Presynaptic modulation by noradrenaline and an opioid of the substance P-induced release of [3H]acetylcholine from the myenteric plexus

Regulatory Peptides, 12 (1985) 317-325

317

Elsevier RPT 00429

Presynaptic modulation by noradrenaline and an opioid of the substance P-induced release of [3H]acetylcholine from the myenteric plexus E.S. Vizi and L. Bartho Institute of Experimental Medicine, Hungarian Academy of Sciences, P.O. Box 67, 1-1-1450Budapest, and Department of Pharmacology, University Medical School of Pecs, 1-1-7643Pecs, Hungary

(Received6 June 1985;revisedmanuscript received 5 September 1985;acceptedfor publication 10 September 1985)

Summary

Substance P (7.5-750 nM) applied in superfusion dose-dependently released 3H from isolated strips of myenteric plexus-longitudinal muscle of the guinea-pig ileum loaded with [3H]choline. Separation of the [3H]acetylcholine and [3H]choline components of the released radioactivity revealed that in response to substance P (SP) administration only the release of [3H]acetylcholine increased above resting level. A slowly developing tachyphylaxis to the effect of SP was observed. Evidence has been obtained that the slow tachyphylaxis developed to the acetylcholine-releasing effect of SP was not due to the exhaustion of releasable acetylcholine pool. Release of acetylcholine by 150 nM SP was completely prevented by tetrodotoxin or in a Ca 2 +-free medium and greatly reduced in the presence of noradrenaline or the opioid receptor agonist (D-Met 2,Pro 5)-enkephalinamide. The effect of noradrenaline and the opioid peptide was apparently prevented by yohimbine and naloxone, respectively. enteric nervous system; (D-Met 2 ,Pro 5)-enkephalinamide; alpha2-adrenoceptors; opioid receptors; acetylcholine release; choline kinase technique

Introduction

There is good evidence to suggest that the undecapeptide substance P (SP) contracts the longitudinal muscle of the guinea-pig small intestine by a direct action on the smooth muscle cells [1-4]. However, myenteric neurons are excited by SP [5]. Pharmacological evidence exists that small, sub-contractile concentrations of SP may 0167-0115/85/$03.30 © 1985ElsevierSciencePublishers B.V. (BiomedicalDivision)

318 facilitate electrically evoked release of acetylcholine (ACh) [6] and that higher concentrations of SP might release ACh on their own [7]. Recently neurochemical evidence has also been presented for the release of radioactivity by SP from myenteric neurones preloaded with labelled choline [8]. The aim of the present study was to provide convincing neurochemical evidence to show (i) whether SP was able to induce a dose-dependent release of [3H]ACh from the myenteric plexus of the guinea-pig ileum in a constant superfusion system; (ii) if so, whether desensitization occurs to this action; (iii) whether activation of opioid receptors or alpha2-adrenoceptors, which is known to reduce electrically evoked ACh release [9], could influence SP-induced liberation of ACh as well.

Materials and Methods

Strips of guinea-pig ileum containing the longitudinal muscle with the myenteric plexus attached were prepared according to Paton and Vizi [10]. Pre-incubation of the tissue with [3H]choline (4/iCi/ml) was carried out as described earlier [11]. Following pre-loading, the strips were constantly superfused at 37°C with Krebs-Henseleit solution (NaC1 113, KC1 4.7, CaC12 2.5, KH2PO4 1.2, MgSO4 1.2, NaHCO3 25 and glucose 11.5 mM) containing hemicholinium-3 (10/~M), at a rate of 1.25 ml/min. Collection of 3-min fractions started 60 min after the pre-loading had been finished. Radioactivity of the samples was determined via liquid-scintillation spectrometry. The release was expressed in percent of resting release (100%) and in Bq/g. At the end of each experiment the radioactive content of the strips was determined as described earlier [12] and was found to be 207 241 + 25 940 Bq/g (n = 24). Labelled choline and ACh were separated quantitatively by reacting choline with choline kinase as suggested by Rand and Johnson [13] and described by Vizi et al. [12]. Drugs were applied via the superfusing solution. Supramaximal stimulation (> 10 V/cm, 1 ms) was applied, as described earlier [10]. Substance P solutions were superfused from polypropylene vessels. Great care was taken to siliconize glassware when SP was perfused. Noradrenaline and the opioid agonist (D-Met2,ProS)-enkephalinamide were administered 12 min before and during SP superfusion; application of the antagonists (yohimbine and naloxone) was started 3 min before the respective agonists. In some cases, at the end of the experiments field stimulation (rectangular impulses of supramaximal voltage, 1 ms duration at a frequency of 1 Hz) was applied for 3 min to check the releasable pool of [aH]ACh. Drugs used were: (D-Met2,ProS)-enkephalinamide (Institute for Drug Research, Budapest, Hungary), 1-noradrenaline bitartrate (BDH), Substance P (Peninsula), tetrodotoxin (Sankyo), yohimbine (Sigma), naloxone-HC1 (donated by Endo Laboratories), physostigmine sulfate (Sigma), [all]choline chloride (Amersham, 15 Ci/ mmol). A statistically significant elevation of [3H]ACh and [all]choline release was assessed using Student's one-sample t-test. When different groups were compared two-sample t-test was used.

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Fig. 1. Concentration-dependent release of 3H by SP (7.5-750 nM, superfused for 9 rain). The average 3H release of two fractions immediately preceding SP administration was considered as basal release. Values obtained after cessation of SP superfusion are depicted with a dotted line (mean q- S.E.M.). Significant elevations of 3H release (P < 0.05 or less) are indicated by dots. For the highest concentration of SP n = 5; in the two other groups n = 6.

Experimental Results

Release of [aH]ACh by SP The release of radioactivity at rest was constant and amounted to 1641 + 51 Bq/g

per 3 min (n = 21). The preparations were exposed to SP 90-120 min after the incubation with [3H]choline had been completed. SP (7.5-750 nM for 9 min) caused a concentration-dependent release of 3H (Fig. 1); an apparently linear TABLE I Radioactivity released as [3H]ACh and [3H]choline from the myenteric plexus preparation at rest and in response to substance P administration n

Resting Substance P (150 nM)

Radioactivity in the sample collected in 3 min Total radioactivity (Bq/g)

[3H]choline (Bq/g)

% of total

[3H]ACh (Bq/g)

% of total

6 6

1 528 + 141 3 346 + 215

751 795

49.2 23.8

777 2 551

50.8 76.2

P

<0.01

>0.5

<0.01

For separation of [3H]ACh and [3H]choline see Rand and Johnson [13] and Vizi et al. [12]; n = number of experiments. Physostigmine sulfate, 10 #M, was present throughout the experiments. Mean + S.E.M. The second 3-rain fractions following the onset of substance P infusion were studied. Note that the release of radioactivity in response to SP is due exclusively to [3H]acetylcholine release. 3H content after (228 500 Bq/g, n = 6) and before (285 610 Bq/g, n = 6) the experiments suggests that 19.9% of the radioactivity was released during our experiments.

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concentration-response relationship was found in this concentration range. The increase in aH outflow induced by 750 nM SP was more than 3-fold. SP (150 nM for 18 min) failed to induce any increase in 3H release in the presence of tetrodotoxin (0.5 aM) or in a calcium-free medium (n = 4 each, data not shown). In order to study the effect of SP on [3H]ACh release in a series of experiments, physostigmine (10 pM) was also added to the perfusion fluid. The resting release of radioactivity (1538 +, 51 Bq/g, n = 35) was relatively constant from experiment to experiment. The released radioactivity, at rest, was about equally divided between [aH]ACh and [all]choline (Table I). When the tissue was exposed to SP (150 nM for 18 min) there was a highly significant increase in total radioactivity. The increase was entirely due to [3H]ACh (Table I), indicating that the radioactivity released by SP was exclusively ACh. Therefore, in further experiments (without physostigrnine) it was understood that the radioactivity released by SP above the resting release is due to [3H]choline liberated by the hydrolysis of the released [aH]ACh. Six preparations were exposed to SP (150 nM) for 45 min. A fairly long-lasting stimulation of 3H release was found; however, this effect invariably faded away within 30 rain (Fig. 2). In 5 of these preparations, still in the presence of SP, 1 Hz electrical stimulation was applied for 3 min, 36 min after superfusion of SP had been started. Electrical stimulation caused a prominent release of 3H in all these preparations, the peak reaching 478 ± 4% (mean ± S.E.M.) of the pre-stimulation value. Thus, considerable amounts of releasable 3H were still present in preparations in which SP could not liberate more ACh. Modulation of SP-indueed ACh release In the presence of (D-Met2,ProS)-enkephalinamide (0.5 pM) or noradrenaline (1 #M) SP (150 nM for 18 min) induced only small, if any, increase in aH output (Fig. 3). Total 3H release (Bq/g) measured during the 18 min superfusion of SP (150 nM) was significantly lower (P < 0.001) than that induced by the same concentration of SP alone (i.e. in the first 18 min of SP application in the experiments shown in Fig.

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Fig. 3. Release of aH by SP (150 nM, superfused for 18 rain) in the presence of (o-Met2,ProS)-enkephalinamide (Enk; n = 6), noradrenaline (NA; n = 5) yohimbine plus noradrenaline (Yoh + NA; n = 5), naloxone plus (D-Met2,ProS)-enkephalinamide (Nal + Enk). Concentrations used were 1 gM for noradrenaline and yohimbine, 0.5 #M for (D-Met2,ProS)-enkephalinamide and 0.3 #M for naloxone. Symbols as in Fig. I. Values are corrected for the decrease in basal release.

2). The inhibitory action of noradrenaline and the opioid peptide was apparently prevented by the alpha2-adrenoceptor blocking drug yohimbine (1 #M) and the opioid antagonist naloxone (0.3 #M), respectively (Fig. 3).

Discussion

Release of [3H]ACh by SP In agreement with the results of Yau and Youther [8], we found that SP was able to induce concentration-dependent release of radioactivity from the myenteric plexus of the guinea-pig ileum preloaded with [3H]choline. With the technique developed for the separation of [aH]ACh and [3H]choline, it was possible to provide direct evidence for the first time that the increase of 3H in response to SP administration was due entirely to [3H]ACh. The release evoked by SP was calcium-dependent and TTX-sensitive. Unlike those of Yau and Youther [8], our results were obtained in a constant superfusion system. This may be an important factor, since Trout and Burleigh [14] argued that release of [3H]ACh from the guinea-pig taenia caeci may be a consequence of the mechanical distortion (contraction) of the tissue when SP is added to the organ bath. While this study was being carried out Fosbraey et al. [15] and Kilbinger et al. [16] also reported release of 3H due to application of SP in superfusion. In the experiments of Yau and Youther [8] lower concentrations of SP were sufficient to evoke a consistent release of ACh than in those studies (including the present one) in which SP was superfused. This may be related to the mode of application; however, tetrodotoxin abolished SP-induced release of ACh both in the present experiments and in those of Yau and Youther [8], but not in the taenia [14].

322 Essentially all the above results indicate that, in the guinea-pig ileum, higher concentrations of SP are needed to evoke ACh release than those sufficient to contract the longitudinal smooth muscle.

Fading of the action of SP In pharmacological experiments the shape of longitudinal contractions of the guinea-pig ileum due to high concentrations of SP varies with the method of recording. Under isotonic conditions the contraction is usually maintained for long periods; this late plateau contraction, unlike the initial peak response, is cholinergic in nature [7,17]. On the other hand, if isometric or semi-isometric (auxotonic) recordings are made the tone of the preparation usually returns to the baseline level within 5-10 min [18-20]. Now it seems clear that intestinal smooth muscle and neurones have SP-sensitive receptors of different characteristics [7,15,16,21,22]. While smooth muscle receptors seem to show rapid desensitization to SP (s.ee among others [18,23-25]), the present experiments indicate a comparatively slow onset of desensitization against the neuronal stimulatory action of SP (150 nM). If the fading itself reflects receptor desensitization, temporary exhaustion of SP-activated ACh stores or other mechanisms remains to be elucidated. It should be noted, however, as a mechanism of fading of SP action. This suggestion was supported by our findings that 1 Hz electrical stimulation was still able to induce a large increase in [aH]ACh release even when the preparations had been rendered insensitive to SP. Modulation of SP-induced ACh release Pharmacological sensitivity of neuropeptide-induced ACh release from enteric neurons (Table I) may be a physiologically relevant factor. Both SP [27] and cholecystokinin-related peptides [28] have been proposed to play a role in the mechanism of peristalsis in the guinea-pig ileum. The strong inhibitory action of noradrenaline and (D-Met2,ProS)-enkephalinamide on SP-induced release of [3H]ACh indicates effective presynaptic modulation of transmitter release through adrenergic and opioid receptors, respectively, from enteric cholinergic neurones activated by SP. That these effects are mediated via specific receptors is further corroborated by the findings that the action of noradrenaline was apparently prevented by yohimbine, an antagonist generally assumed to act at alpha2-adrenoceptor sites and that of (D-Met2,ProS)-enkephalinamide by naloxone. It should be noted that (D-Met2,ProS)-enkephalinamide has a pharmacological profile similar to that of normorphine [29]. Sympathetic alpha receptor stimulation and opioids have a well-known inhibitory action on electrically induced ACh release from enteric neurones [9,30,31] and under physiological conditions there is a non-synaptic interaction between noradrenergic and cholinergic neurones: noradrenaline released from axon terminals inhibits the release of ACh [32]. While electrical stimulation is most likely to excite neurones in a non-selective manner it remains uncertain whether drugs that also induce ACh release, such as ganglionic stimulants or neuropeptides [8,33-35] excite the same or different population of cholinergic neurones. In fact, somatostatin has been reported to inhibit caerulein-, neurotensin- or electrical stimulation-induced release of [3H]ACh [36-37,39]

323

while leaving the ACh-liberating effect of SP uninfluenced [37]. Noradrenaline inhibits ACh release induced by cholecystokinin octapeptide, the pharmacologically related peptide caerulein, or ganglionic stimulants [33-35]. There is pharmacological evidence that opioid receptor activation inhibits release of ACh induced by cholecystokinin-related peptides [38]. Likewise, sustained contraction evoked by high concentrations of SP in the guinea-pig ileum are suppressed by opioid receptor agonists and enhanced by naloxone [17]. Thus, pharmacological and neurochemical data suggest that there is no major difference in the pharmacological sensitivity of ACh release, induced either by SP, cholecystokinin-related peptides or ganglionic stimulants, as far as the modulatory role of adrenoceptors and opioid receptors is concerned. Since TTX prevented the ACh-releasing effect of SP, it seems likely that the site of action of SP is proximal to the axon (e.g. on the axon hillock or on the cell body); it might enhance the firing rate and thereby augment the release of ACh.

Acknowledgements This work was supported by the Scientific Research Council, Ministry of Health, Hungary, Grants No. 06/7-28/559 and 3-04-01014)2/V. (D-Met2,ProS)-Enkephalinamide was a generous gift of Drs. J.I. Szekely and T. Lang (Institute of Drug Research, Budapest, Hungary).

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325 37 Teitelbaum, D.H., O'Dorisio, T.M., Perkins, W.E. and Gaginella, T.S., Somatostatin modulation of peptide-induced acetylcholine release in guinea-pig ileum. Am. J. Physiol., 246 (1984) G509--G514. 38 Zetler, G., Antagonism of cholecystokinin-likepeptides by opioid peptides, morphine and tetrodotoxin, Eur. J. Pharmacol., 60 (1979) 67-77. 39 Vizi, E.S., Horvath, T. and Somogyi, G.T., Evidence that somatostatin releases endogenous substance(s) responsible for its presynaptic inhibitory effect on rat vas deferens and guinea-pig ileum, Neuroendocrinology, 39 (1984) 142-148.