Presynaptic Receptors and Neuronal Transporters: Official Satellite Symposium to the IUPHAR 1990 Congress Held in Rouen, France, on 26–29 June 1990

21:7–30.

Physiological and Pharmacological Relevance of Presynaptic Receptors in Neurotransmission

S.Z. Langer, I. Angel and H. Schoemaker,     Synthélabo Recherche (L.E.R.S.), 58 rue de la Glacière, 75013 Paris, France

ABSTRACT

During the last twenty years, the concept that noradrenaline can regulate its own release through an action on presynaptic autoreceptors, has been confirmed and extended to several neurotransmitters in the periphery and in the central nervous system. In addition, many nerve terminals possess presynaptic heteroreceptors which can be acted upon by transmitters released from adjacent terminals, by cotransmitter neuropeptides or by locally produced or blood-borne endocoids to either inhibit or facilitate transmitter release. In the noradrenergic system, the concept of presynaptic modulation of transmitter release developped in parallel with pharmacological evidence for the existence of α2-and α2-adrenoceptor subtypes. In the meanwhile three different α2-adrenoceptor clones were identified by molecular biologists that may correspond to pharmacologically different subtypes of α2-adrenoceptors. The heterogeneity of presynaptic release-modulating α2-adrenoceptors may offer the opportunity of developping selective drugs with novel and useful therapeutic properties.

KEYWORDS

Presynaptic autoreceptors

Transmitter release

Alpha adrenoceptor subtypes

Presynaptic heteroreceptors

Noradrenaline

Neuropeptide cotransmitters

Introduction

Neurotransmitters can regulate their own release through an action on inhibitory autoreceptors located on the synaptic nerve terminal (for recent reviews see Langer and Lehmann, 1988; Starke et al, 1989). The physiological role of presynaptic inhibitory autoreceptors was first established for noradrenaline in the peripheral nervous system through the demonstration of a negative feed-back mechanism through which this transmitter can modulate its own release (Langer, 1974). The pharmacological possibilities for intervention through selective agonists, partial agonists or antagonists acting on presynaptic, release modulating receptors, resulted from the characterization of the autoreceptor subtype for each of the neurotransmitters. In addition to presynaptic autoreceptors, many nerve terminals possess presynaptic heteroreceptors that are sensitive to endogenous mediators other than the neuron’s own transmitter. Activation of these terminal presynaptic heteroreceptors can either inhibit or facilitate the release of a neurotransmitter. In contrast to presynaptic terminal autoreceptors, the physiological role of presynaptic heteroreceptors remains to be established. Nevertheless, selective agonists or partial agonists should be expected to produce pharmacological effects through the activation of presynaptic heteroreceptors even if these receptors are not involved in a physiologically relevant mechanism modulating transmitter release.

While it is well established that the presynaptic autoreceptor that regulates noradrenaline release corresponds to the α2-subtype (Langer, 1974, 1981), recent molecular biology and receptor binding studies indicate that the α2-adrenoceptor is expressed as three distinct receptor subtypes (Bylund 1988, Harrison et al. 1991). The functional relevance of the three cloned α2-adrenoceptor subtypes is still an open question.

One example of a peripheral neuroeffector junction where α2-adrenoceptors are present both pre-and-postsynaptically is offered by the insulin secreting β-cells of the pancreas. The present article reports the pharmacological profile of SL 84.0418, a pyrrolo-indole derivative with high selectivity for α2-adrenoceptors in the periphery (Langer et al. 1990; Langer and Angel, 1991). This α2-adrenoceptor antagonist is at present being tested in man as a novel therapeutic approach for the treatment of type Π or non-insulin dependent diabetes mellitus (NIDDM).

Materials and Methods

The twitch response of the isolated rat vas deferens was employed to study drug effects at presynaptic α2-adrenoceptors as described by Hicks et al (1985) and the rabbit pulmonary artery was used to determine postsynaptic α2-adrenoceptor antagonism in-vitro according to Schoemaker et al (1989). Drug effects on α2-adrenoceptor agonist-induced hyperglycemia were studied according to Angel et al (1990).

Results

The pyrrolo-indole derivative SL 84.0418 is a potent, competitive antagonist of the inhibition by clonidine of the twitch response of the rat vas deferens and the pA2 is similar to that obtained with idazoxan (Table 1). SL 84.0418 has a chiral center and is a racemic mixture of two enantiomers, SL 86.0715 ((+) enantiomer) and SL 86.0714 ((-) enantiomer). The (+) enantiomer, SL 86.0715 is at least 100-fold more potent than the (-) enantiomer, SL 86.0714 (Table 1).

Table 1

Comparison of the alpha-2 and alpha-1 antagonist properties of SL 84.0418 and its enantiomers with idazoxan

(a)rat vas deferens;

(b)rabbit pulmonary artery

As shown in table 1, SL 84.0418 and its (+) enantiomer, SL 86.0715 are considerably less potent at α,-adrenoceptors, and they possess a selectivity ratio of more than 1000 when their α2-and α1-adrenoceptor antagonist properties are compared under in-vitro conditions.

As shown in Table 2, SL 84.0418 antagonized in a dose-dependent manner the hyperglycemia induced by UK 14304 in mice. This α2-adrenoceptor blocking property resides in the active enantiomer SL 86.0715 (table 2) while SL 86.0714 was much less active in this test (table 2). Consequently both in-vitro and in-vivo SL 84.0418 and 86.0715 elicit α2-adrenoceptor antagonist effects.

Table 2

STEROSPECIFICITY OF SL 84.0418 AGAINST HYPERGLYCEMIA MEDIATED BY ACTIVATION OF ADRENOCEPTORS IN MICE

Discussion

The novel α2-adrenoceptor antagonist SL 84.0418 and its active enantiomer SL 86.0715 represent the most selective compound so far reported in this pharmacological class. From our experimental models it appears that SL 84.0418 possesses similar potency as an antagonist of presynaptic as well as postsynaptic α2-adrenoceptors. For exemple, in the in situ blood perfused dog pancreas SL 84.0418 enhances the release of endogenous noradrenaline during sympathetic nerve stimulation in the same range of doses in which it antagonizes the inhibition of insulin release induced by activation of postsynaptic α2-adrenoceptors (Duval et al., 1991). In contrast to idazoxan, which blocks both central and peripheral α2-adrenoceptors, upon oral administration, SL 84.0418 antagonizes preferentially the responses induced by activation of peripheral α2-adrenoceptors (Langer and Angel, 1991; Angel et al. 1991b). Further to the antagonism of alpha-2 agonist induced hyperglycemia, in the monkey the oral administration of SL 84.0418 or SL 86.0715 reduce in a dose-dependent manner the hyperglycemia induced by an oral glucose load (Hulbron et al, 1990). Similar results were obtained in man, at doses devoid of cardiovascular and other side effects (Bergougnan et al. 1990).

The selectivity of SL 84.0418 for the three reported subtypes of α2-adrenoceptors (Harrison et al. 1991) remains to be established, and it would be of interest to characterize the subtype of α2-adrenoceptor which modulates presynaptically noradrenaline release and the postsynaptic α2-adrenoceptor associated with the inhibition of insulin release in the pancreas.

Conclusions

The development of novel and selective compounds acting preferentially as α2-adrenoceptor antagonists may offer novel therapeutic approaches in the treatment of several diseases. The selective α2-adrenoceptor antagonist SL 84.0418 is a potential drug for the treatment of type Π non insulin dependent diabetes. Different pharmacological interventions are possible at the level of presynaptic release modulating receptors. These compounds may become important pharmacological tools and most significantly, novel and useful therapeutic agents.

Acknowledgement

The authors wish to thank Miss Anny Sebbah for preparing the manuscript.

References

1. ANGEL, I., NIDDAM, R., LANGER, SZ. Involvement of alpha-2 adrenergic receptor subtypes in hyperglycemia. J. Pharmacol. Exp. Ther. 1990; 254:877–882.

2. ANGEL, I., SCHOEMAKER, H., ARBILLA, S., GALZIN, AM, BERRY, C., NIDDAM, R., PIMOULE, C., SEVRIN, M., WICK, A., LANGER, SZ. SL 84.0418: A novel, potent and selective alpha-2 adrenoceptor antagonist: I. In vitro pharmacological profile. J. Pharmacol. Exp. Ther. 1991. [(Submitted).].

3. ANGEL, I., GROSSET, A., PERRAULT, G., SCHOEMAKER, H., LANGER, SZ. SL 84.0418: A new, potent and selective alpha-2 adrenoceptor antagonist with preferential peripheral activity. II: In vivo pharmacological profile. J. Pharmacol. Exp. Ther. 1991. (Submitted).

4. BERGOUGNAN, L., ROSENZWEIG, P., DUCHIER, J., COURNOT, A., BERLIN, I., MORSELLI, PL. SL 84.0418: Clinical and cardiovascular tolerance in healthy young volunteers of a new alpha-2 antagonist with anti-hyperglycaemic properties. Eur. J. Pharmacol. 1990; 183:1015–1016.

5. BYLUND, DB. Subtypes of α2-adrenoceptors: pharmacological and molecular biological evidence converge. TIPS. 1988; 9:356–361.

6. DUVAL, N., ANGEL, I., EON, MT, OBLIN, A., LANGER, SZ. Involvement of alpha-2 and beta-adrenoceptors in insulin release induced by pancreatic nerve stimulation. J. Pharmacol. Exp. Ther. 1991. [(Submitted).].

7. HARRISSON, JK, PEARSON, WR, LYNCH, K. R. Molecular characterization of α1 and α2-adrenoceptors. TIPS. 1991; 12:62–67.

8. HICKS, PE, LANGER, SZ, MACRAE, AD. Differential blocking actions of idazoxan against the inhibitory effects of 6-fluoronoradrenaline and clonidine in the rat vas deferens. Br. J. Pharmacol. 1985; 86:141–150.

9. HULBRON, G., ANGEL, I., OBLIN, A., FRIEDMANN, JC, LANGER, SZ. Antihyperglycaemic effects of the new alpha-2 antagonist SL 84.0418 in the primate (macaca fascicularis) in comparison with idazoxan. Eur. J. Pharmacol. 1990; 183:995–996.

10. LANGER, SZ. Presynaptic regulation of catecholamine release. Biochem. Pharmacol. 1974; 23:1793–1800.

11. LANGER, SZ. Presynaptic regulation of the release of catecholamines. Pharmacol. Rev. 1981; 32:337–362.

12. LANGER, SZ, SCHOEMAKER, H., ANGEL, I., ARBILLA, S., PIMOULE, C., GROSSET, A., PERRAULT, G., SEVRIN, M., WICK, A. SL 84.0418: A new, potent and selective alpha-2 adrenoceptor antagonist with peripheral activity. Eur. J. Pharmacol. 1990; 183(3):802.

13. LANGER, SZ, LEHMAN, J.Trendelenburg U., Weiner N., eds. Presynaptic receptors on catecholamine neurons. Springer Verlag:, 1988:419–507. [Handb. Exp. Pharmacol. 90 (Catecholamines 1)].

14. LANGER, SZ, ANGEL, I. Pre-and postsynaptic alpha-2 adrenoceptors as targets for drug discovery. J. of Neural Transmission. 1991. [(in press).].

15. SCHOEMAKER, H., BLANCHARD, H., PIMOULE, C., LEFEVRE-BORG, F., MANOURY, P., JARDIN, A., LANGER, SZ. Characterization of the effects of alfuzosin on α1-adrenoceptors in the genito-urinary tract. In: Prostate et alpha-bloquants. Amsterdam: Excerpta Medica; 1989:328–343.

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Pre- and Postjunctional Muscarinic Receptors in the Guinea-pig Trachea

H. Kilbinger, D. Wolf and G. D’Agostino,     Department of Pharmacology, University of Mainz, D 6500 Mainz, Germany

ABSTRACT

The effects of M2- and Me-selective muscarinic antagonists on electrically evoked [³H]acetylcholine release and muscle contraction were compared in the isolated guinea-pig trachea. The M2-selective antagonists methoctramine and AF-DX 116 were more potent in enhancing the evoked release than in inhibiting the contractile response. As a consequence of the selective blockade of the inhibitory autoreceptors the evoked muscle contractions were enhanced by low concentrations (0.1 μmol/l) of the M2-selective antagonists. The Me-selective antagonists 4-DAMP, UH-AH 37 and pFHHSiD were more potent in reducing the contraction than in facilitating the evoked release. Surprisingly, HHSiD did not discriminate between pre- and postjunctional receptors. It is concluded that autoinhibition of acetylcholine release is mediated via an M2-like receptor whereas muscle contraction is due to stimulation of an M3-receptor.

KEYWORDS

Trachea

acetylcholine release

contraction

presynaptic autoreceptors

M2 receptors

M3 receptors

INTRODUCTION

The release of acetylcholine from the vagus nerve plays a dominant role in the regulation of airway smooth muscle tone. This release can be inhibited by prejunctional muscarinic autoreceptors as has been demonstrated in functional studies (see Maclagan and Barnes, 1989) and more recently in release experiments (D’Agostino et al., 1990). The objective of the present study was to characterize pharmacologically the muscarinic autoreceptor in the isolated guinea-pig trachea. For this purpose the pre- and postjunctional potencies of muscarinic antagonists were compared on a novel preparation which allows the simultaneous determination of acetylcholine release and smooth muscle contraction (D’Agostino et al., 1990). Muscarinic receptors can be subdivided pharmacologically into at least the three subtypes M1, M2 and Ma (see Hulme et al., 1990). The postjunctional receptor of the guinea-pig trachea has been classified as M3 receptor (Eglen and Whiting, 1988). The drugs tested in this study comprised non-selective (ipratropium), M1-selective (pirenzepine), M2-selective (methoctramine, AF-DX 116), and M3-selective [4-DAMP, UH-AH 37, hexahydrosiladifenidol (HHSiD), p-fluorohexahydrosiladifenidol (pFHHSiD)] muscarinic antagonists.

METHODS

Experiments were performed on a clip-connected epithelium-free trachea strip which has been described in detail recently (D’Agostino et al., 1990). The strip was incubated for 60 min in a 2 ml organ bath with (³H]choline (2.5 μCi/ml) and subsequently superfused with Tyrode solution. After a 90 min washout with medium that contained in addition hemicholinium-3 (10 μmol/l) the superfusate was collected in 3 min fractions, and tritium was determined by liquid scintillation spectrometry. For electrical stimulation square wave pulses of 1 ms duration were applied through platinum electrodes at a frequency of 20 Hz (6 trains of 100 pulses in intervals of 30 s). Five stimulation periods (S1-S5) were carried out in 24 – 27 min intervals on a single trachea preparation. Contractions of the smooth muscle were recorded simultaneously. Muscarinic antagonists were added after S1 to the perfusion medium in cumulatively increasing concentrations so that complete concentration-response curves for pre- and postjunctional effects were obtained in each experiment. For ECso determinations, concentrations of antagonists causing 50% of the maximal increase in evoked [³H]acetylcholine outflow, or inhibiting the evoked contractions by 50% were calculated by linear regression analysis.

RESULTS AND DISCUSSION

All antagonists produced concentration-dependent increases in the electrically evoked outflow of [³H]acetylcholine. None of the compounds affected the basal outflow. The increase in outflow by low concentrations of the M2-selective antagonists methoctramine (0.1 and 1 μmol/l) and AF-DX 116 (0.1 μmol/l) was paralleled by a significant enhancement of the stimulation-evoked contractile response. Higher concentrations of these drugs inhibited the contractions. The M3-selective antagonists UH-AH 37, 4-DAMP and pFHHSiD inhibited the stimulation-evoked contractions already at concentrations that did not yet significantly increase the evoked outflow.

The negative logarithms of the pre- and postjunctional ECso values are given in Table 1. Pirenzepine had similar pre- and postjunctional potencies which indicates that the autoreceptor is not an M1 receptor. M2-selective antagonists (methoctramine, AF-DX 116) were more potent at the prejunctional, and the Ma-selective antagonists UH-AH 37, 4-DAMP and pFHHSiD at the postjunctional receptor. These findings suggest that the muscarinic autoreceptor belongs to the M2 subtype. However, HHSiD did not discriminate between pre- and postjunctional muscarinic receptors, although HHSiD is known to be about 50times more potent at M3 than at M2 receptors (Mutschler and Lambrecht, 1984; Fuder et al., 1985). Moreover, HHSiD and AF-DX 116 were equipotent at the prejunctional muscarinic receptor whereas both antagonists differ by a factor of ten in their affinities to cardiac M2 receptors (pA2 values: HHSiD 6.3, Fuder et al., 1985; AF-DX 116, 7.3, Micheletti et al., 1987). The findings with HHSiD cannot be explained by the occurrence of a mixture of prejunctional M2 and M3 receptors since the other selective antagonists clearly discriminated between both receptor subtypes.

Table 1

Comparison of pre- and postjunctional potencies of muscarinic antagonists in the isolated trachea

For a further analysis the pre- and postjunctional -log ECso values (pD2) were plotted against the pA2 values of the antagonists at M2 and M3 receptors. The left panel of Fig. 1 shows that a significant correlation (r=0.947) exists between the postjunctional pD2 values of the present study and pA2 values obtained at postjunctional M3 receptors. There is, on the other hand, a poor correlation (r=0.482) between the prejunctional pD2 values and pA2 values of the subtype-selective antagonists determined at cardiac M2 receptors (right panel). A significant correlation (r=0.906) is, however, obtained if HHSiD and methoctramine are omitted from the calculation. We therefore conclude that the prejunctional muscarinic receptor in the guinea-pig trachea is not a typical M2 receptor. The classification of muscarinic receptor subtypes has recently been extended by the cloning of 5 distinct subtypes (Bonner, 1989). Binding studies of individual cloned muscarinic receptors showed that the m4 gene product differed from the M2 receptor by its higher affinity to HHSiD and its lower affinity to methoctramine (Buckley et al., 1989). A similar picture is seen in Fig. 1: the prejunctional potency of HHSiD is higher and that of methoctramine is lower than that at a typical M2 receptor. A classification of the autoreceptor as an M4 subtype appears, however, premature until selective antagonists for the M4 receptor become available.

Fig. 1 Left panel: Correlation between postjunctional pD2 values of muscarinic antagonists and pA2 values determined at M3 receptors in guinea-pig trachea. Right panel: Correlation between prejunctional pD2 values of antagonists for the muscarinic receptor in guinea-pig trachea and pA2 values obtained at the atrial M2 receptor. pA2 values are taken from the literature: pirenzepine (PZ) (Eglen and Whiting, 1988; Fuder et al., 1982); methoctramine (MO) (Eglen and Whiting, 1988; Melchiorre, 1988); AF–DX 116 (Micheletti et al., 1987); 4-DAMP (Eglen and Whiting, 1988; Barlow et al., 1976); UH-AH 37 (UH) (Eberlein et al., 1989); pFHHSiD (pF) (Eglen et al., 1990; Lambrecht et al., 1988); HHSiD (HS) (Kilbinger, unpublished; Fuder et al., 1985); ipratropium (IPR) (Van Charldorp et al., 1989).

Acknowledgement

Supported by the Deutsche Forschungsgemeinschaft (Ki 210/6–4).

REFERENCES

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