Distribution of S(−)-zacopride-insensitive [${}^{125}\text{I}$]R(+)-zacopride binding sites in the rat brain and peripheral tissues

Abstract

Increasing evidence indicates that the 5-HT₃ receptor antagonist R(+)-zacopride labels an additional site in brain tissue that is not sensitive to 5-HT (non-5-HT R(+)-zacopride site, R(+)-site). Since the levels of R(+)-sites in the brain are relatively low, the present studies explored the use of [${}^{125}\text{I}$]R(+)-zacopride to label the R(+)-site; the incorporation of an [${}^{125}\text{I}$] atom considerably increasing the specific activity of the radioligand relative to [${}^3\text{H}$]R(+)-zacopride that has been utilised previously. Competition experiments with [${}^{125}\text{I}$]R(+)-zacopride (1.0 nM) binding to rat whole brain homogenates, in the presence of the 5-HT₃ receptor antagonist granisetron (1.0 μM), identified that R(+)-zacopride and prazosin bound to two sites (pIC₅₀: 7.59 and 5.28, respectively, for R(+)-zacopride; 6.75 and 4.42, respectively, for prazosin) whereas S(−)-zacopride and mianserin possessed relatively low affinity (pIC₅₀: 4.37 and 3.80, respectively) while (−)sulpiride and 5-HT failed to compete for [${}^{125}\text{I}$]R(+)-zacopride binding at concentrations up to 10 μM. Autoradiographic radioligand binding studies using [${}^{125}\text{I}$]R(+)-zacopride (0.5 nM) identified a heterogeneous distribution of specific binding sites (defined by unlabelled R(+)-zacopride, 1.0 μM) throughout the rat brain. In the presence of a saturating concentration of granisetron (1.0 μM), highest levels of specific [${}^{125}\text{I}$]R(+)-zacopride binding sites (defined by R(+)-zacopride, 1.0 μM; R(+)-site), were detected in the olfactory tubercle, thalamus, corpus callosum, colliculus, dorsal and median raphe nucleus, spinal cord and the pons (8.0–13.0 fmol/mg). Moderate densities of R(+)-sites were located in the striatum, nucleus accumbens, substantia nigra, ventral tegmental area, globus pallidus, septal nuclei, frontal cortex and cerebellum (2.0–7.9 fmol/mg). In the hippocampus, amygdala and cortical areas, R(+)-site levels were low but detectable (0.1–1.9 fmol/mg). [${}^{125}\text{I}$]R(+)-zacopride labelled R(+)-sites were also detected in some rat peripheral tissues, for instance kidney cortex, adrenal gland and liver (2.4–6.8 fmol/mg). The present results indicate that specific non-5-HT [${}^{125}\text{I}$]R(+)-zacopride sites are heterogeneously distributed throughout the rat brain and are expressed in various peripheral tissues.

Introduction

In common with many other potent 5-HT₃ receptor antagonists, racemic zacopride modulates animal behaviour (Costall et al., 1990; Barnes et al., 1992c; Bentley and Barnes, 1995). The availability of S(−) and R(+)-zacopride, however, demonstrated that the stereoisomers of zacopride displayed a differential profile of activity in preclinical behavioural models (Barnes et al., 1990). For instance, R(+)-zacopride possesses anxiolytic-like activities at low microgram doses whereas effective doses of S(−)-zacopride are at least 4 orders of magnitude higher (Barnes et al., 1990; Young and Johnson, 1991). R(+)-zacopride is also more potent than S(−)-zacopride to improve the cognitive performance of laboratory animals (Barnes et al., 1990). However, the S(−)isomer of zacopride is more potent than the R(+)isomer to inhibit emesis induced by chemo- and radio-therapy (Andrews et al., 1988; Sancilio et al., 1991) and to antagonise the increased locomotor activity of laboratory animals induced by intra-nucleus accumbens administration of dopaminomimetics (Barnes et al., 1990). This behavioural pharmacological profile does not, however, correlate with the ability of the isomers of zacopride to antagonise the 5-HT₃ receptors nor agonise the 5-HT₄ receptor. Thus S(−)-zacopride displays some 10–20 fold higher affinity/potency than R(+)-zacopride for these two 5-HT receptor subtypes (Young and Johnson, 1990; Baxter et al., 1991). Using the radioligand binding technique, an additional recognition site for [${}^3\text{H}$]R(+)-zacopride has been identified within both rat and mouse brain which displays low nanomolar affinity for R(+)-zacopride whilst only micromolar affinity for S(−)-zacopride (Barnes et al., 1990). Subsequent studies have demonstrated that this site is unlikely to be associated with a 5-HT receptor since 5-HT itself displays virtually no affinity for this site (Kidd et al., 1992). Indeed, a range of structurally distinct pharmacologically active compounds fail to display more than micromolar affinity for the S(−)-zacopride insensitive R(+)-zacopride site (non-5-HT R(+)-zacopride site, R(+)-site) (Kidd et al., 1992), with only the R(+)stereoisomers of some structural analogues of zacopride displaying sub-micromolar affinity (Kidd et al., 1992).

Microdialysis studies have demonstrated that the non-5-HT R(+)-site may have a functional correlate since the release of 5-HT in the rat frontal cortex has been shown to be modulated by the isomers of zacopride with a similar profile to their ability to interact with the R(+)-site (Barnes et al., 1992d). In an attempt to further investigate the R(+)-site, we presently report the autoradiographic distribution of [${}^{125}\text{I}$]R(+)-zacopride binding to the R(+)-site in the brain and various peripheral tissues of the rat. The incorporation of an [${}^{125}\text{I}$]atom into this molecule increases the specific activity of the radioligand by over 20 fold which facilitates the detection of the R(+)-site in animal tissues where it is present at relatively low levels.