351
J. Physiol. (1979), 292, pp. 351-361 With 6 text-ftgure Printed in Great Britain
EFFECTS OF ATROPINE ON SECRETION AND MOTILITY IN ISOLATED GASTRIC MUCOSA AND ATTACHED MUSCULARIS EXTERNA FROM FERRET AND CAT
BY S. H. ROTHt, B. SCHOFIELD* AND J. C. YATES* From the * Division of Medical Physiology and Division t of Pharrnacology and Therapeutics, University of Calgary, Calgary, Alberta, Canada (Received 8 Augwst 1978) SUMMARY
1. A combined in vitro preparation of gastric mucosa and adjacent muscle from young ferrets and kittens has been used to study the effects of atropine on acid secretion and motility produced by acetylcholine (ACh) and pentagastrin. 2. The minimal dose of atropine required to abolish a maximum secretary response to ACh also prevented the associated motility response. This dose of atropine also blocked the motility response to pentagastrin, but was without influence on the secretary effect of this agent. A 103 times larger dose of atropine reduced the secretary effect of pentagastrin by half, probably not by anti-muscarinic effect. The results exclude the possibility that the acid secretary response to pentagastrin necessarily involves a cholinergic receptor. 3. The results support the view that the response of the fundic smooth muscle to pentagastrin depends on the excitation of cholinergic nerves. 4. No evidence has been found of any cholinergic component in the acid secretary response to pentagastrin. In assessing the significance of this result, however, it must be remembered that the Auerbach plexus has been removed over the major part of the mucosa, and the Meissner plexus deprived of input and probably damaged. 5. The results are compatible with the hypothesis that the depressant effect of atropine on acid secretion produced by gastrin and its derivatives is due to the elimination of a cholinergic potentiating influence arising in the intramural plexuses. The residual Meissner plexus elements in this in vitro preparation appear inadequate to sustain this effect. INTRODUCTION
There has been long controversy regarding the action of atropine on the acid responses produced by gastrin and its derivatives. Originally Komarov (1942) observed that acute surgical preparations in whole anaesthetized cats showed no effect of atropine on the secretary response to his gastrin extracts. However Linde (1950) reported that atropine depressed but did not abolish, the secretary response to a gastrin preparation in Pavlov pouch dogs. Gregory & Tracy (1961) found that near Reprints: Dr B. Schofield, Division of Medical Physiology, Faculty of Medicine, University of Calgary, Health Sciences Centre, Calgary T2N 1N4, Alberta, Canada. 0022-3751/79/3830-0678 601.50 © 1979 The Physiological Society.
S. H. ROTH, B. SCHOFIELD AND J. C. YATES maximal rates of secretion from gastric fistula dogs to s.c. injections of purified gastrin were reduced by 50 % following 1 mg atropine i.v. while in denervated gastric pouches the same dose almost abolished the response. However, the results of Komarov were confirmed by Blair, Harper, Lake & Reed (1961). Since then it has been clearly established that while the inhibitory effect of atropine is seen in intact and chronic animal preparations it is absent from acute surgical preparations. This effect is species independent and Schofield, Tepperman & Davison (1975) have shown that absence of the atropine effect is not a consequence of anaesthesia alone, but of the acute preparation. There have been few studies in chronic animal preparations designed to investigate the role of the intramural plexuses in gastrin produced acid responses. Davison, Fahrner, Schofield & Tepperman (1974) showed in vagally denervated gastric pouches in dogs that hexamethonium did not interfere with gastrin responses which were powerfully depressed by small doses of atropine. This they interpreted as meaning that only terminal elements of the plexuses were involved in any potentiating effect. Against this background, studies of the effect of atropine on secretary effects of gastrin on isolated tissue would be of considerable interest. The acutely prepared whole animal preparation is, from many points of view, an unsatisfactory one, since its homoeostatic mechanisms will be reacting to the effects of recent surgery. An isolated fundic preparation which permits the simultaneous study of both secretary and motility responses has been developed recently by Yates, Schofield & Roth(1978). The present work reports results obtained using this preparation to study the action of atropine on motility and secretion and to define the role of cholinergic influences on both processes in the same experiment. 352
METHODS The apparatus and general procedures for setting up the preparation have already been described in detail (Yates et al. 1978). Briefly it consisted of a circular portion of fundic mucosa mounted, secretary surface inside, on the end of a tubular acrylic chamber. The muscularis externa was removed save for a strip 2 mm wide across the diameter orientated in the direction of the circular muscle fibres. One end of this strip was attached to a force transducer. The preparations were placed in Krebs Henseleit solution at 37 'C, and acid secreted on the mucosal side estimated by pH-stat titration to pH 7-0 in unbuffered 5 % dextrose. The experiments have been carried out primarily on ferret tissue, but similar results are given with kitten material. Base lines for secretion and motility were obtained over a 16 min period before adding drugs. All drugs were added to the nutrient solution and are expressed as final concentration in the nutrient bath. All data represent the results of paired experiments in which the mucosal preparation from one half of the stomach is atropinized 16 min before adding secretion agonists to the nutrient bath, while the other half of the stomach provides control data on the response to the agonist in the absence of atropine. The following changes in muscle activity have been analysed: (1) mean basal tension as an index of tone, and (2) mean amplitude of spontaneous contractions, referred to as contractility. To achieve normalization of the data the changes in both aspects produced by the addition of agonists to the medium have been expressed as percent changes from the values existing before the addition.
ATROPINE ON SECRETION AND MOTILITY IN VITRO
353
RESULTS
Our previous study (Yates et al. 1978) showed a maximum acid secretary response was elicited in the isolated ferret fundic mucosal preparations with 104 M-ACh. Fig. 1 shows mean acid and motility responses to this level of ACh stimulation 5-0
c 40 c
1OM- i ACh
_-
.Q " a E 3-0 :., 2-0 a,
IF
a 10 ZL1us
I
TI-
I
-
-IL-
I--- -L---I- I-
260 I 220
=+ -c:r-11880 a,. C
0 140
100 60
C= I
0
m
roi Ir. C.E
4) 4..
4-
0
cola e
-16
0
-8
8 Time (min)
I
I
16
I
I
24
I
I
32
Fig. 1. Acid secretion and motility responses of isolated ferret fundic mucosae to 1O4M-ACh in the presence (- -) and absence (S 5) of 1O6M-atropine. These data are the mean + S.E. of eight paired experiments. -
(10-4 M) in four experiments. A significant increase in the rate of acid secretion was evident within the first 4 min following addition of ACh to the nutrient solution. The secretary rate reached a plateau within 12-16 min which was sustained thereafter. Pretreatment of the test half of each preparation with 10-6 M-atropine 16 min before adding ACh completely abolished the increase in the rate of acid secretion in response to
10-4
M-ACh.
The contractility increased by approximately 250 % within the first min following addition of 10-4 M-ACh but this increase was not sustained and had declined to control levels by 4 min. On the other hand, there was a powerful increase in basal I2
PHY 2(2
S. H. ROTH, B. SCHOFIELD AND J. C. YATES tension in response to 10-4 M-ACh which was sustained. Both of these changes in the behaviour of the fundic smooth muscle were prevented by 10-6 M-atropine. Like ACh, pentagastrin produces a powerful secretary and motility response in the isolated ferret fundic mucosal preparation. A pentagastrin concentration in the 354
0 .
E
i *._~
< cr
._-
_1 1E UU
U.._
o
r c
X ro .
-) co at c-
Time (min) Fig. 2. Acid secretion and motility responses of isolated ferret fundic mucosae to ) of 1O4M-atropine, 6-5 x 10-7M-pentagastrin in the presence --- -) and absence ( and in the presence of 1O-M-atropine (-* -). These data are the mean + s.E. of twelve paired experiments.
nutrient bath of 6-5 x 10-7 M produces a sustained increase in the rate of acid secretion and an alteration in smooth muscle contractility similar to that seen with ACh stimulation (Fig. 2). Changes in basal tension produced by pentagastrin were initially larger but of much shorter duration than those produced by ACh. Pretreatment of the test half of pentagastrin stimulated preparations with 10-6 Matropine for 16 min had no effect on the acid secretary response to pentagastrin but abolished the contractility response and almost abolished the increase in basal
ATROPINE ON SECRETION AND MOTILITY IN VITRO 355 tension in response to pentagastrin. A considerably higher dose of atropine of 10-3 M was effective in significantly reducing the acid secretary response to pentagastrin to about one half. 5-0
K
,.' 4-0
E
LI .> 3-C
I-
0
F 1*0 F
C)
o- 2*0
01
A:
V0
a
I
YI/
I
10-8
10-s [ACh] (M)
10-6
I10-4
I 10-3
Fig. 3. Log dose-response curve for ACh as a stimulus for acid secretion in the presence (A/ - -A ) and absence (* -@) of 10-M-atropine. The mean secretory rate (+ s.E.) measured from 16 to 32 min after stimulation was initiated is plotted against ACh concentration in the serosal solution. 200 -a
r
150
c
0.
is
l00F
01
o
4)
50F
of
CL 0 -
-0
-50 1(11
F I
lo-8
II .~~~~~~ I,
a
10-5
10-6 (ACh]
10-4
10-3
(M)
Fig. 4. Log dose-response curve for ACh as a stimulus of gastric smooth muscle A) and absence (@-@contractility in the presence (A ) of 10M-atropine. The mean change (± S.E. in force of contractions at the peak of the response following stimulation is plotted against ACh concentrations in the serosal solution.
To assess the likely significance of the inhibitory effects in relation to the specific antimuscarinic effect of atropine, dose-response curves for both secretary and motility effects of ACh and pentagastrin were constructed in the presence and absence of 10-6 M-atropine. A dose-response curve for acid secretion in response to ACh is shown in Fig. 3. 12-2
356 S. H. ROTH, B. SCHOFIELD AND J. C. YATES The threshold for an acid secretary response to ACh stimulation was found to occur at a concentration in the bath of between 10-6 and 10-5 M. A maximum secretary response of 4-4 jequiv acid/cm2. hr was observed at 10-4M-ACh, and the response declined again to a mean of 2.5 /tequiv/cm2 . hr at a concentration of 10-3 M. As can be seen in Fig. 3, a bath concentration of 10-6 M-atropine in the test half of each pair was found to produce a parallel shift of the ACh dose-response curve of 2 log units to the right with the result that the effects of an ACh concentration of 10-4 M which otherwise produces a maximal secretary response, were completely abolished. 60
5-0 E
40* a)
30 ~ 0 --
-
-
-
-
-
2-0 1.0
i 9 10-0 10 10-7 10-8 [Pentagastrin] (M X 6 51) Fig. 5. Log dose-response curve for pentagastrin as a stimulus of acid secretion in the 0) of 1 OM-atropine, and in the presence of presence (A - - - A) and absence ( 10-3M-atropine (0 - - - 0). The mean secretary rate ( + S.E.) measured from 16 to 32 min after stimulation was initiated is plotted against pentagastrin concentration in the
10-13
10-12
10-
1
serosal solution.
Fig. 4 shows the maximum change in contractility following addition of ACh plotted against ACh concentration. The threshold concentration for an increase in muscle activity was 10-8 M-ACh with the greatest increase in activity seen at concentrations of 10-6 M and higher. There did not appear to be any reduction in the peak response of the gastric smooth muscle at concentrations of up to 10-3 M-ACh. Smooth muscle responses to stimulation by ACh concentrations of 10-4 M and lower were found to be abolished by 10-6 M-atropine and the response to 10-3 M-ACh was significantly reduced. The threshold at which an acid secretary response to pentagastrin occurs falls between 10-12 and 10-11 M (Fig. 5). A maximal increase in the rate of acid output was seen at a bath concentration of 10-9 M but the peak response remained well above threshold at concentrations up to 10-7 M. The addition of 10-6 M-atropine produced no significant effect on the acid secretion dose response curve. The effect of 10-3 Matropine was to produce an inhibition of about 50 % at all values of pentagastrin dose. A clearly defined plateau was not observed for muscle contractility in the dose range employed in these studies, and the response was still rising at 10-7 M (Fig. 6). 10-6 Matropine completely abolished the response of the gastric smooth muscle to pentagastrin stimulation.
ATROPINE ON SECRETION AND MOTILITY IN VITRO 357 To exclude the possibility that the absence of an effect of moderate atropine concentrations on acid secretary response to pentagastrin was peculiar to the ferret, similar experiments using paired mucosae from kittens were performed. These results are summarized in Table 1. The stable choline ester Urecholine was used in place of 200 r CA c
150
0.
F
an
0
100o 01 O
W-
50 Q) .
CL
0t 0
OF
A-
- - - - - -
-a--
- --
+--,
-50 F
~~~~~~~~~~~~~ -100 10-l1 10-10 10 9 10-8 10-7 [Pentagastrin] (M X 6 51) Fig. 6. Log dose-response curve for pentagastrin as a stimulus of gastric smooth muscle contractility in the presence (A ---A) and absence (@ *A) of 10Matropine. The mean change (± S.E.) in force of contractions at the peak of the response following stimulation is plotted against pentagastrin concentration in the serosal solution. 10-13
lo-l2
TABLE 1. Acid secretion and muscle activity responses of isolated kitten fundic mucosae to urecholine and pentagastrin in the presence and absence of atropine
Muscle activity (% at peak)
Stimulant Control 104 mi-Urecholine
Atropine
No. of expts. 4
104 m 6-5 x 10-7 m-pentagastrin
10-6 M
3 3 3 3
H+ secretion
(#uequiv/cm2 . hr) 1*1±0-3 3-5±0-6 1-3±0-7 4-8±0-6 4.4+0-2
Basal tension 5+7 450 + 8 5 + 10 192 + 14 0+0
Contractility 0+3 435 ± 25 10 + 10 170±30 10+4
ACh in these experiments as ACh has been shown to be less effective than Urecholine for obtaining stimulation of secretion in the kitten preparation (Yates et al. 1978). A Urecholine concentration of 10-4 M was found to produce an increase in rate of acid secretion to 3-5 + 0-6 flequiv H+/cm2 . hr, a sustained 435 % increase in contractility and a 45000 increase in the basal tension of the smooth muscle strip. All of these responses were totally abolished in the presence of 10-6 mI-atropine. Pentagastrin (6.5 x 10-7 M) resulted in an acid secretion rate of 4-8 + 0-6 jtequiv H+/cm2.hr, a maximum increase in contractility of 1700% and an increase in basal tension to the smooth muscle strip of 192 %. The acid secretary response to pentagastrin was not
358
S. H. ROTH, B. SCHOFIELD AND J. C. YATES significantly altered by 10- -m-atropine, but the changes in fundic smooth muscle activity were completely prevented. DISCUSSION
These results show that a concentration of atropine (10-6 A) which was just adequate to eliminate the maximum acid secretary response to ACh also prevented the associated muscle response. The dose-response curve shift in both cases was compatible with competitive inhibition. This concentration of atropine also abolished the motor effects of pentagastrin, confirming in this preparation the claim that they may be exerted through excitation of cholinergic nerves (Vizi, Bertaccini, Impicciatore & Knoll, 1973). Under these conditions however, there was no effect on the acid secretary response to pentagastrin. The fact that similar results were obtained in both kitten and ferret mucosal preparations eliminates the possibility of a species dependent phenomenon in the latter animal which has not been used frequently in gastrointestinal investigation. Suppression of acid responses to gastrin by atropine in the conscious cat has been demonstrated by Emas (1967). The behaviour of these isolated mammalian mucosal preparations has thus been shown to be similar to that reported for isolated amphibian mucosa (Thorpe & Durbin, 1972; Urushibara, Davidson & Thompson, 1968). These results stand in very marked contrast to the situation in intact mammalian preparations in which the acid secretary response to gastrin is depressed by atropine, even in vagally denervated mucosa. Since the action of atropine is thought to depend on the elimination of cholinergic effects from the intramural plexuses, it is necessary to examine the state of these structures in the experimental model. Cook, Kowalewski & Daniel (1974) have shown in the isolated vascular perfused dog stomach that the contractile response of the fundic muscle to intra-arterial pentagastrin is cholinergically mediated and may involve some degree of presynaptic stimulation. The pathway for this response is intact in our preparation, since in the region of the muscle strip there has been no interference with either of the the plexuses and both are presumably functional. Over the greater part of the mucosa, however, a different situation exists. The Auerbach plexus has been totally removed and the Meissner submucous plexus has sustained loss of input and possibly some degree of damage. The cells of this plexus in the stomach are relatively fewer in number and dispersed through the submucous layer (Al-Nuaimi, 1966). Some of them might be damaged during the dissection of our mucosal preparation, and their input connexions would be certainly disrupted. Thus the major area of mucosa is likely to have reduced connexions with fully active nerve cells. It is very unlikely that the small area under the muscle strip would itself influence significantly the over-all secretary result. Thus it would seem that the absence of a depression of secretion in this preparation at low atropine dosage is explained satisfactorily on the basis of removal, damage to, and consequent reduced activity in those elements of the intramural plexuses concerned in any cholinergic component in the secretary response to gastrin. This conclusion renders questionable the interpretation placed on their hexamethonium results by Davison et al. (1974), i.e. that only terminal elements of the
ATROPINE ON SECRETION AND MOTILITY IN VITRO 359 Meissner plexus could be involved in the atropine sensitive component of the gastrin acid response. A clear alternative possibility is that muscarinic rather than nicotinic synapses might be concerned in any influence of these plexuses on gastrin responses. Davison et al. (1974) also devised a means of blocking the terminal secretary stimulant nervous connexions to the parietal cells by a topical local anaesthetic applied to the pouch mucosa. This process did not significantly reduce to gastrin stimulation as compared with the almost 90 % reduction produced by atropine. In view of the very small dose of atropine used in their experiments, they felt that the possibility of a non-muscarinic action of atropine was unlikely. In the current investigation two dose levels of atropine were employed, one chosen as just adequate to abolish a maximum secretary response to ACh. Its effects on the ACh dose-response curve were such as to suggest a specific effect and it was without action on pentagastrin secretary responses. A much larger concentration reduced the acid response to about half but the dose-response curves suggested a non-competitive influence. Davison et al. (1974) attempted to explain the apparent continuance of a cholinergic contribution in the presence of anaesthetized plexus terminals by suggesting that quantal release of ACh which would still occur from these blocked fibres might be sufficient to sustain a necessary level of potentiation. The current study shows that under in vitro conditions such terminals as are present in the mucosa stripped from its muscle are not adequate to sustain any such effect. In view of the very different preparations and conditions in these two investigations it is clear that contradictions between them will be resolved only by further investigation in experimental models intermediate between the in vitro mucosa stripped of its muscle coat and the conscious Heidenhain pouch animal. The results with the present model are clear. They show abolition of the motor response to pentgastrin by atropine but no effect of the same concentration on the acid secretary response. Thus they exclude in this preparation, the possibility of a major effect of pentagastrin on acid secretion being exerted through cholinergic nerve terminals as suggested by Bennett (1965). They are compatible with the wellestablished concept of three separate receptors on the parietal cell for gastrin, ACh and histamine respectively. The work of Soll (1978a, b) on isolated parietal cells has recently strongly reinforced this concept. Soll has also shown that in this preparation atropine acts specifically against cholinergic stimulation only, and metiamide only against histamine. Although the results obtained with this preparation superficially resemble those seen in whole anaesthetized animals prepared by acute surgery, i.e. absence of atropine inhibition of the acid response to gastrin derivatives, the resemblance does not provide an explanation for the latter effect. The current hypothesis regarding the situation in these preparations is that the intramural plexus cells responsible for cholinergic potentiation of the gastrin response are depressed, possibly as a result of sympathetic activity from homeostatic responses to the surgery (Schofield et al. 1975). The results emphasize the need, however, for studies on intermediate isolated preparations with full intramural innervation but without the active homeostatic responses of the whole animal in order to establish that the cholinergic potentiating influence of the plexuses is present in them. To summarize, the effects of atropine on pentagastrin induced acid secretion have
S. H. ROTH, B. SCHOFIELD AND J. C. YATES been shown to be absent from partially denervated ferret and kitten gastric mucosa, though under the same experimental conditions the simultaneous motility response in a fully innervated attached strip of muscularis externa was prevented. The results thus confirm the role of a cholinergic pathway as the mediator for a contractile response to pentagastrin in fundic muscle in these species, but also indicate that the secretary effects of pentagastrin in this preparation are not mediated via a cholinergic receptor on the parietal cell. They are compatible with the concept that the intact stomachs the depressant effect of atropine on the acid secretary response to gastrin derivatives is due to the elimination of a cholinergically mediated potentiating influence from the intramural plexuses. In the two species studied the nervous elements left after removal of the muscularis externa are apparently inadequate to sustain the effect. 360
We are grateful to Ayerst Laboratories, Saint Laurent 381, Montreal, Quebec, for a generous supply of pentagastrin. This work was supported by Canadian M.R.C. grant MA-4322. REFERENCES AI-NuMm, K. A. H. (1966). Intrinsic innervation of the stomach in relation to the control of gastric secretion. Ph.D. thesis, University of Newcastle-upon-Tyne, p. 94. BE:NYETT, A. (1965). Effect of gastrin on isolated smooth muscle preparations. Nature, Lond. 208, 170-173. BLAmR, E. L., HARPER, A. A., LAKE, H. J. & REED, D. J. (1961). The effect of atropine upon gastrin-stimulated acid gastric secretion. J. Phy~iol. 159, 72-73P. COOK, M. A., KowALswsxl, K. & DANIEL, E. E. (1974). The electrical and mechanical activity recorded from the isolated perfused canine stomach: the effect of some G.I. polypeptides. In Proceedings of the [Vth International Sympo8ium on Gaetrointes8tinal Motility. Vancouver: Mitchell. DAVISON, J. S., FAHKNER, P. C., SCHOFIELD, B. & TEPPERMA, B. L. (1974). The role of the intramural cholinergic innervation in the response of the parietal cell to gastrin derivatives. Can. J. Phy-iol. Pharmacol. 52, 469-482. EXAs, S. (1967). In Ga8tric Secretion, Mechanism8 and Control, Proceedings of a Symposium, Edmonton, p. 279 (Discussion), ed. ScHNITKA, J. K., GILBERT, J. A. & HARISON, R. C.
Oxford: Pergamon. GREGORY, R. A. & TRACY, J. .H. (1961). The preparation and properties of gastrn. J. PhlyJiol. 156, 523-543. KoxARov, S. H. (1942). Studies on gastrin. II. Physiological properties of the specific gastric secretagogue of the pyloric mucous membrane. Revue can. Biol. 1, 377. LINDE, S. (1950). Studies oI the stimulation mechanism of gastric secretion. Acta physiol. scand. 21, Suppl. 74. SCHOrIxLD, B., TEPPERMAN, B. L. & DAVISON, J. S. (1975). The effects of atropine on the response to pentagastrin of anaesthetised and conscious dog preparations. Rend. Ga8troenterol. 7, 147-152. SoLL, A. H. (1978a). The actions of secretagogues on oxygen uptake by isolated mammalian parietal cells. J. dlin. Invest. 61, 370-380. SoiL, A. H. (1978b). The interaction of histamine with gastrin and carbamyl choline on oxygen uptake by isolated mammalian parietal cells. J. clin. Invest. 61, 381-389. THORPE, C. C. & DuRBIN, R. P. (1972). Effects of atropine on acid secretion by isolated frog gastric mucosa. Ga8troenterology 62, 1153-1158. URuSHIBARA, O., DAVIDSON, W. D. & THOMPSON, J. C. (1968). Effect of nerve function inhibitors on pentagastrin stimulated acid secretion by isolated bullfrog gastric mucosa. Surg. Forum 19, 279-280.
ATROPINE ON SECRETION AND MOTILITY IN VITRO
361
Vizi, S. E., BERTACCINi, J., IMPICCIATORE, M. & KNOLL, J. (1973). Evidence that ACh release by gastrin and related polypeptides contributes to their effect on gastointestinal motility. Gastroenterology 64, 268-277. YATES, J. C., SCHOFIELD, B. & Romr, S. H. (1978). Acid secretion and motility of isolated mammalian gastric mucosa and attached muscularis externa. Am. J. Physiol. 234, E319-326.
J. Physiol. (1979), 292, pp. 351-361 With 6 text-ftgure Printed in Great Britain
EFFECTS OF ATROPINE ON SECRETION AND MOTILITY IN ISOLATED GASTRIC MUCOSA AND ATTACHED MUSCULARIS EXTERNA FROM FERRET AND CAT
BY S. H. ROTHt, B. SCHOFIELD* AND J. C. YATES* From the * Division of Medical Physiology and Division t of Pharrnacology and Therapeutics, University of Calgary, Calgary, Alberta, Canada (Received 8 Augwst 1978) SUMMARY
1. A combined in vitro preparation of gastric mucosa and adjacent muscle from young ferrets and kittens has been used to study the effects of atropine on acid secretion and motility produced by acetylcholine (ACh) and pentagastrin. 2. The minimal dose of atropine required to abolish a maximum secretary response to ACh also prevented the associated motility response. This dose of atropine also blocked the motility response to pentagastrin, but was without influence on the secretary effect of this agent. A 103 times larger dose of atropine reduced the secretary effect of pentagastrin by half, probably not by anti-muscarinic effect. The results exclude the possibility that the acid secretary response to pentagastrin necessarily involves a cholinergic receptor. 3. The results support the view that the response of the fundic smooth muscle to pentagastrin depends on the excitation of cholinergic nerves. 4. No evidence has been found of any cholinergic component in the acid secretary response to pentagastrin. In assessing the significance of this result, however, it must be remembered that the Auerbach plexus has been removed over the major part of the mucosa, and the Meissner plexus deprived of input and probably damaged. 5. The results are compatible with the hypothesis that the depressant effect of atropine on acid secretion produced by gastrin and its derivatives is due to the elimination of a cholinergic potentiating influence arising in the intramural plexuses. The residual Meissner plexus elements in this in vitro preparation appear inadequate to sustain this effect. INTRODUCTION
There has been long controversy regarding the action of atropine on the acid responses produced by gastrin and its derivatives. Originally Komarov (1942) observed that acute surgical preparations in whole anaesthetized cats showed no effect of atropine on the secretary response to his gastrin extracts. However Linde (1950) reported that atropine depressed but did not abolish, the secretary response to a gastrin preparation in Pavlov pouch dogs. Gregory & Tracy (1961) found that near Reprints: Dr B. Schofield, Division of Medical Physiology, Faculty of Medicine, University of Calgary, Health Sciences Centre, Calgary T2N 1N4, Alberta, Canada. 0022-3751/79/3830-0678 601.50 © 1979 The Physiological Society.
S. H. ROTH, B. SCHOFIELD AND J. C. YATES maximal rates of secretion from gastric fistula dogs to s.c. injections of purified gastrin were reduced by 50 % following 1 mg atropine i.v. while in denervated gastric pouches the same dose almost abolished the response. However, the results of Komarov were confirmed by Blair, Harper, Lake & Reed (1961). Since then it has been clearly established that while the inhibitory effect of atropine is seen in intact and chronic animal preparations it is absent from acute surgical preparations. This effect is species independent and Schofield, Tepperman & Davison (1975) have shown that absence of the atropine effect is not a consequence of anaesthesia alone, but of the acute preparation. There have been few studies in chronic animal preparations designed to investigate the role of the intramural plexuses in gastrin produced acid responses. Davison, Fahrner, Schofield & Tepperman (1974) showed in vagally denervated gastric pouches in dogs that hexamethonium did not interfere with gastrin responses which were powerfully depressed by small doses of atropine. This they interpreted as meaning that only terminal elements of the plexuses were involved in any potentiating effect. Against this background, studies of the effect of atropine on secretary effects of gastrin on isolated tissue would be of considerable interest. The acutely prepared whole animal preparation is, from many points of view, an unsatisfactory one, since its homoeostatic mechanisms will be reacting to the effects of recent surgery. An isolated fundic preparation which permits the simultaneous study of both secretary and motility responses has been developed recently by Yates, Schofield & Roth(1978). The present work reports results obtained using this preparation to study the action of atropine on motility and secretion and to define the role of cholinergic influences on both processes in the same experiment. 352
METHODS The apparatus and general procedures for setting up the preparation have already been described in detail (Yates et al. 1978). Briefly it consisted of a circular portion of fundic mucosa mounted, secretary surface inside, on the end of a tubular acrylic chamber. The muscularis externa was removed save for a strip 2 mm wide across the diameter orientated in the direction of the circular muscle fibres. One end of this strip was attached to a force transducer. The preparations were placed in Krebs Henseleit solution at 37 'C, and acid secreted on the mucosal side estimated by pH-stat titration to pH 7-0 in unbuffered 5 % dextrose. The experiments have been carried out primarily on ferret tissue, but similar results are given with kitten material. Base lines for secretion and motility were obtained over a 16 min period before adding drugs. All drugs were added to the nutrient solution and are expressed as final concentration in the nutrient bath. All data represent the results of paired experiments in which the mucosal preparation from one half of the stomach is atropinized 16 min before adding secretion agonists to the nutrient bath, while the other half of the stomach provides control data on the response to the agonist in the absence of atropine. The following changes in muscle activity have been analysed: (1) mean basal tension as an index of tone, and (2) mean amplitude of spontaneous contractions, referred to as contractility. To achieve normalization of the data the changes in both aspects produced by the addition of agonists to the medium have been expressed as percent changes from the values existing before the addition.
ATROPINE ON SECRETION AND MOTILITY IN VITRO
353
RESULTS
Our previous study (Yates et al. 1978) showed a maximum acid secretary response was elicited in the isolated ferret fundic mucosal preparations with 104 M-ACh. Fig. 1 shows mean acid and motility responses to this level of ACh stimulation 5-0
c 40 c
1OM- i ACh
_-
.Q " a E 3-0 :., 2-0 a,
IF
a 10 ZL1us
I
TI-
I
-
-IL-
I--- -L---I- I-
260 I 220
=+ -c:r-11880 a,. C
0 140
100 60
C= I
0
m
roi Ir. C.E
4) 4..
4-
0
cola e
-16
0
-8
8 Time (min)
I
I
16
I
I
24
I
I
32
Fig. 1. Acid secretion and motility responses of isolated ferret fundic mucosae to 1O4M-ACh in the presence (- -) and absence (S 5) of 1O6M-atropine. These data are the mean + S.E. of eight paired experiments. -
(10-4 M) in four experiments. A significant increase in the rate of acid secretion was evident within the first 4 min following addition of ACh to the nutrient solution. The secretary rate reached a plateau within 12-16 min which was sustained thereafter. Pretreatment of the test half of each preparation with 10-6 M-atropine 16 min before adding ACh completely abolished the increase in the rate of acid secretion in response to
10-4
M-ACh.
The contractility increased by approximately 250 % within the first min following addition of 10-4 M-ACh but this increase was not sustained and had declined to control levels by 4 min. On the other hand, there was a powerful increase in basal I2
PHY 2(2
S. H. ROTH, B. SCHOFIELD AND J. C. YATES tension in response to 10-4 M-ACh which was sustained. Both of these changes in the behaviour of the fundic smooth muscle were prevented by 10-6 M-atropine. Like ACh, pentagastrin produces a powerful secretary and motility response in the isolated ferret fundic mucosal preparation. A pentagastrin concentration in the 354
0 .
E
i *._~
< cr
._-
_1 1E UU
U.._
o
r c
X ro .
-) co at c-
Time (min) Fig. 2. Acid secretion and motility responses of isolated ferret fundic mucosae to ) of 1O4M-atropine, 6-5 x 10-7M-pentagastrin in the presence --- -) and absence ( and in the presence of 1O-M-atropine (-* -). These data are the mean + s.E. of twelve paired experiments.
nutrient bath of 6-5 x 10-7 M produces a sustained increase in the rate of acid secretion and an alteration in smooth muscle contractility similar to that seen with ACh stimulation (Fig. 2). Changes in basal tension produced by pentagastrin were initially larger but of much shorter duration than those produced by ACh. Pretreatment of the test half of pentagastrin stimulated preparations with 10-6 Matropine for 16 min had no effect on the acid secretary response to pentagastrin but abolished the contractility response and almost abolished the increase in basal
ATROPINE ON SECRETION AND MOTILITY IN VITRO 355 tension in response to pentagastrin. A considerably higher dose of atropine of 10-3 M was effective in significantly reducing the acid secretary response to pentagastrin to about one half. 5-0
K
,.' 4-0
E
LI .> 3-C
I-
0
F 1*0 F
C)
o- 2*0
01
A:
V0
a
I
YI/
I
10-8
10-s [ACh] (M)
10-6
I10-4
I 10-3
Fig. 3. Log dose-response curve for ACh as a stimulus for acid secretion in the presence (A/ - -A ) and absence (* -@) of 10-M-atropine. The mean secretory rate (+ s.E.) measured from 16 to 32 min after stimulation was initiated is plotted against ACh concentration in the serosal solution. 200 -a
r
150
c
0.
is
l00F
01
o
4)
50F
of
CL 0 -
-0
-50 1(11
F I
lo-8
II .~~~~~~ I,
a
10-5
10-6 (ACh]
10-4
10-3
(M)
Fig. 4. Log dose-response curve for ACh as a stimulus of gastric smooth muscle A) and absence (@-@contractility in the presence (A ) of 10M-atropine. The mean change (± S.E. in force of contractions at the peak of the response following stimulation is plotted against ACh concentrations in the serosal solution.
To assess the likely significance of the inhibitory effects in relation to the specific antimuscarinic effect of atropine, dose-response curves for both secretary and motility effects of ACh and pentagastrin were constructed in the presence and absence of 10-6 M-atropine. A dose-response curve for acid secretion in response to ACh is shown in Fig. 3. 12-2
356 S. H. ROTH, B. SCHOFIELD AND J. C. YATES The threshold for an acid secretary response to ACh stimulation was found to occur at a concentration in the bath of between 10-6 and 10-5 M. A maximum secretary response of 4-4 jequiv acid/cm2. hr was observed at 10-4M-ACh, and the response declined again to a mean of 2.5 /tequiv/cm2 . hr at a concentration of 10-3 M. As can be seen in Fig. 3, a bath concentration of 10-6 M-atropine in the test half of each pair was found to produce a parallel shift of the ACh dose-response curve of 2 log units to the right with the result that the effects of an ACh concentration of 10-4 M which otherwise produces a maximal secretary response, were completely abolished. 60
5-0 E
40* a)
30 ~ 0 --
-
-
-
-
-
2-0 1.0
i 9 10-0 10 10-7 10-8 [Pentagastrin] (M X 6 51) Fig. 5. Log dose-response curve for pentagastrin as a stimulus of acid secretion in the 0) of 1 OM-atropine, and in the presence of presence (A - - - A) and absence ( 10-3M-atropine (0 - - - 0). The mean secretary rate ( + S.E.) measured from 16 to 32 min after stimulation was initiated is plotted against pentagastrin concentration in the
10-13
10-12
10-
1
serosal solution.
Fig. 4 shows the maximum change in contractility following addition of ACh plotted against ACh concentration. The threshold concentration for an increase in muscle activity was 10-8 M-ACh with the greatest increase in activity seen at concentrations of 10-6 M and higher. There did not appear to be any reduction in the peak response of the gastric smooth muscle at concentrations of up to 10-3 M-ACh. Smooth muscle responses to stimulation by ACh concentrations of 10-4 M and lower were found to be abolished by 10-6 M-atropine and the response to 10-3 M-ACh was significantly reduced. The threshold at which an acid secretary response to pentagastrin occurs falls between 10-12 and 10-11 M (Fig. 5). A maximal increase in the rate of acid output was seen at a bath concentration of 10-9 M but the peak response remained well above threshold at concentrations up to 10-7 M. The addition of 10-6 M-atropine produced no significant effect on the acid secretion dose response curve. The effect of 10-3 Matropine was to produce an inhibition of about 50 % at all values of pentagastrin dose. A clearly defined plateau was not observed for muscle contractility in the dose range employed in these studies, and the response was still rising at 10-7 M (Fig. 6). 10-6 Matropine completely abolished the response of the gastric smooth muscle to pentagastrin stimulation.
ATROPINE ON SECRETION AND MOTILITY IN VITRO 357 To exclude the possibility that the absence of an effect of moderate atropine concentrations on acid secretary response to pentagastrin was peculiar to the ferret, similar experiments using paired mucosae from kittens were performed. These results are summarized in Table 1. The stable choline ester Urecholine was used in place of 200 r CA c
150
0.
F
an
0
100o 01 O
W-
50 Q) .
CL
0t 0
OF
A-
- - - - - -
-a--
- --
+--,
-50 F
~~~~~~~~~~~~~ -100 10-l1 10-10 10 9 10-8 10-7 [Pentagastrin] (M X 6 51) Fig. 6. Log dose-response curve for pentagastrin as a stimulus of gastric smooth muscle contractility in the presence (A ---A) and absence (@ *A) of 10Matropine. The mean change (± S.E.) in force of contractions at the peak of the response following stimulation is plotted against pentagastrin concentration in the serosal solution. 10-13
lo-l2
TABLE 1. Acid secretion and muscle activity responses of isolated kitten fundic mucosae to urecholine and pentagastrin in the presence and absence of atropine
Muscle activity (% at peak)
Stimulant Control 104 mi-Urecholine
Atropine
No. of expts. 4
104 m 6-5 x 10-7 m-pentagastrin
10-6 M
3 3 3 3
H+ secretion
(#uequiv/cm2 . hr) 1*1±0-3 3-5±0-6 1-3±0-7 4-8±0-6 4.4+0-2
Basal tension 5+7 450 + 8 5 + 10 192 + 14 0+0
Contractility 0+3 435 ± 25 10 + 10 170±30 10+4
ACh in these experiments as ACh has been shown to be less effective than Urecholine for obtaining stimulation of secretion in the kitten preparation (Yates et al. 1978). A Urecholine concentration of 10-4 M was found to produce an increase in rate of acid secretion to 3-5 + 0-6 flequiv H+/cm2 . hr, a sustained 435 % increase in contractility and a 45000 increase in the basal tension of the smooth muscle strip. All of these responses were totally abolished in the presence of 10-6 mI-atropine. Pentagastrin (6.5 x 10-7 M) resulted in an acid secretion rate of 4-8 + 0-6 jtequiv H+/cm2.hr, a maximum increase in contractility of 1700% and an increase in basal tension to the smooth muscle strip of 192 %. The acid secretary response to pentagastrin was not
358
S. H. ROTH, B. SCHOFIELD AND J. C. YATES significantly altered by 10- -m-atropine, but the changes in fundic smooth muscle activity were completely prevented. DISCUSSION
These results show that a concentration of atropine (10-6 A) which was just adequate to eliminate the maximum acid secretary response to ACh also prevented the associated muscle response. The dose-response curve shift in both cases was compatible with competitive inhibition. This concentration of atropine also abolished the motor effects of pentagastrin, confirming in this preparation the claim that they may be exerted through excitation of cholinergic nerves (Vizi, Bertaccini, Impicciatore & Knoll, 1973). Under these conditions however, there was no effect on the acid secretary response to pentagastrin. The fact that similar results were obtained in both kitten and ferret mucosal preparations eliminates the possibility of a species dependent phenomenon in the latter animal which has not been used frequently in gastrointestinal investigation. Suppression of acid responses to gastrin by atropine in the conscious cat has been demonstrated by Emas (1967). The behaviour of these isolated mammalian mucosal preparations has thus been shown to be similar to that reported for isolated amphibian mucosa (Thorpe & Durbin, 1972; Urushibara, Davidson & Thompson, 1968). These results stand in very marked contrast to the situation in intact mammalian preparations in which the acid secretary response to gastrin is depressed by atropine, even in vagally denervated mucosa. Since the action of atropine is thought to depend on the elimination of cholinergic effects from the intramural plexuses, it is necessary to examine the state of these structures in the experimental model. Cook, Kowalewski & Daniel (1974) have shown in the isolated vascular perfused dog stomach that the contractile response of the fundic muscle to intra-arterial pentagastrin is cholinergically mediated and may involve some degree of presynaptic stimulation. The pathway for this response is intact in our preparation, since in the region of the muscle strip there has been no interference with either of the the plexuses and both are presumably functional. Over the greater part of the mucosa, however, a different situation exists. The Auerbach plexus has been totally removed and the Meissner submucous plexus has sustained loss of input and possibly some degree of damage. The cells of this plexus in the stomach are relatively fewer in number and dispersed through the submucous layer (Al-Nuaimi, 1966). Some of them might be damaged during the dissection of our mucosal preparation, and their input connexions would be certainly disrupted. Thus the major area of mucosa is likely to have reduced connexions with fully active nerve cells. It is very unlikely that the small area under the muscle strip would itself influence significantly the over-all secretary result. Thus it would seem that the absence of a depression of secretion in this preparation at low atropine dosage is explained satisfactorily on the basis of removal, damage to, and consequent reduced activity in those elements of the intramural plexuses concerned in any cholinergic component in the secretary response to gastrin. This conclusion renders questionable the interpretation placed on their hexamethonium results by Davison et al. (1974), i.e. that only terminal elements of the
ATROPINE ON SECRETION AND MOTILITY IN VITRO 359 Meissner plexus could be involved in the atropine sensitive component of the gastrin acid response. A clear alternative possibility is that muscarinic rather than nicotinic synapses might be concerned in any influence of these plexuses on gastrin responses. Davison et al. (1974) also devised a means of blocking the terminal secretary stimulant nervous connexions to the parietal cells by a topical local anaesthetic applied to the pouch mucosa. This process did not significantly reduce to gastrin stimulation as compared with the almost 90 % reduction produced by atropine. In view of the very small dose of atropine used in their experiments, they felt that the possibility of a non-muscarinic action of atropine was unlikely. In the current investigation two dose levels of atropine were employed, one chosen as just adequate to abolish a maximum secretary response to ACh. Its effects on the ACh dose-response curve were such as to suggest a specific effect and it was without action on pentagastrin secretary responses. A much larger concentration reduced the acid response to about half but the dose-response curves suggested a non-competitive influence. Davison et al. (1974) attempted to explain the apparent continuance of a cholinergic contribution in the presence of anaesthetized plexus terminals by suggesting that quantal release of ACh which would still occur from these blocked fibres might be sufficient to sustain a necessary level of potentiation. The current study shows that under in vitro conditions such terminals as are present in the mucosa stripped from its muscle are not adequate to sustain any such effect. In view of the very different preparations and conditions in these two investigations it is clear that contradictions between them will be resolved only by further investigation in experimental models intermediate between the in vitro mucosa stripped of its muscle coat and the conscious Heidenhain pouch animal. The results with the present model are clear. They show abolition of the motor response to pentgastrin by atropine but no effect of the same concentration on the acid secretary response. Thus they exclude in this preparation, the possibility of a major effect of pentagastrin on acid secretion being exerted through cholinergic nerve terminals as suggested by Bennett (1965). They are compatible with the wellestablished concept of three separate receptors on the parietal cell for gastrin, ACh and histamine respectively. The work of Soll (1978a, b) on isolated parietal cells has recently strongly reinforced this concept. Soll has also shown that in this preparation atropine acts specifically against cholinergic stimulation only, and metiamide only against histamine. Although the results obtained with this preparation superficially resemble those seen in whole anaesthetized animals prepared by acute surgery, i.e. absence of atropine inhibition of the acid response to gastrin derivatives, the resemblance does not provide an explanation for the latter effect. The current hypothesis regarding the situation in these preparations is that the intramural plexus cells responsible for cholinergic potentiation of the gastrin response are depressed, possibly as a result of sympathetic activity from homeostatic responses to the surgery (Schofield et al. 1975). The results emphasize the need, however, for studies on intermediate isolated preparations with full intramural innervation but without the active homeostatic responses of the whole animal in order to establish that the cholinergic potentiating influence of the plexuses is present in them. To summarize, the effects of atropine on pentagastrin induced acid secretion have
S. H. ROTH, B. SCHOFIELD AND J. C. YATES been shown to be absent from partially denervated ferret and kitten gastric mucosa, though under the same experimental conditions the simultaneous motility response in a fully innervated attached strip of muscularis externa was prevented. The results thus confirm the role of a cholinergic pathway as the mediator for a contractile response to pentagastrin in fundic muscle in these species, but also indicate that the secretary effects of pentagastrin in this preparation are not mediated via a cholinergic receptor on the parietal cell. They are compatible with the concept that the intact stomachs the depressant effect of atropine on the acid secretary response to gastrin derivatives is due to the elimination of a cholinergically mediated potentiating influence from the intramural plexuses. In the two species studied the nervous elements left after removal of the muscularis externa are apparently inadequate to sustain the effect. 360
We are grateful to Ayerst Laboratories, Saint Laurent 381, Montreal, Quebec, for a generous supply of pentagastrin. This work was supported by Canadian M.R.C. grant MA-4322. REFERENCES AI-NuMm, K. A. H. (1966). Intrinsic innervation of the stomach in relation to the control of gastric secretion. Ph.D. thesis, University of Newcastle-upon-Tyne, p. 94. BE:NYETT, A. (1965). Effect of gastrin on isolated smooth muscle preparations. Nature, Lond. 208, 170-173. BLAmR, E. L., HARPER, A. A., LAKE, H. J. & REED, D. J. (1961). The effect of atropine upon gastrin-stimulated acid gastric secretion. J. Phy~iol. 159, 72-73P. COOK, M. A., KowALswsxl, K. & DANIEL, E. E. (1974). The electrical and mechanical activity recorded from the isolated perfused canine stomach: the effect of some G.I. polypeptides. In Proceedings of the [Vth International Sympo8ium on Gaetrointes8tinal Motility. Vancouver: Mitchell. DAVISON, J. S., FAHKNER, P. C., SCHOFIELD, B. & TEPPERMA, B. L. (1974). The role of the intramural cholinergic innervation in the response of the parietal cell to gastrin derivatives. Can. J. Phy-iol. Pharmacol. 52, 469-482. EXAs, S. (1967). In Ga8tric Secretion, Mechanism8 and Control, Proceedings of a Symposium, Edmonton, p. 279 (Discussion), ed. ScHNITKA, J. K., GILBERT, J. A. & HARISON, R. C.
Oxford: Pergamon. GREGORY, R. A. & TRACY, J. .H. (1961). The preparation and properties of gastrn. J. PhlyJiol. 156, 523-543. KoxARov, S. H. (1942). Studies on gastrin. II. Physiological properties of the specific gastric secretagogue of the pyloric mucous membrane. Revue can. Biol. 1, 377. LINDE, S. (1950). Studies oI the stimulation mechanism of gastric secretion. Acta physiol. scand. 21, Suppl. 74. SCHOrIxLD, B., TEPPERMAN, B. L. & DAVISON, J. S. (1975). The effects of atropine on the response to pentagastrin of anaesthetised and conscious dog preparations. Rend. Ga8troenterol. 7, 147-152. SoLL, A. H. (1978a). The actions of secretagogues on oxygen uptake by isolated mammalian parietal cells. J. dlin. Invest. 61, 370-380. SoiL, A. H. (1978b). The interaction of histamine with gastrin and carbamyl choline on oxygen uptake by isolated mammalian parietal cells. J. clin. Invest. 61, 381-389. THORPE, C. C. & DuRBIN, R. P. (1972). Effects of atropine on acid secretion by isolated frog gastric mucosa. Ga8troenterology 62, 1153-1158. URuSHIBARA, O., DAVIDSON, W. D. & THOMPSON, J. C. (1968). Effect of nerve function inhibitors on pentagastrin stimulated acid secretion by isolated bullfrog gastric mucosa. Surg. Forum 19, 279-280.
ATROPINE ON SECRETION AND MOTILITY IN VITRO
361
Vizi, S. E., BERTACCINi, J., IMPICCIATORE, M. & KNOLL, J. (1973). Evidence that ACh release by gastrin and related polypeptides contributes to their effect on gastointestinal motility. Gastroenterology 64, 268-277. YATES, J. C., SCHOFIELD, B. & Romr, S. H. (1978). Acid secretion and motility of isolated mammalian gastric mucosa and attached muscularis externa. Am. J. Physiol. 234, E319-326.
