Brief Communication The interaction of immunomodulator muramyl dipeptide with peripheral 5-HT receptors: overview of the current state

Abstract

Immunomodulator muramyl dipeptide (MDP) exerts also pronounced neuropharmacological activities which are probably mediated by an interaction with 5-HT receptors. Some of these effects are considered as undesirable by its clinical use. More precise information concerning MDP effects on 5-HT receptors with respect to their many subtypes could result from studies using isolated organs in vitro. Earlier conducted studies of this type provided data that are concisely overviewed and reinterpreted here from the view of current 5-HT receptor classification. Since new 5-HT receptor types have emerged recently, new studies are under way. The results might contribute to the development of novel immunomodulatory drugs devoid of adverse effects.

Introduction

Muramyl peptides range among the most effective recognized substances with immunoadjuvant and immunostimulatory properties. The representative drug MDP, a synthetic analogue of bacterial peptidoglycans, surprisingly possesses besides its immunomodulatory effect also pronounced neuropharmacological activities. Some of these effects were described by research workers of our institute, such as the effects on sleep, analgesic properties or influence on behaviour (Masek, 1986; Masek et al., 1989). It was found that the immunopharmacological as well as neuropharmacological effects of MDP and some of its analogues can be significantly influenced by the substances which have relation to the serotoninergic system. Our group was successful in proving the ability of muramyl peptide group of substances interaction with 5-HT receptors more than twenty years ago. The results of independent studies by American groups (Silverman et al., 1985; Karnovsky, 1986; Silverman et al., 1989; Root-Bernstein and Westall, 1990; Silverman et al., 1990) have confirmed and further broadened these our findings. The participation of serotoninergic structures was also confirmed by binding studies performed by French scientists (Fillion et al., 1989). An important link between the serotoninergic nervous system and immune response was further highlighted by the finding that on one hand central neurotransmitters dopamine, norepinephrine and serotonin participate in immune response (Devoino et al., 1994), on the other immunomodulators of muramyl peptide group increase serotoninergic turnover in the brain (Masek, 1986).

Some neurobiological activities of model substance, MDP, are considered as undesirable by its clinical use. It is pyrogenic, it not only induces SWS sleep (Polanski and Karnovsky, 1992), but in some doses also inhibits REM sleep (Masek, 1986). It is known that partly these undesirable properties may be caused by interleukin-1, the substance with pyrogenic and somnogenic characteristics (Krueger et al., 1984a), which is released from macrophages, as well as by other cytokines. The substances might be released by MDP not only from macrophages, but also in the CNS, where the site of their release is much less clear. The mechanisms involved in the neuropharmacological effects of MDP through cytokines are however not known. The fact that by chemical modification of the MDP molecule it is possible to separate different neuropharmacological effects (Krueger et al., 1984b) suggests that different mechanisms and neurotransmitters could be involved. The activation of 5-HT receptors in MDP effects is supported by a number of data from both in vivo as well as in vitro experiments (Kadlec et al., 1984; Masek, 1986; Masek et al., 1989; Slansky et al., 1994; Slansky et al., 1996). The effects of MDP on temperature and sleep can be inhibited by the administration of PCPA ( p-chlorophenylalanine) (Masek et al., 1978), a compound which inhibits the synthesis of 5-HT. It was further proven that SWS sleep is influenced by 5-HT_1A, 5-HT₂ and 5-HT₃ receptors (Dzoljic et al., 1992; Ponzoni et al., 1993; Sharpley et al., 1994; Ponzoni et al., 1995) and that nonspecific 5-HT_1/2 antagonist methysergide inhibited hyperthermia induced in the monkey hypothalamus by prostaglandins (Simpson et al., 1994). If it could be found whether and to which extent the activation of respective subtypes of 5-HT receptors may correlate with pyrogenic and somnogenic effects of MDP, it might be possible to purposefully design optimal substances devoid of the above mentioned adverse effects.

Nowadays, serotoninergic system is considered as probably the most complicated and the most complex system of all transmitter systems at all. So far, fourteen subtypes of 5-HT receptors have been discovered. In accordance to the structure, the second messenger system coupling and responses to agonists and antagonists are divided into seven groups which are indexed from 5-HT₁ to 5-HT₇; groups 5-HT₁, 5-HT₂ and 5-HT₅ are further subdivided (Martin and Humphrey, 1994; Hoyer and Martin, 1996). While 5-HT₂ receptors are coupled to the transductional system of phospholipase C and 5-HT₃ receptors are ligand-gated cation channels, the 5-HT_1A receptors are negatively and 5-HT₄, 5-HT₆ and 5-HT₇ positively coupled to adenylate cyclase. The transductional system for 5-HT₅ receptors is not known so far. In addition to this, the existence of 5-HT₅, 5-HT₆ receptors was not proven in the periphery. Nevertheless, remaining 5-HT receptor subtypes can be investigated in, for example, guinea-pig gastrointestinal tract without any greater difficulties.

For definite characterization of the 5-HT effect and modulation by substances of immunomodulatory origin, it is very necessary to try to define the type and subtype of 5-HT receptor which mediates the effect. This article aims therefore at possible reinterpretation of some earlier results from view of up-to-date 5-HT receptor classification and current knowledge of 5-HT receptor subtype localization and function in various peripheral tissues.

The common binding sites for both 5-HT and MDP were demonstrated on myelin basic protein, LHRH and MSH-ACTH 4–10 (Root-Bernstein and Westall, 1990). Of course, these binding sites have, functionally, nothing to do with genuine 5-HT receptors; however, this observation helped to establish an important link between the immune and nervous systems and prompted further research aimed at the possible interaction between muramyl peptides and 5-HT receptors themselves. Muramyl peptides were observed to interact with 5-HT receptors on macrophages (Silverman et al., 1985; Karnovsky, 1986; Sternberg et al., 1986) which are endowed with 5-HT₂ receptors (Arzt et al., 1991); the respective subtype has not yet been determined. MDP was active on human platelets (Polanski and Karnovsky, 1992) endowed with 5-HT_2A receptors (Cook et al., 1994). Muramyl peptides further interacted with 5-HT receptors of rat C6 glioma cells (Silverman et al., 1989) that should also be of 5-HT_2A subtype (Elliott et al., 1995).

Our laboratory studied the possible interaction of MDP with the serotoninergic nervous system using isolated nerve-muscle preparations placed in an organ bath. It was found that higher MDP concentrations (1–100 mM) evoked contractions in the isolated neuromuscular preparations sensitive to 5-HT, while lower MDP concentrations (50–500 nM) modulated (potentiated) 5-HT evoked contractions or relaxations (Kadlec et al., 1984; Slansky et al., 1994), suggesting a possible interaction with postsynaptic 5-HT receptors of the smooth muscle. These observations are interesting with respect to the possible mechanism of MDP action on 5-HT receptors. Since MDP evoked direct contractions with far lower potency than 5-HT itself, one would consider MDP as a classical partial agonist; if so, low modulatory MDP concentrations should rather decrease than increase the effect of full agonist 5-HT. However, the opposite was the case. Therefore it seems that the effects of low and high MDP concentrations are mediated by different mechanisms.

With regard to the type of 5-HT receptors involved, the most interesting results were achieved with guinea-pig ileum, rat stomach strip, rat vas deferens, guinea-pig colon and carp stomach strip.

In guinea-pig ileum, electrically evoked neurogenic cholinergic twitches of myenteric plexus-longitudinal muscle (MPLM) strip were augmented in the presence of low concentrations of MDP (Kadlec et al., 1984). The effect of MDP switched from twitch potentiation to clear inhibition in the presence of mixed 5-HT_1A agonist/5-HT₂ antagonist lisuride. This observation suggests the possible involvement of 5-HT_1A as well as 5-HT₄ receptor types in the effect of MDP. It is now well established that enteric 5-HT_1A and 5-HT₄ receptors function to inhibit and facilitate transmitter release, respectively (Kilbinger and Pfeuffer-Friederich, 1985; Pan and Galligan, 1994). However, it was demonstrated that muramyl dipeptides do not bind to 5-HT_1A binding sites on bovine cortical membrane preparations (Takeuchi et al., 1990). Any other information concerning possible interaction of MDP with peripheral 5-HT receptors coupled to adenylyl-cyclase (i.e. 5-HT_1A, 5-HT₄ or 5-HT₇) is lacking to date.

It was found that directly evoked contractions of rat stomach strip by the presence of high MDP concentrations were reduced in the concomitant presence of unspecific 5-HT₂ antagonists (lisuride, methysergide, methiothepine and cyproheptadine). MDP also made this preparation more sensitive to directly evoked 5-HT contractions (Kadlec et al., 1984). 5-HT_2B receptor subtype has been recently described to be responsible for the contractions of this preparation (Martin and Humphrey, 1994).

In rat vas deferens, the enhancement of electrically evoked neurogenic twitches observed in the presence of 5-HT was further potentiated in the concomitant presence of low concentrations of MDP; this effect was blocked by 5-HT₃ antagonists (Slansky et al., 1994).

In guinea-pig proximal colon, MDP did not increase 5-HT contraction of the preparation (Slansky et al., 1994). This contraction consists of a direct (tetrodotoxin-resistant) and indirect (tetrodotoxin-sensitive) component, the former being mediated mainly via 5-HT₂ receptors, probably of 5-HT_2A type (Engel et al., 1984; Humphrey, 1984; Kojima and Shimo, 1986). However, it should not be concluded that MDP lacks any modulatory effect on 5-HT_2A receptors since it is well known that the contraction tends to fade rapidly following only a few repeated applications (Costa and Furness, 1979) due to 5-HT₂ receptor long-lasting desensitization (Rahman and Neuman, 1993); guinea-pig proximal colon therefore may not represent an ideal preparation for this kind of study. On the other hand, MDP increased 5-HT neurogenic relaxation which is probably mediated by 5-HT₃ receptors (Bogers et al., 1991; Slansky et al., 1994; Woollard et al., 1994; Sevcik et al., 1996; Sevcik et al., 1998).

Finally, in carp stomach MDP potentiated 5-HT inhibition of electrically evoked neurogenic twitches; this inhibition probably consists in nitric oxide release from interneurones endowed with 5-HT₃ receptors (Slansky et al., 1996).

The data presented above are summarized in Table 1. It seems that MDP interactions with 5-HT receptors might be rather complex and not only confined to some particular 5-HT receptor type. In our opinion, a new and exciting area of research opens since the possible participation of, in particular, recently discovered 5-HT receptor subtypes in such interactions has not been systematically investigated yet. This research, which is currently under way in our laboratory, might contribute to the development of novel immunomodulatory substances having a better pharmacological profile than drugs used so far.