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The distribution and some pharmacological properties of centrally located dextromethorphan high-affinity binding sites were investigated by in vitro autoradiography.
Sodium chloride (50 mm) induced a 7 to 12 fold increase in dextromethorphan binding to rat brain in all areas tested. The effect of sodium was concentration-dependent with a higher dose (120 mm) exerting a smaller effect on binding.
[3H]-dextromethorphan binding in the presence of sodium was inhibited in the presence of the anticonvulsant phenytoin at a concentration of 100 μm, while the σ ligand (+)-3-(-3-hydroxyphenyl)-N-(1-propyl)pipendine ((+)-PPP) had no effect on the binding, suggesting an interaction with the DM2 site.
The distribution of the sodium-dependent binding identified in this study correlated significantly with the distribution of the selective 5-HT uptake inhibitor [3H]-paroxetine, and paroxetine and dextromethorphan mutually displaced their binding at concentrations in the low nanomolar range.
These data show that dextromethorphan and paroxetine share a sodium-dependent high affinity binding site in rat brain, and suggest that dextromethorphan might interact, in the presence of sodium, with the 5-HT uptake mechanism in rat brain.
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The functional role of these dextromethorphan high affinity binding sites has yet to be fully elucidated, but the selective displacement of dextromethorphan from these sites by other centrally acting antitussives such as 3-substituted 17-methyl morphinan analogues, and the efficacy of these substances as neuroprotectants and anticonvulsants (Newman et al., 1992) suggest their involvement in the pharmacological actions of dextromethorphan and its derivatives.
Phenytoin and ropizine have been shown to produce a concentration-dependent allosteric increase in the affinity of dextromethorphan to a subpopulation of dextromethorphan high affinity binding sites in guinea-pig (Musacchio et al., 1988) and rat brain homogenates (Klein & Musacchio, 1992). The subsequent observation of a differential effect of phenytoin and other anticonvulsants on dextromethorphan binding in different areas of guinea-pig brain (Canoll et al., 1990) in an autoradiographical study and the computer-assisted modelling of homologous and heterologous competition studies between dextromethorphan and the σ ligand (+)-3-(-3-hydro-xyphenyl)-N-(1-propyl)piperidine ((+)-PPP) (Zhou et al., 1991; Klein & Musacchio, 1992), has led to a model in which dextromethorphan would be binding in guinea-pig and rat brain to two high affinity and one low affinity site. One of the two high affinity sites, termed DM1 by Musacchio et al. (1989), is allosterically enhanced by phenytoin and ropizine (Musacchio et al., 1988; Klein & Musacchio, 1992) and could therefore be involved in the anticonvulsant action of dextromethorphan. The DM1 site has properties in common with the binding site for (+)-PPP, and should therefore be regarded as a DM1/σ1 site (Klein & Musacchio, 1990). By comparison, (+)-PPP only shows a micromolar affinity for the other high affinity binding site (Zhou & Musacchio, 1991; Klein & Musacchio, 1992). DM2, therefore does not represent a σ site, and binding to this site is inhibited (Canoll et al., 1990; Klein & Musacchio, 1992) by phenytoin and ropizine.
SKF 525-A, a wide spectrum inhibitor of cytochrome P-450 (Schenkman et al., 1972), and GBR 12909, a dopamine uptake inhibitor with high affinity for cytochrome P450IID1 (Niznik et al., 1990), have been shown to displace dextromethorphan binding from the DM1/σ1, site (Klein et al., 1991), and similar findings have suggested that this site can be identified with a metabolic site.
Antitussive properties have never been described for σ site ligands, thus the DM2 high affinity site may play a key role in the antitussive action of dextromethorphan. The DM2site is relatively less abundant in guinea-pig than in rat brain, where it represents the majority of the binding (Klein & Musacchio, 1992). Most of the behavioural tests that have highlighted the antitussive, anticonvulsant and neuroprotectant properties of dextromethorphan have been conducted in the rat. This prompted us to characterize in more detail dextromethorphan binding in rat brain, in order to elucidate the identity of the DM2 binding site in this species.
As part of the characterization we have considered the possibility that dextromethorphan might interact with the uptake process for 5-hydroxytryptamine (5-HT). α(—)-Trans-4 - (p-fluorophenyl)-3-[3,4 - methylenedioxyphenoxy-methyl]pi-peridine (paroxetine) is a very potent and selective inhibitor of sodium-dependent 5-HT uptake in vitro (Buus-Lassen, 1978; Habert et al., 1985) and in vivo (Graham & Langer, 1988). We have therefore used it in the present study to examine the possible link between dextromethorphan and the sodium-dependent neuronal 5-HT uptake mechanism.
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The present work provides autoradiographical evidence for the existence of a sodium-dependent fraction of dextromethorphan binding to rat brain. This sodium-dependent binding, characterized for the first time in this study, constitutes the majority of dextromethorphan binding to rat brain, and shares most of the pharmacological properties with the DM2 site identified in previous studies (Zhou et al., 1991; Klein & Musacchio, 1992).
In particular, the sodium-dependent binding was unaffected by a concentration of (+)-PPP equal to 4 times the Kd value for the common DM1/σ1 site in the rat (Klein & Musacchio, 1992), and it was only minimally affected (8 and 11% reduction respectively) by concentrations of SKF 525-A and GBR 12909 equal to three times their Kd for the DM1/σ1 site (Klein et al., 1991). Dextromethorphan binding in guinea-pig brain has been shown to be reduced by about 50% by these two cytochrome P450 inhibitors (Klein et al., 1991).
The present study showed for the sodium-dependent binding of dextromethorphan an average Kd of 14.7 nM; that is appreciably lower than the Kd for the DM1/σ1 site in rat brain (47 nM, Klein & Musacchio, 1992). This difference in Kd between the DM1/σ1 and the DM2 site might also be reflected in the higher value of Kd in the cerebellum. The cerebellum was the only area in which dextromethorphan binding was not affected by sodium, and could therefore present relatively high levels of DM1/σ1 binding.
Phenytoin significantly decreased the sodium-dependent binding of dextromethorphan. This anticonvulsant was shown to interact with the voltage-dependent sodium channel in rat brain (Francis & McIntyre-Burnahm, 1992), and its effect on sodium-dependent binding might be either the result of a direct competition with dextromethorphan or an indirect effect following the action on the sodium channel.
The proposed identity of the sodium-dependent dextromethorphan binding with 5-HT uptake and the requirement of external sodium for this process seem to suggest a direct interaction of phenytoin with the dextromethorphan binding site or to a connected allosteric site.
Previous studies on the distribution and characteristics of dextromethorphan binding to guinea-pig and rat brain have all been conducted in sodium phosphate 50 mM (Musacchio et al., 1988; Klein & Musacchio, 1992), and therefore reflect the properties of both the sodium-dependent and non-dependent binding site.
A specific effect of sodium following the addition of sodium chloride to the incubation buffer is supported by the analogous effect on binding exerted by sodium phosphate (data not shown).
This effect of sodium led us to investigate the possible interaction of dextromethorphan with a sodium-dependent uptake mechanism. The particularly high levels of sodium-dependent dextromethorphan binding in the medial mammil-lary, ventral tegmental area and dorsal raphe prompted us to examine the relationship between dextromethorphan binding and the 5-HT uptake mechanism.
The significant colocalization between dextromethorphan binding and the selective 5-HT uptake inhibitor paroxetine, and their mutual displacement strongly suggest an interaction of dextromethorphan at this site. Competition of these two ligands at a metabolic site is very unlikely, since the two cy-tochrome P450 inhibitors did not affect the majority of binding, and this enzyme is implicated in the main metabolic pathway of both drugs.
Analysis of the dextromethorphan saturation curve suggests binding to one site only. This conclusion is contradicted by evidence emerging from the present study such as the residual binding in the absence of sodium, that follows a different pattern of distribution, and the heterogeneous effect of different pharmacological agents in different brain areas.
Binding to the cerebellum for example, was not potentiated by sodium ions, was reduced by (+)-PPP and paroxetine had no effect on the levels of binding. The cerebellum could therefore express particularly high levels of sodium-independent binding, that appears to have the same pharmacological profile as the DM1/σ1 site.
This heterogeneity in the modulation of dextromethorphan binding to rat brain revealed in this study suggests an unequal distribution of the sodium-dependent and non-dependent binding sites, as confirmed by the two different patterns of distribution for [H]-dextromethorphan obtained in the absence and presence of sodium.
The binding properties in different brain areas therefore appeared to be determined by the relative proportion of sodium-dependent binding, which was influenced by paroxetine, and sodium-independent binding which corresponded to the previously identified DM1/σ1 site.
Since σ ligands have never been shown to possess any an-titussive activity, the sodium-dependent site identified in this study as the DM2 site may represent a good candidate for a role in cough modulation, and its possible identity with 5-HT uptake may provide an important clue to the mechanism of action of the antitussive effect of dextromethorphan.
5-HT has been demonstrated to play a key role in the antitussive mechanism of morphine, dihydrocodeine and dextromethorphan (Kamei et al., 1987; 1988), and a further study showed an increase in the release of 5-HT from the nucleus of the solitary tract in the rat following dextromethorphan administration (Kamei et al., 1992). Since this nucleus represents an important relay in the modulation of cough, a local effect of dextromethorphan through DM2 sites could be implicated.
Other evidence for the possible functional effects of the interaction of dextromethorphan with 5-HT uptake have appeared in the literature: Ahtee (1975) demonstrated inhibition of 5-HT uptake in human platelets by nanomolar concentrations of the antitussive agent, and Finnegan et al. (1991) demonstrated a protective effect of dextromethorphan on the acute depletion of 5-HT produced by the administration of p-chloroamphetamine in rats.
An action of dextromethorphan at 5-HT uptake sites in man is also supported by evidence of adverse reactions witnessed by clinicians following the coadministration of dextromethorphan and monoamine oxidase (MAO) inhibitors (Sovner & Wolfe, 1988; Nierenberg & Semprebon, 1993). The symptoms described in these reports correspond to the central nervous system 5-HT syndrome, which is a condition resulting from the interaction of drugs which enhance 5-hydroxy-tryptaminergic tone at receptors in the central nervous system. The same effect following the administration of MAO inhibitors and dextromethorphan has been observed in rabbits (Sinclair, 1976) and ascribed to the inhibition of 5-HT uptake (Sinclair & Lo, 1976).
Further studies are required to establish unequivocally the identity of the sodium-dependent dextromethorphan high affinity binding site with the 5-HT uptake mechanism, but the commonality between a subpopulation of dextromethorphan binding sites and paroxetine binding might lead the way to new possible applications for 5-HT uptake inhibitors and dextromethorphan.