Effect of Metoclopramide on Guinea Pig Stomach

Transcription

1 GASTROENTEROLOGY 76: , 1979 Effect of Metoclopramide on Guinea Pig Stomach Critical Dependence on Intrinsic Stores of Acety lcholine A. M. HAY, B.Sc., M.B., F.R.C.S., and W. K. MAN, Ph.D. Department of Surgery, Royal Postgraduate Medical School. Hammersmith Hospital. London, England This study is concerned with an investigation of the hypothesis that metoclopramide enhances the contractile activity of the guinea pig antrum by increasing acetylcholine release from the postganglionic cholinergic nerve ending. Longitudinal muscle strips were stimulated repetitively (200 f.lsec pulses, 20 Hz, supramaximal current) for 2 hr in the presence of hemicholinium-3 (200 f.lm). Pretreatment in this manner produced a mean reduction of 50% in the acetylcholine content, when compared with that in repetitively ~timulated control strips not incubated with hemicholinium-3. In the hemicholinium-3- treated strips the normal excitatory response to metoclopramide (60 f.lm) was prevented. Only a small reduction in response to metoclopramide was detected in strips which were incubated with hemicholinium-3 but not stimulated repetitively, and in which there was no significant change in acetylcholine content. The stimulant effect of metoclopramide depends, therefore, upon maintenance of intrinsic stores of acetylcholine. There are now many reports" 2 of the potent effects of metoclopramide (MCP) on human gastrointestinal motility, but uncertainty still exists in regard to its precise mode of action. Recently published data from this laboratory3 have raised, on the basis of indirect evidence, the possibility that MCP may act, in part, at least, by increasing the amount of acetylcholine (ACh) released by nerve impulses arriving at Received May 15, Accepted October 24, Address requests for reprints to: A. M. Hay, F.R.C.S., Department of Surgery, Royal Postgraduate Medical School, Hammersmith Hospital, Du Cane Road, London, W12 OHS, England. This study was supported by a grant from Beecham Research Laboratories by the American Gastroenterological Association /79/ $02.00 the postganglionic cholinergic nerve terminal. Direct testing of this hypothesis has been made difficult by the discovery of an unexpected inhibitory interaction between MCP and anticholinesterase drugs,3 making conventional techniques for the measurement of ACh release inappropriate. We have, therefore, adopted an oblique approach to the problem. If the hypothesis of increased ACh release is correct, there should be a relationship between the response to MCP and the availability of ACh for release from postganglionic nerve endings. Depletion of transmitter stores of ACh should reduce or abolish the response. This paper is concerned with the results of a series of experiments in which muscle strips prepared from the guinea pig antrum were depleted of ACh by pretreatment with the choline analogue hemicholinium-3 (HC-3) and their subsequent responsiveness to MCP measured. Some of the data in this paper have been presented in proceedings form" Materials and Methods Male Duncan-Hartley guinea pigs, weighing g, were killed by stunning and bleeding. The techniques involved in the preparation and preservation of longitudinal muscle strips-composed predominantly of antral tissue-and the mounting of such strips in a superfusion apparatus, in which isometric contractile activity can be recorded, have been described previously.3 The majority of muscle strips mounted in this way exhibit, after an equilibration period of min, spontaneous rhythmic contractile activity of remarkable stability.3 Quantification of contractile activity was effected by the measurement of the amplitude of each separate contraction relative to the baseline tone (1-3 g force) of the preparation and computation of the mean peak contractile force (g) for consecutive 2 min periods. Only those experiments in which entirely stable contractile activity was observed, and in which no appreciable change in tone or

2 March 1979 METOCLOPRAMIDE AND ACETYLCHOLINE STORES 493 contractile rate occurred in response to MCP, have provided that data reported in this paper. Transmural Stimulation Selective stimulation 5 of neural as opposed to muscular elements in the stomach strips was effected by the passage of square-wave pulses of 200 /-lsec duration at supramaximal current strength from a constant current source through stainless steel clips gripping each end of the muscle strip. Stimulus parameters were continuously monitored on a cathode ray oscilloscope. Determination of Acetylcholine Content of Muscle Strips If the ACh content of a particular muscle strip was to be assayed, the strip was rapidly removed from the superfusion apparatus, and the ends retained within the suspensory clips were cut away. The residual tissue, weighing mg, was homogenized in 2 ml freshly prepared ice-cold trichloroacetic acid (TCA) (10% wtjvol, made up in 0.9% wtjvol NaCI). The homogenate was allowed to stand on ice for 60 min and was then centrifuged at 40,000 g at 4 C for 10 min. The resulting pellet was resuspended in 1 ml ice-cold TCA and centrifuged as above. The supernatants were combined and passed through a periodide precipitation procedure 6 by means of which a nearly pure solution of ACh was obtained. The mean recovery of ACh passed through extraction, and periodide precipitation procedures were 92 ± 7% (SEM) in seven experiments. The ACh concentration of the solution derived from the periodide precipitation procedure was determined by bioassay, using sensitized guinea pig ileum,' mounted in the superfusion apparatus under isometric conditions, as the test system. The bioassay was conducted according to a 4 x 4 Latin square design, and the potency ratio of standard/unknown solutions was calculated by analysis of variance." The stimulant activity of the assay solutions on the sensitized ileum was always completely reversed by the introduction of hyoscine (22.8 nm) into the superfusate. Various quaternary ammonium compounds (notably choline and tetramethylammonium) other than ACh were present in the solutions for assay but not in sufficient amounts to interfere. Drugs and Chemicals The following drugs and chemicals were used: acetylcholine chloride (Sigma Chemical Co., St Louis, Mo.), choline chloride (Sigma), hemicholinium-3 dibromide (Aldrich Chemical Co., Inc., Milwaukee, Wis.), hyoscine hydrobromide (BDH Chemicals Ltd., Poole, England), and metoclopramide hydrochloride (Beecham Research Laboratories, London, England). Drug concentrations are expressed as molar values. Statistical Methods The Mann-Whitney U test" has been used wherever calculations of significance levels for observed differences between data groups have been made. Results Thirty-three muscle strips were studied. After a preliminary stabilization period of min, each strip entered one or another of the following four experimental modes: 1. Six strips were superfused for 150 min in Krebs solution only, followed by 20 min exposure to MCP (60 pm). ACh assay was performed on five of these strips. 2. Eight strips underwent prolonged transmural stimulation (PTM) at 20 Hz for 120 min (in Krebs only), followed by a 30-min recovery period (in Krebs only) before 20 min exposure to MCP (60 pm). ACh assay was performed on seven of these strips. 3. Ten strips were treated as in mode 1 above, except that HC-3 (200 flm) was present throughout the whole superfusion period. ACh assay was performed on five of these strips. 4. Nine strips were treated as in mode 2 above, except that HC-3 (200 flm) was present throughout. ACh assay was performed on eight of these strips. Effect of Hemicholinium-3 (200 flm) on Acetylcholine Content (Table 1) A significant reduction in ACh content was detected in those strips undergoing PTM in the presence of HC-3. In eight such strips, there was a mean reduction of 53% (P = 0.001) and 50% (P = 0.001) in ACh content when compared, respectively, with five strips undergoing superfusion with Krebs solution alone and seven strips undergoing PTM in the absence of HC-3. Prolonged transmural stimulation alone did not affect ACh stores, and in. five strips merely superfused with HC-3, there was a statistically nonsignificant reduction of 13% in ACh content when compared with five strips superfused with Krebs solution only. Effect of Hemicholinium-3 (200 pm) on Response to Metoclopramide (60 flm) The effect of MCP on spontaneous contractile activity in each of the four experimental situations is shown in Figure 1. In the three groups in which responsiveness to MCP was retained, there was, in keeping with earlier observations,3 a plateau in the increase in contractile amplitude after approximately a 10-min exposure to MCP. It is apparent from Figure 1, and from the upper trace in Figure 2, that PTM alone did not alter responsiveness to MCP, as measured at plateau, when compared with the curve obtained from those strips merely superfused

3 494 HAY AND MAN GASTROENTEROLOGY Vol. 76, No.3 Table 1. Effect of Hemicholinium-3 (200 I'M) and Prolonged Transmural Stimulation (20 Hz, 120 min), Alone and in Combination, on Acetylcholine Content of Muscle Strips Experimental Illode o 1 (Krebs' only) 2 (PTM) 3 (HC-3) 4 (HC-3 + PTM) No. of strips ACh content, nm/g wet wt b 24.7 ± ± ± ± 1.6 < 'ep = <-- 'ep = _ 'cns ~ Refer to text. b Mean ± SEM. C Mann-Whitney U Test. with Krebs solution. The apparent discrepancy in the rising phase of the two curves is not statistically significant and may be attributed to undue influence upon the mean value for the merely superfused strips of one highly responsive preparation (>300% increase at 2 min). Superfusion with HC-3 produced a consistent, small reduction in the response to MCP when compared with the two sets of strips superfused with Krebs solution alone (one undergoing PTM, the other not). The difference is not, at any stage, statistically significant. In those preparations subjected to PTM and HC-3, i.e., in the group revealing a significant reduction in ACh stores, the mean contractile amplitude during exposure to MCP showed no change from that observed before addition of MCP to the superfusate. In three of the nine muscle strips in this group, MCP produced a small «150%), slowly developing increase in contractile amplitude. In the remainder, there was either no change or a slight reduction in contractile activity, as illustrated in the lower trace of Figure 2. The important contrast to be made (see Discussion below), is that between this group and the group just superfused with HC-3. The difference between the two curves is statistically significant throughout the 20 min period. Figure 2 shows how the normal, predominantly contractile response 3 to a 10-sec train of electrical stimuli at an impulse frequency of 5 Hz is reversed to one of relaxation in the preparation previously subjected to PTM in the presence of HC-3. Conversion of this kind was seen in eight (all those tested) of the nine preparations in this group and in none of the eight strips undergoing PTM in Krebs solution alone. Discussion A number of studies lo - 14 have demonstrated how HC-3, in combination with repetitive electrical stimulation, may be used to deplete the stores of ACh at peripheral cholinergic synapses. It is probable that such depletion is produced by the combined inhibition of a high affinity transport system for precursor choline uptake and of choline acetyltransferase activity within the nerve terminal In our experiments, significant ACh depletion was observed, as expected, in only those muscle strips which had been subjected to PTM in the presence of HC-3. The degree of depletion (mean value 50-53%) 200 " u ::;: e.!? 160 1l '" c ~ e 120 c ill E 80 4 I Duration 01 exposure 10 MCP (min) Figure 1. Effect of hemicholinium-3 (HC-3) (200 I'M) and prolonged transmural stimulation (PTM) (20 Hz, for 2 hr), alone and in combination, on the response to a 20-min exposure to metoclopramide (MCP) (60 I'M). Vertical scale: mean peak contractile amplitude for consecutive 2 min periods expressed as a percentage of mean peak contractile amplitude in 7-10 min periods immediately preceding exposure to MCP. Horizontal scale: duration of exposure to MCP in min. Open circles: mean values for six preparations superfused with Krebs solution alone. Filled circles: mean values for eight preparations subjected to PTM alone. Open squares: mean values for 10 preparations exposed to HC-3 alone. Filled squares: mean values for nine preparations exposed to PTM and HC-3. Vertical bars: SEM. The letters a-d indicate p values for differences between individual points of the lowermost two curves (a, P = 0.05; b, 0.02 < P < 0.05; c, < P < 0.02; and d, P < 0.002). 20

4 March 1979 METOCLOPRAMIDE AND ACETYLCHOLINE STORES 495 MCP 60 ~ Control S MCP 60 IIA HC-3 4 s Figure 2. Effect of prolonged transmural stimulation (PTM) (20 Hz. 120 min) in the presence of hemicholinium-3 (HC-3) (200 pm) on the response to (a) a short train of transmural electrical stimuli. and (b) metoc1opramide (MCP) (60 pm). Horizontal calibration: 1 min. Vertical calibration: contractile force (g). Upper trace: recording from a strip which had previously been subjected to PTM in Krebs solution only. Lower trace: recording from a strip subjected previously to PTM in the presence of HC-3. S: transmural stimulation for 10 sec at 5 Hz. MCP added to superfusate at arrow and continuously thereafter. is in reasonable agreement with the results reported in the two studies of peripheral cholinergic synapses in which ACh pool size has been measured. 1O 14 Studies on the superior cervical ganglion of the cat" O. 14 have indicated that ACh is stored in the preganglionic nerve terminals in two forms. Between 18% and 50% of ACh is immediately available for release. whereas the remainder is in a form that can only very slowly be made available for release. The readily available store may be rapidly depleted by stimulation (in the presence of HC-3) of the preganglionic nerve at impulse frequencies of Hz. so that within min there is no detectable transmitter r e lease. lo. l~ Thus. a nerve ending may contain around 50% of its normal ACh store and yet be unable to engage in synaptic transmission. In this way may be explained our observation of conversion of the normal contractile response, induced by a short train of electrical stimuli, to one of relaxation. This occurred in eight preparations in which the ACh store had been depleted by a mean value of 50%. The same phenomenon is seen in preparations exposed to hyoscine 3 20 and indicates the uncovering of inhibitory neuromuscular systems by the abolition or blockade of synchronously activated cholinergic processes. We may reasonably conclude, therefore. that in the majority of those muscle strips exposed to PTM in the presence of HC-3. the postganglionic nerve endings were unable to release ACh for purposes of neuromuscular transmission. In designing these experiments we were careful to allow for a possible postsynaptic effect of HC-3. Bertolini et al. 21 have demonstrated an atropine-like effect of HC-3 on the guinea pig ileum, and, recently, a similar effect has been detected in rat ileum. 22 It seems probable that the small reduction in response to MCP seen in those muscle strips merely superfused with HC-3 (if, indeed, there is a true reduction) may be attributed to a postsynaptic effect of this kind, because there is no significant reduction in the ACh stores of these strips. The slight reduction in contractile amplitude produced by MCP in some of the strips exposed to PTM in the presence of HC-3-as seen, for example, in the lower trace of Figure 2--deserves some comment. Several authors have described how higher concentrations of MCP (300 /LM and above) can exert an inhibitory effect on smooth muscle activity. This effect, thought to be a nonspecific depressant action of the drug, would presumably be apparent, albeit to a lesser degree, at lower concentrations of MCP were it not for its simultaneously exerted stimulant effects. Removal of the drug's potent stimulant properties by the highly specific process of ACh depletion thus unmasks the depressant properties, which, at the lower concentration of 60 /LM. are relatively weak. We have found that PTM alone does not alter the normal stimulant response to MCP, and thus the comparison of MCP-induced responses in muscle strips merely superfused with HC-3 (to allow for any postsynaptic effect of HC-3) with those in strips

5 496 HAY AND MAN GASTROENTEROLOGY Vol. 76, No.3 subjected to PTM in the presence of HC-3 would appear to be valid. It is essentially a comparison between nerve endings able to liberate ACh and those unable to do so. Prevention of the normal response to MCP in the latter reveals, therefore, a clearcut dependence of MCP on the availability of releasable ACh for its' stimulant effect. Considered in isolation, these experiments can not be said to exclude an alternative hypothesis of sensitization of muscarinic receptors by MCP!3-25 However, an earlier study from this laboratory has demonstrated how, in the guinea pig antral preparation, at any rate, the apparent sensitization of muscarinic receptors by MCP is artefactual and can be explained on the basis of a ganglionic effect of ACh. In this context, the findings of this study must be taken as further support for an hypothesis of increased ACh release from the postganglionic nerve ending. Conversion of the normal contractile response induced by a short train of transmural electrical stimuli to one of relaxation in the ACh-depleted strips only provides evidence, of course, of functionally significant depletion at the postganglionic nerve terminal. It is likely that similar levels of depletion were achieved at preganglionic cholinergic nerve endings, and, thus, it is possible that depletion at these sites contributed to prevention of the normal response to MCP. The effect of MCP on the guinea pig antrum is unaffected, however, by the presence of hexamethonium or tetrodotoxin," and, thus, while not denying the possibility of a significant more proximal effect of MCP, restriction of the authors' considerations to cholinergic processes at the myoneural junction seems reasonable at the present time. References 1. Robinson OPW: Metoclopramide-a new pharmacological approach? 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