrin- and vagus-stimulated secretion (Grossman and Konturek, 1974; Carter et al., 1974; Sch/5/Sn and Olbe, 1977; Uvn~is-Wallensten and Andersson, 1977; Olbe et al., 1978; Daly et al., 1981). As a result two * To whom all correspondence should be addressed Farmakologxska lnstltUtlonen, S61vegatan 10, S-223 62 Lund, Sweden
concepts have evolved, both of which place histamine m the role of a major physiological excitant of the parietal cells (cf. Black et al., 1978). One concept links gastrin to histamine by postulating that the parietal cells possess receptors for both gastrin and histamine interacting in such a way that occupation of histamine receptors permits gastnn to act (Grossman and Konturek, 1974; Soll, 1978). According to the other concept the parietal cells would have receptors for histamine only and gastrin (and acetylcholine) would act by releasing histamine from a local gastric store (Macintosh, 1938; Code, 1965; Kahlson et al., 1964; 1973; Kasbekar et al., 1969; Svensson, 1973; Rangachari, 1975; Bergqvist and Obrink, 1979; Bergqvist et al., 1980; Ekblad 1980, 1985; Angus
0014-2999/87/$03 50 © 1987 Elsevier Science Pubhshers B V (Biomedical Dxvxslon)
226
and Black, 1982; Black et al., 1985). This latter concept explains the general inhibitory effect of H2-receptor antagonists but does not explain why anticholinergic agents also inhibit both basal and stimulated acid secretion (cf. Brodie, 1966; Johansson et al., 1971; Szelenyi, 1980; HS.kanson et al., 1981). It has recently been suggested that the acetylchohne receptor important in acid secretion is present in neurons in the ganglia of the gut wall rather than within parietal cell membranes (see Hammer 1980; 1982; Rosenfeld, 1982; Feldman, 1984). This suggestion is based on studies with plrenzepine, an antlmuscannic agent which discriminates between two distinct subclasses of muscarlnic receptors. Those receptors having high affinity for pirenzepine are termed M 1 and those displaying a lower affinity for the drug are denoted M 2. Unlike pirenzepine, atropine blocks both types of muscarinic receptors with equal effectiveness. Histamine has been claimed to act as the final common mediator of acid secretion, based on the well-documented ability of H2-receptor antagonists to block both basal and stimulated acid secretion (Svensson, 1973; see also Ekblad, 1985). However, according to the same reasoning acetylcholine could be a candidate as final common mediator xf anticholinergics also inhibit basal and stimulated acid secretion. These two claims are obviously in conflict. The complication would not arise if it could be shown that anticholanergics inhibit acid secretion by mechanisms not involving muscarinic receptors. Realizing this, Johansson et al. (1971) suggested that atropine acted in a non-specific way to suppress acid secretion. Previous studies on the acid inhibitory effects of atropine in conscious rats involved no attempts at characterizing the receptor involved (Johansson et al., 1971; Hgtkanson et al., 1981), and studies with pirenzeplne either involved no attempt to characterize the receptor (Okabe et al., 1982) or were done with pylorus-ligated rats rather than fistula rats (Parry and Heathcote, 1982). The present report therefore analyzes the effects of atropine and pirenzepine and of one histamine H2-receptor antagonist (cimetidine) on basal as well as histamine- and pentagastrin-stimulated acid secretion
in the conscious gastric fistula rat. The antisecretagogue effects of atropine and pirenzepine were compared with their pupil dilating effects.
2. Materials and methods
Male Sprague-Dawley rats (200-270 g body weight) obtained from the same breeder were fed a standard diet of rat food pellets and tap water. Plastic gastric fistulas (Bel et al., 1966) were implanted in the rumen of 149 rats, permitting repeated studies of gastric acid secretion in the same group of rats for periods of up to 5 or 6 weeks. Each rat was also fitted with an indwelling chronic jugular vein catheter. The rats were placed in Bollman-type restraining cages for a few hours daily for at least two weeks in order to familiarize them with the experimental conditions. They were fasted for 24 h before acid secretion experiments. After the rats had been immobilized in the restraining cages, the gastric fistulas were opened and the stomachs were rinsed with warm saline and drained for 1 h. In studxes on the effects of anti-secretagogues (cimetldine, atropine or pirenzepine) on basal acid secretion, gastric juice was collected in 1 h portions for 2 h, the antisecretagogue was then admimstered and the collection of gastric juice was usually continued for 4 h. The acid secretion collected during the second hour of basal secretion was used as baseline for evaluating the effect of the anti-secretagogue. The protocol in studies of the anti-secretagogue effect upon stimulated acid secretion was as follows: after 2 h of basal secretion, pentagastrin (Peptavlon~; 'ICI, Macclesfield, England) (10 /~g/kg per h) or histamine dihydrochloride (BDH, Poole, England) (3 m g / k g per h) in saline was infused intravenously (i.v.) at a constant rate of 2.9 m l / h and acid secretion was collected in 1 h periods for 6 h. The doses used were previously found to give a maximal acid response (Vallgren et al., 1983). The acid output during infusion of pentagastrin or histamine reached a stable plateau for a minimum of 6 h (Ekelund et al., 1984). The anti-secretagogue was given after 1 h of basal and 1-2 h of stimulated secretion; the collection of gastric juice was continued for 3-4 h and the effects of the
227 anti-secretagogues were expressed as inhibition of the stimulated acid output (corrected for basal secretion unless otherwise stated). Atropine s u l p h a t e ( A C O , S t o c k h o l m , Sweden) and pirenzepine dihydrochlonde (Dr. Karl Thomae G m b H , Biberach/Riss, F R G ) were given by continuous i.v. infusion along with either histamine or pentagastrin and the dose of anti-secretagogue was increased stepwlse every hour. A total of 4-5 doses were given. Cimetidine hydrochloride (Smxth, Kline & French, Wellwyn Garden City, England) was injected intraperitoneally (i.p.). Only one dose was given to each rat. The doses of the anti-secretagogues refer to the weight of the free base. Acid output was determined by titration with 0.02 N N a O H with phenolphthalein as indicator. In the atropine- and pirenzepine-treated rats the pupil diameter was assessed at the end of each 1 h period by photographic recording (Nikonmat EL with two extension rings PK-2) of the pupil under standardized light conditions and subsequent measurement of the diameter on the print. Unless otherwtse stated, dilatation was expressed as the change in pupil diameter (basal value subtracted). The relative potency of clmetidine, atropine and pirenzepine in the various experiments was assessed from dose-response curves by calculation
of the EDs0 value (the concentraUon giving half maximal inhibition) by linear regression analysis (least square method) of each log dose-response curve using a response interval of 20-80% of the response range. The mean EDs0 value and S.E.M. for each experimental group were calculated. The significance of differences between EDs0 values was assessed by means of Student's t-test for unpaired observations.
Acid secret=on %of BAO 100 -
Acid secretion %of MAO 100
50-
Acid secretion % of MAO 100" ~ ~ 1 3 171
ED50 =35".13 (6)
3. Results
3.1. Effect of ctrnettdine The dose-response curves presented in fig. 1 show clmetidine to be equally potent in blocking basal, pentagastrin- or histamine-stimulated acid secretion, as reflected by comparable EDs0 values (fig. 1). Each rat received one dose of cimetidine i.p. and the maximal attenuation of acid secretion was noted for each dose and was used for calculating the EDs0.
3.2. Effect of atropine Atropine inhibited basal and pentagastrin- as well as histamine-stimulated acid secretion with
50, ~ 7 2 . - 1 8 ( 5 ) 50o
0
~
; 1'0 30 100 Cimetldme mg/kg
O-
1
3 10 3'0 100 Cimehdme rng/kg
0
i
3 10 30 100 Clrnehdme r~g/kg
Fig. 1 Clmetldme dose-response curves showing the mtubmon of acid secretion m conscious gastric fistula rats. (A) Basal secretxon, (B) pentagastnn-stlmulated secretion (increase over basal), (C) lustarmne-stimulated secreuon (increase over basal). The secretagogues were refused xv for 6 h. Cmaetldine was injected 1.p. (one dose per ammal) after 2 h of pentagastnn of lustanune infusion. Acad secretion before clrnetldine was taken as 100 and the effect of clrnetldme dunng the hour of maximal mtubxtlon (the first or second hour) was calculated and expressed as % of the starting value. BAO, basal acid output, MAO, maximal acxd output (corrected for basal secretion). Each value zs the mean of 6-8 experiments. Verttcal bars show the S.E.M. The EDs0 values are gwen as rng/kg body weight; they do not differ slgmficantlybetween the expertrnental groups.
228
/k Pupil diameter
Pupul
dmmeter (ram) o ~ .4
Acid secretion
%of BAO
Acid secretion
(ram)
0
/
Acid secrehon %of MAO 0
B
100
~I
3 2
5I" ~"
~ Pupil
dmrneter (ram) 0 -4
100
-3 -2
50.
.1 r
,
,
,
10
100 1000
Atropine
lug/ kg / h
0
.1
.
1 10
100 1000
Atropme,pg
0
/ kg / h
0"
1'0 100 1000 0 Atropine/ug/kg/h
Fig 2 Cumulatave atropine dose-response curves showang the inhibition of acid secretion and the pupil dflataUon m conscious gastrac fistula rats (A) Basal secreUon, (B) pentagastrm-stlmulated secreUon (increase over basal), (C) htstanune-sUmulated secretion (Increase over basal). The secretagogues were refused LV. for 7-8 h Atropine was gaven 1.v after 1-2 h of pentagastrln or histamine infusion The atropine dose was increased stepwlse 3 or 4 times. Each dose was adrmnlstered for 1 h. Acid secretxon before atropine was taken as 100 and the effect of each dose of atropine was calculated and expressed as % of the starting value BAO, basal acid output, MAO, maxamal acid output (corrected for basal secretion) The pupd dmmeter was measured and the dllatauon (change in dmmeter) was expressed m m m Each value xs the mean of 8-15 experiments Vertxcal bars show the S.E.M
TABLE 1 EDs0 values for atropine for antl-secretagogue effect and pupd ddataUon m conscious fistula rats The EDso values 5: S.E.M were calculated by regressaon analys~s from the dose-response curves m fig 2 The results give the mhabmon of basal and sumulated acid output corrected for basal secretion, or of pupil ddatatlon with the basal value subtracted Values m parentheses were calculated from dose-response curves of acid mtub~t~on uncorrected for basal secreuon or of pupd dilatation uncorrected for the basal pupd dmmeter There was no significant difference between EDs0 values for pupil dilatation m the various groups. There was a s~gmfcant difference, however, between EDs0 values when the antl-secretagogue effect of atropine was compared m tustarrune-st~mulated and non-sUmulated rats (P < 0 001), and moreover, there was a significant difference between the EDs0 values for antl-secretagogue and pupil dilating effect of atropine m non-sumulated rats (P < 0 001) (Student's t-test) Secretagogue
EDso for antl-secretagogue effect (/Lg/kg per h)
EDs0 for pupd dilatation ( / t g / k g per h)
None Pentagasmn Histamine
415:14 n=15 6 8 5:2 1 (20 3 5 : 4 7) n = 8 14 5 + 2 2 (42 0 + 11 4) n = 10
2 0 0 + 1 6 ( 1 6 0 + 0 8 ) n =11 27 1 5:2.3 (24 8 5:2 8) n = 10 29.8 5:3 2 (21 1 + 3 1) n = 9
TABLE 2 EDso values for plrenzepme for the ant~-secretagogue effect and pupd dllatauon in conscious fstula rats EDs0 values 5: S.E.M were calculated from the expenments shown m fig 3 For details of the calculations see table 1. There was no statistically slgmflcant difference m EDs0 values between the basal and the sUmulated groups with respect to either anU-secretagogue effect or pupil ddatatlon The EDs0 values for antl-secretagogue effects &ffered greatly from those for pupil ddatatlon m all three groups (P < 0 001) (Student's t-test) Secretagogue
EDso for ant~-secretagogue effect ( m g / k g per h)
EDs0 for pupal ddatatlon ( m g / k g per h)
None Pentagastrln Histamine
0 057 5:0 030 n = 11 0095+0059(028 +011) n=7 0 036 + 0 011 (0 086 + 0 038) n = 6
5 9 + 0 2 (3.7 + 0.1 ) n = 6 56+02(34+01) n=6 6 8 + 0 4 (3 9 5: 0.2) n = 6
229 A Pupd
Amd secretion % of BAO
diameter
Z~ Pupil dmmeter (ram} O
secretion
(mini 0
O
Acid % of MAO 100"~
1OO -
A Pupil dmmeter
Acid
secretion
(ram)
% of MAO
O
100" '3
"3
2
,2
50
50-
50-
it
.1 .0
O. O01 O1
1
10
100
Pirenzepme m g / k g / h
0
~ 0.01 01
-0 1
10 100
Pwenzepme m g / k g / h
,1
0-
O 001 01
1
10 100
Pirenzepme m g / k g / h
Fig 3 Cumulative pirenzeplne dose-response curves showing the inhibition of acid secretion and the pupil dilatation xn conscious gastric fistula rats. (A) Basal secretion, (B) pentagastrin-stlmulated secreuon (over basal secretion), (C) histarmne-stxmulated secreuon (over basal secretion) The secretagogues were infused i v. for 7-8 h Plrenzeplne was gwen i v after 1-2 h of pentagastnn or histamine infusion. The pxrenzeplne dose was increased stepw~se 3 or 4 times Each dose was administered for 1 h. Acid secretion prior to plrenzeplne was taken as 100 and the effect of each dose of plrenzepine was calculated as % of the starting value BAO, basal acid output, MAO, maximal acid output (corrected for basal secretion) The pupd dmmeter was measured and the ddatatlon (change in diameter) was expressed in mm Each value is the mean of 6-11 experiments Vertical bars give the S E M
about the same potency, as reflected by their similar (but not Identical) EDs0 values (fig. 2 and table 1). The design of these experiments was such that cumulative dose-response curves were obtained; each dose was administered i.v. for 1 h. A total of 4-5 doses were gwen to each rat. The dose-response curves for pupil dilatation were notably steep compared with those for anti-secretagogue effect but the EDs0 values for blockade of acid secretion and for dilatation of the pupal were m the same range (table 1).
3.3. Effect of plrenzepme Pirenzeplne inhibited basal, pentagastrin- or htstamme-stlmulated acid secreUon with similar potency (fig. 3). The design of these experiments was identical to that described for the atropine experiments. Pirenzepine was much more potent to block acid secretion than to block the muscarinic receptor of the iris sphincter muscle as is evident from a comparison of the widely differing ED50 values (table 2).
4. Discussion
Clmetldlne blocked basal, histamine- and pentagastrin-stimulated acid secretion in conscious
gastric fistula rats with the same potency. The available evidence suggests that this and other H2-receptor antagonists interact selectively with the Hz-receptors of the parietal cells and do not suppress the secretory process by non-specific means (Tepperman et al., 1979). Thus, the involvement of histamine in both basal and gastrin-stimulated acid secretion seems beyond dispute. Atropine, a non-selective anti-muscarlnic drug, also blocked basal, histamine- or pentagastrinstimulated acid secretion. The EDs0 values for acid inhibition and pupil dilatation were in the same range. These observations are in line with previous results for basal acid secretion reported by Brodle (1966) who administered antlcholinergtcs by the peroral or subcutaneous route. Hence, there appears to be no evidence to support the claim that atropine suppresses acid secretion through a nonspecific action (Johansson et al., 1971). Pirenzepine, a selective muscarinlc Mi-receptor blocking agent (Hammer et al., 1980), blocked basal and stimulated acid secretion with approximately the same potency. It was, however, more potent to block acid secretion than to cause pupil dilatation (see also Parry and Heathcote, 1982), indicating the involvement of M]-receptors in the process of acid secretion and Mz-receptors in the control of pupil size. The physiological regulation of gastric ac]d
230 secretion involves at least three effectors, gastrin, histamine and acetylcholine. The pathways whereby these three effectors influence the parietal cells have not been identified and even the precise localization of their respective receptors remains to be established. The present findings suggest a complex interaction of mechanisms involving H 2receptors and Ml-receptors in both basal and stimulated acid secretion. Histamine H2-receptors have been demonstrated on the parietal cells (Soil, 1978; 1979; 1980). Thus, histamine could act directly on these cells but could also act indirectly by exciting neurons in intramural ganglia (cf. Paton and Vane, 1963). The localization of the muscarinic Ml-type receptors and the gastrin receptors is unknown. There is some evidence that the parietal cells have gastrin receptors (Soil, 1980) but both M 1 receptors and gastrin receptors may, in addition, be associated with structures other than the parietal cells e.g. endocrine (enterochromaffin-like) cells, mast cells or intramural ganglia. Muscarimc receptors on parietal cells (Ecknauer et al., 1980) are thought to be of the M2-type (Rosenfeld, 1982). Conceivably, the pirenzeplnesensitive Ml-receptors are located in the intramural ganglia, winch are known to play a key role in the control of acid secretion (see e.g. HLkanson et al., 1980; Szelenyl, 1980; Alumets et al., 1982). The effect of pentagastrin was abolished by blockade of either H2-receptors or Ml-receptors. Consequently, if gastrln acts directly on the parietal cell it does so in concert with histamine and acetylcholine. If it acts indirectly it does so through the activation of both histaminergic and cholinergic pathways. If a similar reasoning is applied it appears that basal secretion also depends on histaminergic and cholinergic pathways. The vagal nerve plays a crucial part in the control of acid secretmn in the rat. Bilateral subdiaphragmatic vagal denervation in conscious fistula rats promptly lowers basal acid output to a fraction of the pre-denervatmn level and renders the parietal cells more or less unresponsive to circulating stimuli (Vallgren et al., 1983). The suppression appears to be permanent. However, vagal stimuli do not reach the parietal cells directly but are
amplified in the intramural ganglia (Stach et al., 1975). Hence, most if not all nerve endings acting on the oxyntic glands originate in intramural nerve cell bodies. Angus and Black (1982) showed the H2-receptor antagonists blocked the acid secretory response to electrical field stimulation of the perfused rat stomach, an effect suggesting that histamine was being released by excitation of intramural neurons. Since atropine also blocked the response to field stimulation, acetylcholine was probably being released too. The response to electrical stimulation was thought to be neuraUy mediated since it was abolished by the neurotoxic agent tetrodotoxin. It was thus concluded that electrical stimulation releases histamine locally, involving muscarinic receptors present within an as yet unidentified histamine-storing cell. Evidence to date indicates that the acid output in the rat depends upon intramural 'transducer' systems that respond to gastrin and vagal excitation. It is likely that the intramural ganglia represent such a transducer system. In the absence of a vagal input or following M 1- or H2-receptor blockade these intramural transducers cease to signal. As a result the parietal cells no longer respond to gastrin or to vagal excitation. The function of the parietal cells under basal as well as stimulated conditions appears to depend upon a normal impulse flow in intramural transducer systems and upon a continuous vagal drive. It may be argued that only experiments with isolated parietal cells will explain the physiological control of acid secretion. In our view this argument is not valid because there is mounting evidence that the physioloDcally important targets for gastrin, acetylcholine and histamine in the pathways of acid secretion are not the parietal cells only but intramural transducer systems represented by e.g., intramural neurons. Hence, the action and interactions of gastrin, acetylcholine and histamine must be studied in a bioassay system which responds to each of these agonists in ways which are compatible with their effects in intact animals. Only systems in which the mucosal architecture and nerve supply are intact realize this objective (see Black and Shankley, 1985).
231
Acknowledgements Grant support from the Swedish MRC (04X-1007), the PMalsson Foundation and the Medical Faculty, Umverslty of Lund, Lund, Sweden We are grateful to Professor C. Rerup for statistical assistance
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232 Rangachan, P K , 1975, Hlstarnlne release by gastric stxmulants, Nature 253, 53 Rosenfeld, G C., 1982, Pirenzepine (LS 519) A weak lntubltor aod secretion by isolated rat parietal cells, European J. armacol 86, 99 Schoon, I.-M. and L. Olbe, 1977, Effect of clmeUdme on chohnerg~c reflex stimulation of gastric acid secretion in duodenal ulcer patients, in Clmeudme Proc. Second Int Syrup Histamine HE-Receptor Antagomsts, eds. W L Burland and M.A. Slmkans (Excerpta Medxca. Amsterdam-Oxford) p. 207 Soil, A H , 1978, The Interaction of hastarmne with gastrln and carbamylchohne on oxygen uptake by isolated mammalian parietal cells, J Chn. Invest. 61,381. Soll, A H., 1979, Secretagogue interactions on the camne isolated parietal cell, In' Gastrms and the Vagus, eds J F Rehfeld and E Amdrup (Acadermc Press, London) p. 149. Soll, A.H., 1980, Secretagogue stimulation of l'~C-arrunopyrme accumulation by isolated canine parietal cells, Am J Physiol 238, G366. Stach, W, R. Weiss and R Radke, 1975, Lcht- und
~
elektronenmlkroskoptsche Untersuchungen uber die Nervengeflechte der Magenschlelmhaut und deren Bezaehungen zu Beleg- und Mastzellen, Dtsch. Z Verdau Stoffwechselkr 35, 205. Svensson, S.E, 1973, Mobilization of gastric mucosal hlstamane as related to gastric secretion, in. Int. Symp. Htstamane HE-Receptor Antagomsts, eds. C J Wood and M.A. Slmkms (Sm_lth, Khne & French) p. 361 Szelenyl, I, 1980, Chohnergtc components in the gastnn pathway of gastric acid stimulation, Expenentla 36, 973 Tepperman, B L, E D Jacobson and G C Rosenfeld, 1979, Hlstamme HE-receptors in the gastric mucosa: role in acid secretion, Life Sci 24, 2301 Uvnas-Wallensten, K and A Andersson, 1977, Effect of atropine and metlamlde on vagally-mduced gastric aod secretion and gastnn release m anaesthetized cats, Acta Physlol Scand 99, 496 Vallgren, S, M. Ekelund and R Hdlkanson, 1983, Mechamsm of inhibition of gastric acid secretion by vagal denervatlon m the rat, Acta Physlol Scand 119, 77
225
Elsewer EJP 00787
Effects of cimetidine, atropine and pirenzepine on basal and stimulated gastric acid secretion in the rat M. Ekelund 1, R. H~kanson 2,, and S. Vallgren 1 Departments of ] Surgery and "-Pharmacology, Unwerstty of Lund, Lund, Sweden Recexved 11 September 1986, revised MS received 16 December 1986, accepted 24 March 1987
The gastric ant~-secretagogue effects of clmetxdme (a histamine H2-receptor antagomst) and of atropine (a non-selecUve muscarlnic receptor antagomst) and p~renzepme (a selective muscarlmc Mx-receptor antagomst) were examined in conscious gastric fistula rats both under basal condiuons and after stimulauon with maxamal doses of pentagastrin and histamine. Cimeudme blocked basal as well as sUmulated acid secretion. The c~metldine dose-response curves and the calculated EDso values were s~malar in the different experimental SltUaUons. Atropine blocked equally effecuvely the basal and the stimulated acid secreuon. The antisecretagogue and pupal ddatmg effects were compared. The EDs0 values for the anU-secretagogue effect and for the pupal dilating effect were m the same range though not ldenucal. P~renzepme blocked acid secretion, whether basal or sUmulated, with s~milar potency. It was much more potent to block acid secretion than to cause pupd dilatation The greater potency of p~renzeplne to block acid secretion than to cause pupil ddatat~on suggests that the chohnerg~c pathway of acid secretion revolves neuronal muscarimc M~-receptors within the intramural gangha of the stomach wall. In conclusion, clmetldme, atropine and pirenzepme effectwely blocked basal as well as pentagastrin- and tustamine-sumulated acid secretxon, indicating that both bastamine and acetylcholine are ~mportant m the control of the parietal cell. Histamine has been cla~med to be the final common chermcal mediator of acid secretion Thts wew is at odds w~th the fact that muscannic blockang agents also inhabit basal and stimulated acid secreUon. H2-receptor antagomsts; Atropine; Plrenzepine; AnUchohnerg~c drugs, Gastric acid secretion
1. Introduction Histamine H2-receptor blocking agents antagonize the effects of histamine on gastric acid secretion in a competitive manner (Black et al., 1972). However, histamine Hz-receptor antagonists inhibit not only the histamine-stimulated acid secretion but also the basal, gastrin- and vagus-stimulated secretion (Grossman and Konturek, 1974; Carter et al., 1974; Sch/5/Sn and Olbe, 1977; Uvn~is-Wallensten and Andersson, 1977; Olbe et al., 1978; Daly et al., 1981). As a result two * To whom all correspondence should be addressed Farmakologxska lnstltUtlonen, S61vegatan 10, S-223 62 Lund, Sweden
concepts have evolved, both of which place histamine m the role of a major physiological excitant of the parietal cells (cf. Black et al., 1978). One concept links gastrin to histamine by postulating that the parietal cells possess receptors for both gastrin and histamine interacting in such a way that occupation of histamine receptors permits gastnn to act (Grossman and Konturek, 1974; Soll, 1978). According to the other concept the parietal cells would have receptors for histamine only and gastrin (and acetylcholine) would act by releasing histamine from a local gastric store (Macintosh, 1938; Code, 1965; Kahlson et al., 1964; 1973; Kasbekar et al., 1969; Svensson, 1973; Rangachari, 1975; Bergqvist and Obrink, 1979; Bergqvist et al., 1980; Ekblad 1980, 1985; Angus
0014-2999/87/$03 50 © 1987 Elsevier Science Pubhshers B V (Biomedical Dxvxslon)
226
and Black, 1982; Black et al., 1985). This latter concept explains the general inhibitory effect of H2-receptor antagonists but does not explain why anticholinergic agents also inhibit both basal and stimulated acid secretion (cf. Brodie, 1966; Johansson et al., 1971; Szelenyi, 1980; HS.kanson et al., 1981). It has recently been suggested that the acetylchohne receptor important in acid secretion is present in neurons in the ganglia of the gut wall rather than within parietal cell membranes (see Hammer 1980; 1982; Rosenfeld, 1982; Feldman, 1984). This suggestion is based on studies with plrenzepine, an antlmuscannic agent which discriminates between two distinct subclasses of muscarlnic receptors. Those receptors having high affinity for pirenzepine are termed M 1 and those displaying a lower affinity for the drug are denoted M 2. Unlike pirenzepine, atropine blocks both types of muscarinic receptors with equal effectiveness. Histamine has been claimed to act as the final common mediator of acid secretion, based on the well-documented ability of H2-receptor antagonists to block both basal and stimulated acid secretion (Svensson, 1973; see also Ekblad, 1985). However, according to the same reasoning acetylcholine could be a candidate as final common mediator xf anticholinergics also inhibit basal and stimulated acid secretion. These two claims are obviously in conflict. The complication would not arise if it could be shown that anticholanergics inhibit acid secretion by mechanisms not involving muscarinic receptors. Realizing this, Johansson et al. (1971) suggested that atropine acted in a non-specific way to suppress acid secretion. Previous studies on the acid inhibitory effects of atropine in conscious rats involved no attempts at characterizing the receptor involved (Johansson et al., 1971; Hgtkanson et al., 1981), and studies with pirenzeplne either involved no attempt to characterize the receptor (Okabe et al., 1982) or were done with pylorus-ligated rats rather than fistula rats (Parry and Heathcote, 1982). The present report therefore analyzes the effects of atropine and pirenzepine and of one histamine H2-receptor antagonist (cimetidine) on basal as well as histamine- and pentagastrin-stimulated acid secretion
in the conscious gastric fistula rat. The antisecretagogue effects of atropine and pirenzepine were compared with their pupil dilating effects.
2. Materials and methods
Male Sprague-Dawley rats (200-270 g body weight) obtained from the same breeder were fed a standard diet of rat food pellets and tap water. Plastic gastric fistulas (Bel et al., 1966) were implanted in the rumen of 149 rats, permitting repeated studies of gastric acid secretion in the same group of rats for periods of up to 5 or 6 weeks. Each rat was also fitted with an indwelling chronic jugular vein catheter. The rats were placed in Bollman-type restraining cages for a few hours daily for at least two weeks in order to familiarize them with the experimental conditions. They were fasted for 24 h before acid secretion experiments. After the rats had been immobilized in the restraining cages, the gastric fistulas were opened and the stomachs were rinsed with warm saline and drained for 1 h. In studxes on the effects of anti-secretagogues (cimetldine, atropine or pirenzepine) on basal acid secretion, gastric juice was collected in 1 h portions for 2 h, the antisecretagogue was then admimstered and the collection of gastric juice was usually continued for 4 h. The acid secretion collected during the second hour of basal secretion was used as baseline for evaluating the effect of the anti-secretagogue. The protocol in studies of the anti-secretagogue effect upon stimulated acid secretion was as follows: after 2 h of basal secretion, pentagastrin (Peptavlon~; 'ICI, Macclesfield, England) (10 /~g/kg per h) or histamine dihydrochloride (BDH, Poole, England) (3 m g / k g per h) in saline was infused intravenously (i.v.) at a constant rate of 2.9 m l / h and acid secretion was collected in 1 h periods for 6 h. The doses used were previously found to give a maximal acid response (Vallgren et al., 1983). The acid output during infusion of pentagastrin or histamine reached a stable plateau for a minimum of 6 h (Ekelund et al., 1984). The anti-secretagogue was given after 1 h of basal and 1-2 h of stimulated secretion; the collection of gastric juice was continued for 3-4 h and the effects of the
227 anti-secretagogues were expressed as inhibition of the stimulated acid output (corrected for basal secretion unless otherwise stated). Atropine s u l p h a t e ( A C O , S t o c k h o l m , Sweden) and pirenzepine dihydrochlonde (Dr. Karl Thomae G m b H , Biberach/Riss, F R G ) were given by continuous i.v. infusion along with either histamine or pentagastrin and the dose of anti-secretagogue was increased stepwlse every hour. A total of 4-5 doses were given. Cimetidine hydrochloride (Smxth, Kline & French, Wellwyn Garden City, England) was injected intraperitoneally (i.p.). Only one dose was given to each rat. The doses of the anti-secretagogues refer to the weight of the free base. Acid output was determined by titration with 0.02 N N a O H with phenolphthalein as indicator. In the atropine- and pirenzepine-treated rats the pupil diameter was assessed at the end of each 1 h period by photographic recording (Nikonmat EL with two extension rings PK-2) of the pupil under standardized light conditions and subsequent measurement of the diameter on the print. Unless otherwtse stated, dilatation was expressed as the change in pupil diameter (basal value subtracted). The relative potency of clmetidine, atropine and pirenzepine in the various experiments was assessed from dose-response curves by calculation
of the EDs0 value (the concentraUon giving half maximal inhibition) by linear regression analysis (least square method) of each log dose-response curve using a response interval of 20-80% of the response range. The mean EDs0 value and S.E.M. for each experimental group were calculated. The significance of differences between EDs0 values was assessed by means of Student's t-test for unpaired observations.
Acid secret=on %of BAO 100 -
Acid secretion %of MAO 100
50-
Acid secretion % of MAO 100" ~ ~ 1 3 171
ED50 =35".13 (6)
3. Results
3.1. Effect of ctrnettdine The dose-response curves presented in fig. 1 show clmetidine to be equally potent in blocking basal, pentagastrin- or histamine-stimulated acid secretion, as reflected by comparable EDs0 values (fig. 1). Each rat received one dose of cimetidine i.p. and the maximal attenuation of acid secretion was noted for each dose and was used for calculating the EDs0.
3.2. Effect of atropine Atropine inhibited basal and pentagastrin- as well as histamine-stimulated acid secretion with
50, ~ 7 2 . - 1 8 ( 5 ) 50o
0
~
; 1'0 30 100 Cimetldme mg/kg
O-
1
3 10 3'0 100 Cimehdme rng/kg
0
i
3 10 30 100 Clrnehdme r~g/kg
Fig. 1 Clmetldme dose-response curves showing the mtubmon of acid secretion m conscious gastric fistula rats. (A) Basal secretxon, (B) pentagastnn-stlmulated secretion (increase over basal), (C) lustarmne-stimulated secreuon (increase over basal). The secretagogues were refused xv for 6 h. Cmaetldine was injected 1.p. (one dose per ammal) after 2 h of pentagastnn of lustanune infusion. Acad secretion before clrnetldine was taken as 100 and the effect of clrnetldme dunng the hour of maximal mtubxtlon (the first or second hour) was calculated and expressed as % of the starting value. BAO, basal acid output, MAO, maximal acxd output (corrected for basal secretion). Each value zs the mean of 6-8 experiments. Verttcal bars show the S.E.M. The EDs0 values are gwen as rng/kg body weight; they do not differ slgmficantlybetween the expertrnental groups.
228
/k Pupil diameter
Pupul
dmmeter (ram) o ~ .4
Acid secretion
%of BAO
Acid secretion
(ram)
0
/
Acid secrehon %of MAO 0
B
100
~I
3 2
5I" ~"
~ Pupil
dmrneter (ram) 0 -4
100
-3 -2
50.
.1 r
,
,
,
10
100 1000
Atropine
lug/ kg / h
0
.1
.
1 10
100 1000
Atropme,pg
0
/ kg / h
0"
1'0 100 1000 0 Atropine/ug/kg/h
Fig 2 Cumulatave atropine dose-response curves showang the inhibition of acid secretion and the pupil dflataUon m conscious gastrac fistula rats (A) Basal secreUon, (B) pentagastrm-stlmulated secreUon (increase over basal), (C) htstanune-sUmulated secretion (Increase over basal). The secretagogues were refused LV. for 7-8 h Atropine was gaven 1.v after 1-2 h of pentagastrln or histamine infusion The atropine dose was increased stepwlse 3 or 4 times. Each dose was adrmnlstered for 1 h. Acid secretxon before atropine was taken as 100 and the effect of each dose of atropine was calculated and expressed as % of the starting value BAO, basal acid output, MAO, maxamal acid output (corrected for basal secretion) The pupd dmmeter was measured and the dllatauon (change in dmmeter) was expressed m m m Each value xs the mean of 8-15 experiments Vertxcal bars show the S.E.M
TABLE 1 EDs0 values for atropine for antl-secretagogue effect and pupd ddataUon m conscious fistula rats The EDso values 5: S.E.M were calculated by regressaon analys~s from the dose-response curves m fig 2 The results give the mhabmon of basal and sumulated acid output corrected for basal secretion, or of pupil ddatatlon with the basal value subtracted Values m parentheses were calculated from dose-response curves of acid mtub~t~on uncorrected for basal secreuon or of pupd dilatation uncorrected for the basal pupd dmmeter There was no significant difference between EDs0 values for pupil dilatation m the various groups. There was a s~gmfcant difference, however, between EDs0 values when the antl-secretagogue effect of atropine was compared m tustarrune-st~mulated and non-sUmulated rats (P < 0 001), and moreover, there was a significant difference between the EDs0 values for antl-secretagogue and pupil dilating effect of atropine m non-sumulated rats (P < 0 001) (Student's t-test) Secretagogue
EDso for antl-secretagogue effect (/Lg/kg per h)
EDs0 for pupd dilatation ( / t g / k g per h)
None Pentagasmn Histamine
415:14 n=15 6 8 5:2 1 (20 3 5 : 4 7) n = 8 14 5 + 2 2 (42 0 + 11 4) n = 10
2 0 0 + 1 6 ( 1 6 0 + 0 8 ) n =11 27 1 5:2.3 (24 8 5:2 8) n = 10 29.8 5:3 2 (21 1 + 3 1) n = 9
TABLE 2 EDso values for plrenzepme for the ant~-secretagogue effect and pupd dllatauon in conscious fstula rats EDs0 values 5: S.E.M were calculated from the expenments shown m fig 3 For details of the calculations see table 1. There was no statistically slgmflcant difference m EDs0 values between the basal and the sUmulated groups with respect to either anU-secretagogue effect or pupil ddatatlon The EDs0 values for antl-secretagogue effects &ffered greatly from those for pupil ddatatlon m all three groups (P < 0 001) (Student's t-test) Secretagogue
EDso for ant~-secretagogue effect ( m g / k g per h)
EDs0 for pupal ddatatlon ( m g / k g per h)
None Pentagastrln Histamine
0 057 5:0 030 n = 11 0095+0059(028 +011) n=7 0 036 + 0 011 (0 086 + 0 038) n = 6
5 9 + 0 2 (3.7 + 0.1 ) n = 6 56+02(34+01) n=6 6 8 + 0 4 (3 9 5: 0.2) n = 6
229 A Pupd
Amd secretion % of BAO
diameter
Z~ Pupil dmmeter (ram} O
secretion
(mini 0
O
Acid % of MAO 100"~
1OO -
A Pupil dmmeter
Acid
secretion
(ram)
% of MAO
O
100" '3
"3
2
,2
50
50-
50-
it
.1 .0
O. O01 O1
1
10
100
Pirenzepme m g / k g / h
0
~ 0.01 01
-0 1
10 100
Pwenzepme m g / k g / h
,1
0-
O 001 01
1
10 100
Pirenzepme m g / k g / h
Fig 3 Cumulative pirenzeplne dose-response curves showing the inhibition of acid secretion and the pupil dilatation xn conscious gastric fistula rats. (A) Basal secretion, (B) pentagastrin-stlmulated secreuon (over basal secretion), (C) histarmne-stxmulated secreuon (over basal secretion) The secretagogues were infused i v. for 7-8 h Plrenzeplne was gwen i v after 1-2 h of pentagastnn or histamine infusion. The pxrenzeplne dose was increased stepw~se 3 or 4 times Each dose was administered for 1 h. Acid secretion prior to plrenzeplne was taken as 100 and the effect of each dose of plrenzepine was calculated as % of the starting value BAO, basal acid output, MAO, maximal acid output (corrected for basal secretion) The pupd dmmeter was measured and the ddatatlon (change in diameter) was expressed in mm Each value is the mean of 6-11 experiments Vertical bars give the S E M
about the same potency, as reflected by their similar (but not Identical) EDs0 values (fig. 2 and table 1). The design of these experiments was such that cumulative dose-response curves were obtained; each dose was administered i.v. for 1 h. A total of 4-5 doses were gwen to each rat. The dose-response curves for pupil dilatation were notably steep compared with those for anti-secretagogue effect but the EDs0 values for blockade of acid secretion and for dilatation of the pupal were m the same range (table 1).
3.3. Effect of plrenzepme Pirenzeplne inhibited basal, pentagastrin- or htstamme-stlmulated acid secreUon with similar potency (fig. 3). The design of these experiments was identical to that described for the atropine experiments. Pirenzepine was much more potent to block acid secretion than to block the muscarinic receptor of the iris sphincter muscle as is evident from a comparison of the widely differing ED50 values (table 2).
4. Discussion
Clmetldlne blocked basal, histamine- and pentagastrin-stimulated acid secretion in conscious
gastric fistula rats with the same potency. The available evidence suggests that this and other H2-receptor antagonists interact selectively with the Hz-receptors of the parietal cells and do not suppress the secretory process by non-specific means (Tepperman et al., 1979). Thus, the involvement of histamine in both basal and gastrin-stimulated acid secretion seems beyond dispute. Atropine, a non-selective anti-muscarlnic drug, also blocked basal, histamine- or pentagastrinstimulated acid secretion. The EDs0 values for acid inhibition and pupil dilatation were in the same range. These observations are in line with previous results for basal acid secretion reported by Brodle (1966) who administered antlcholinergtcs by the peroral or subcutaneous route. Hence, there appears to be no evidence to support the claim that atropine suppresses acid secretion through a nonspecific action (Johansson et al., 1971). Pirenzepine, a selective muscarinlc Mi-receptor blocking agent (Hammer et al., 1980), blocked basal and stimulated acid secretion with approximately the same potency. It was, however, more potent to block acid secretion than to cause pupil dilatation (see also Parry and Heathcote, 1982), indicating the involvement of M]-receptors in the process of acid secretion and Mz-receptors in the control of pupil size. The physiological regulation of gastric ac]d
230 secretion involves at least three effectors, gastrin, histamine and acetylcholine. The pathways whereby these three effectors influence the parietal cells have not been identified and even the precise localization of their respective receptors remains to be established. The present findings suggest a complex interaction of mechanisms involving H 2receptors and Ml-receptors in both basal and stimulated acid secretion. Histamine H2-receptors have been demonstrated on the parietal cells (Soil, 1978; 1979; 1980). Thus, histamine could act directly on these cells but could also act indirectly by exciting neurons in intramural ganglia (cf. Paton and Vane, 1963). The localization of the muscarinic Ml-type receptors and the gastrin receptors is unknown. There is some evidence that the parietal cells have gastrin receptors (Soil, 1980) but both M 1 receptors and gastrin receptors may, in addition, be associated with structures other than the parietal cells e.g. endocrine (enterochromaffin-like) cells, mast cells or intramural ganglia. Muscarimc receptors on parietal cells (Ecknauer et al., 1980) are thought to be of the M2-type (Rosenfeld, 1982). Conceivably, the pirenzeplnesensitive Ml-receptors are located in the intramural ganglia, winch are known to play a key role in the control of acid secretion (see e.g. HLkanson et al., 1980; Szelenyl, 1980; Alumets et al., 1982). The effect of pentagastrin was abolished by blockade of either H2-receptors or Ml-receptors. Consequently, if gastrln acts directly on the parietal cell it does so in concert with histamine and acetylcholine. If it acts indirectly it does so through the activation of both histaminergic and cholinergic pathways. If a similar reasoning is applied it appears that basal secretion also depends on histaminergic and cholinergic pathways. The vagal nerve plays a crucial part in the control of acid secretmn in the rat. Bilateral subdiaphragmatic vagal denervation in conscious fistula rats promptly lowers basal acid output to a fraction of the pre-denervatmn level and renders the parietal cells more or less unresponsive to circulating stimuli (Vallgren et al., 1983). The suppression appears to be permanent. However, vagal stimuli do not reach the parietal cells directly but are
amplified in the intramural ganglia (Stach et al., 1975). Hence, most if not all nerve endings acting on the oxyntic glands originate in intramural nerve cell bodies. Angus and Black (1982) showed the H2-receptor antagonists blocked the acid secretory response to electrical field stimulation of the perfused rat stomach, an effect suggesting that histamine was being released by excitation of intramural neurons. Since atropine also blocked the response to field stimulation, acetylcholine was probably being released too. The response to electrical stimulation was thought to be neuraUy mediated since it was abolished by the neurotoxic agent tetrodotoxin. It was thus concluded that electrical stimulation releases histamine locally, involving muscarinic receptors present within an as yet unidentified histamine-storing cell. Evidence to date indicates that the acid output in the rat depends upon intramural 'transducer' systems that respond to gastrin and vagal excitation. It is likely that the intramural ganglia represent such a transducer system. In the absence of a vagal input or following M 1- or H2-receptor blockade these intramural transducers cease to signal. As a result the parietal cells no longer respond to gastrin or to vagal excitation. The function of the parietal cells under basal as well as stimulated conditions appears to depend upon a normal impulse flow in intramural transducer systems and upon a continuous vagal drive. It may be argued that only experiments with isolated parietal cells will explain the physiological control of acid secretion. In our view this argument is not valid because there is mounting evidence that the physioloDcally important targets for gastrin, acetylcholine and histamine in the pathways of acid secretion are not the parietal cells only but intramural transducer systems represented by e.g., intramural neurons. Hence, the action and interactions of gastrin, acetylcholine and histamine must be studied in a bioassay system which responds to each of these agonists in ways which are compatible with their effects in intact animals. Only systems in which the mucosal architecture and nerve supply are intact realize this objective (see Black and Shankley, 1985).
231
Acknowledgements Grant support from the Swedish MRC (04X-1007), the PMalsson Foundation and the Medical Faculty, Umverslty of Lund, Lund, Sweden We are grateful to Professor C. Rerup for statistical assistance
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