Characterisation and localisation of [3H]2-(2-benzofuranyl)-2-imidazoline binding in rat brain: a selective ligand for imidazoline I2 receptors

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Abstract

In rat whole brain homogenates, saturation binding analysis revealed that both [3H]2-BFI (2-(2-benzofuranyl)-2-imidazoline) and [3H]idazoxan (in the presence of 5 μM rauwolscine) bound with high affinity to an apparent single population of sites. However, the Kd for [3H]2-BFI (1.74±0.14 nM) was significantly (P<0.01) less than that for [3H]idazoxan (10.4±2.68 nM). In competition studies idazoxan, 2-BFI, BU224 (2-(4,5-dihydroimidaz-2-yl)-quinoline), amiloride and guanabenz displayed high affinity (Ki values=7.32, 1.71, 2.08, 21.80 and 14.90 nM, respectively) for 70–80% of sites, and low μM affinity for the remaining 20–30% of sites labelled by [3H]2-BFI. In contrast, several α2-adrenoceptor, imidazoline I1 receptor and histamine receptor ligands exhibited only micromolar affinity for the [3H]2-BFI labelled site. Quantitative receptor autoradiography revealed high binding by [3H]2-BFI to discrete brain nuclei, notably the area postrema, interpeduncular nucleus, arcuate nucleus, mammillary peduncle, ependyma and pineal gland. These data indicate that [3H]2-BFI recognises imidazoline I2 receptors in rat brain with higher affinity and selectivity than [3H]idazoxan and thus represents a superior radioligand to [3H]idazoxan for the study of imidazoline I2 receptors.

Introduction

Extensive examination of the binding characteristics and distribution of the imidazoline radioligands [3H]clonidine, [3H]p-aminoclonidine and [3H]idazoxan, as well as the biochemical and functional effects of imidazolines and related compounds has led to a widely accepted view that at least two principle classes of imidazoline (I) receptors now exist, designated I1 and I2 (for review see Parini et al., 1996). Imidazoline I1 receptors are recognised as those labelled by [3H]clonidine and [3H]p-aminoclonidine and imidazoline I2 receptors are recognised as those labelled by [3H]idazoxan (Ernsberger et al., 1987; Kamisaki et al., 1990; Mallard et al., 1992). Both imidazoline I1 and I2 receptors show a high nanomolar affinity for cirazoline and idazoxan, but are distinguished from each other by the low affinity (>micromolar) of clonidine, rilmenidine, moxonidine, efaroxan, imidazole-4-acetic acid and cimetidine for the imidazoline I2 receptor and the low affinity (μM) of the guanadides, guanabenz and amiloride, for the imidazoline I1 receptor. Moreover, Ernsberger further subdivided the imidazoline I2 receptor into I2A and I2B according to differential affinity for amiloride in different species and tissues (see Parini et al., 1996). Biochemical studies have purified various imidazoline binding proteins of 27–85 kDa, from several tissues and species, which clearly indicate the existence of a heterogeneity of imidazoline binding proteins, which is in agreement with previous pharmacological studies (Wang et al., 1992; Limon et al., 1992; Lanier et al., 1993; Escribá et al., 1994, Escribá et al., 1995; Greney et al., 1994). Interestingly, some of these groups have more recently disclosed an association of imidazoline I2 receptors and imidazoline receptor binding proteins with monoamine oxidase (for review see Parini et al., 1996). Furthermore binding and functional studies have revealed the existence of additional imidazoline receptors which cannot be grouped into I1, I2A or I2B subtypes (Chan et al., 1994; Olmos et al., 1994a; King et al., 1992, King et al., 1995).

For many years [3H]idazoxan has proved invaluable as the radioligand for the study of both α2-adrenoceptors and imidazoline I2 receptors in rat brain (Boyajian et al., 1987; Bruning et al., 1987; Mallard et al., 1992; Olmos et al., 1996). This dual identity of idazoxan has limited the characterisation of imidazoline I2 receptors, as the inclusion of α2-adrenoceptor antagonists is required to study imidazoline I2 receptors in isolation (Mallard et al., 1992). More recently new selective radioligands for imidazoline I2 receptors have been developed. The azido derivative of cirazoline, AZIPI (2,[3-amino-4-{125I}iodophenoxy]methyl imidazoline), has been used to purify three different putative imidazoline binding proteins of three different apparent molecular weights, two of which correspond with the molecular weights of monoamine oxidase-A and monoamine oxidase-B (Lanier et al., 1993) and whose distribution mirrors that of monoamine oxidase-B in rat brain (Saura et al., 1992). Also [3H]RS-45041-190 (4-chloro-2-(imidazolin-2-yl)-isoindolene) has been characterised and identified as a radioligand that has a higher affinity and selectivity for imidazoline I2 receptors in rat kidney, compared with non-adrenoceptor [3H]idazoxan binding in rat kidney and brain (Mackinnon et al., 1995). Furthermore, these workers have revealed a correlation of >0.95 between the affinities of several competitors for [3H]RS-45041-190 binding in rat kidney and non-adrenoceptor [3H]idazoxan binding in rat kidney and brain. In addition the distribution of [3H]RS-45041-190 binding in rat brain has been shown to substantially overlap the previously reported distribution of [3H]idazoxan labelling of imidazoline I2 receptors in rat brain (Mallard et al., 1992).

The 2-benzofuranyl-derivative of idazoxan, 2-BFI (2-(2-benzofuranyl)-2-imidazoline) has been found to possess a high affinity for imidazoline I2 receptors and has an improved I2: α2 selectivity profile in rat brain, such that it is >1500-fold selective (Hudson et al., 1997). [3H]2-BFI has recently been characterised as a selective ligand for imidazoline I2 receptors in rabbit brain and kidney, and found to be a superior ligand to [3H]idazoxan for the study of these receptors (Lione et al., 1996; Hosseini et al., 1997; Alemany and Garcı́a-Sevilla, 1997). We have now characterised the binding of [3H]2-BFI in rat brain, and used quantitative autoradiography to confirm the distribution of central I2 receptors in this species (Mallard et al., 1992).

Section snippets

Membrane preparation

Male Wistar rats (240–300 g) were sacrificed by stunning followed by decapitation. Whole brains were immediately removed over ice and homogenised in 10 volumes (w/v) of buffered sucrose (0.32 M in 50 mM Tris–HCl, pH 7.4 at 4°C) using a motor driven Teflon-glass homogeniser. The homogenate was centrifuged at 1000×g for 10 min at 4°C. The resultant supernatants were pooled and recentrifuged at 32 000×g for 20 min at 4°C. The supernatants were discarded and each pellet resuspended in 10 volumes of

Kinetics binding studies

The association of [3H]2-BFI (1 nM) binding to rat whole brain membranes was rapid, with equilibrium being reached within 20 min and remained at equilibrium for a further 2 h (Fig. 1). Consequently an incubation time of 40 min was chosen for future experiments. On the addition of 10 μM 6-fluoro-idazoxan, the specific binding of [3H]2-BFI was rapidly and fully dissociated (Fig. 1). Four independent studies followed the association and dissociation time course of [3H]2-BFI. Based on the F-test,

Discussion

Until 1995 [3H]idazoxan had been the only radioligand commercially available to study imidazoline I2 receptors, albeit indirectly, as such studies were complicated by the interaction of [3H]idazoxan with α2-adrenoceptors. In 1995, 2-BFI was reported to have a higher affinity for imidazoline I2 receptors and a >1000-fold selectivity for imidazoline I2 receptors vs. α2-adrenoceptors in mammalian brain, compared with idazoxan (see Hudson et al., 1997). More recently, we have reported [3H]2-BFI to

Acknowledgements

The authors wish to thank Dr. Pat Towers and Dr. Gareth Ellis at Amersham International (Cardiff) for the kind gift of [3H]2-BFI and Professor John Lewis at Bristol for the synthesis of 2-BFI and BU224. Dr. L.A. Lione was supported by an MRC collaborative award with SmithKline Beecham. Dr. A.L. Hudson and this research are supported by the Wellcome Trust.

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