Pharmacology and subcellular distribution of [3H]rilmenidine binding sites in rat brain

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Abstract

We have previously reported that in rat brain membranes, [3H]rilmenidine, in addition to labelling α2-adrenoceptors and the I2B-subtype of imidazoline receptor binding site (I2B-RBS), may label an additional I-RBS population, distinct from previously classified I1-RBS and I2-RBS. In this study, using crude or fractionated rat brain membranes we examined the possible association of [3H]rilmenidine-labelled I-RBS with the A- and B-isoforms of monoamine oxidase (MAO) by studying the inhibition of [3H]rilmenidine binding by a number of MAO inhibitors; and comparing the maximal binding density (Bmax) and subcellular distribution of [3H]rilmenidine binding sites with that of MAO-A and MAO-B catalytic sites labelled by [3H]RO41-1049 and [3H]RO19-6327 and I2-RBS labelled by [3H]2-BFI. Inhibition of [3H]rilmenidine binding by all MAO inhibitors tested produced very shallow curves (slope 0.29–0.56). Clorgyline and moclobemide (selective MAO-A inhibitors) displayed moderate affinities (60–140 nM), while pargyline (non-selective MAO-inhibitor), RO41-1049 (selective MAO-A inhibitor) and RO19-6327 (selective MAO-B inhibitor) exhibited very low affinities (>2 μM) for 50–75% of [3H]rilmenidine-labelled I-RBS in crude brain membranes and even lower affinity for the remaining binding. Under identical buffer conditions, the Bmax of [3H]rilmenidine-labelled I-RBS (1.45±0.14 pmol/mg protein) was considerably lower than those of MAO-A (13.10±0.15 pmol/mg) and MAO-B (10.35±0.50 pmol/mg) sites. These results suggest that [3H]rilmenidine does not interact directly with the active catalytic site of either MAO enzyme and could at best only associate with a subpopulation of MAO molecules. Binding studies on five fractions of rat cortex homogenates—nuclear (N), heavy (M) and light (L) mitochondrial, microsomal non-mitochondrial (P), and soluble cytosolic (S) fractions—revealed that 45% of total [3H]rilmenidine binding was present in the P fraction cf. 20 and 23% in the M and L fractions, in contrast to [3H]RO19-6327 and [3H]2-BFI which bound 11–13% in the P fraction and 36–38% and 35–44% in the M and L fractions, respectively. Binding of all ligands in the N fraction was 6–15% of total. These studies reveal that [3H]rilmenidine-labelled I-RBS, unlike the I2-RBS, are not predominantly associated with mitochondrial fractions containing the MAO enzymes (and cytochrome oxidase activity), but appear to be distributed in both the mitochondrial and plasma membrane fractions in rat cerebral cortex.

Introduction

Imidazoline receptor binding sites (I-RBS) are defined as a range of heterogeneous proteins that can be labelled by various [3H]imidazoline ligands and are currently divided into two types—I1- and I2-RBS. The I1-RBS are reportedly labelled by [3H]clonidine and [3H]p-aminoclonidine and the I2-RBS by [3H]idazoxan and chemically-related compounds (Reis et al., 1992; Hosseini et al., 1997). I2-RBS have been further divided into I2A- and I2B-RBS which have high (nM) and low (μM) affinity, respectively for the guanidine diuretic, amiloride. We have previously reported that the oxazoline, [3H]rilmenidine, which is believed by many to be an I1-RBS-selective compound (Ernsberger et al., 1987, Ernsberger et al., 1990; van Zwieten, 1997), labels a population of I-RBS in the rat that are distinct from the I1-RBS, and more similar to I2B-RBS in rat brain and kidney (King et al., 1992, King et al., 1995a, King et al., 1995b).

Several studies have described the isolation of I-RBS proteins from different tissue sources and many laboratories have studied the association of I-RBS with monoamine oxidase (MAO) enzymes. A 43-kDa protein isolated from human brain (Greney et al., 1994) which is non-mitochondrial and proposed to be a cell-surface, G-protein-coupled receptor has been described as being an I1-RBS (Ernsberger and Shen, 1997). Although there is no evidence of association of this I1-RBS protein with MAO enzymes studies of platelet membranes from patients with depression, a condition which may involve changes in MAO activity, revealed increased levels of I1-RBS densities labelled by [3H]p-aminoclonidine and decreased levels following tricyclic antidepressant therapy (Piletz et al., 1990, Piletz et al., 1991).

The association of the I2-RBS labelled by [3H]idazoxan and MAO enzymes has been widely studied following the discovery of similarities in tissue distribution between the two proteins. Earlier subcellular fractionation studies in rabbit and human liver suggested that the major localization of the I2-RBS was to the outer mitochondrial membrane (Tesson et al., 1991). A similar co-localization has been reported in human and rabbit brain and kidney (Tesson and Parini, 1991, Tesson et al., 1992, Tesson et al., 1995). Autoradiographic mapping studies using [3H]lazabemide (RO19-6327) (Saura et al., 1992) and [3H]idazoxan (Mallard et al., 1992) describe similar distributions of MAO-B and I2-RBS in rat brain with a positive correlation between the regional distribution of the two proteins (Mallard et al., 1992). High levels of I2-RBS have been reported in dopamine-rich areas of the human brain which are rich in MAO-B (De Vos et al., 1991) and the I2-RBS is present in astrocytes which contain high concentrations of MAO-B (Sastre and Garcı́a-Sevilla, 1993; Westlund et al., 1993). Other evidence suggests that although I2-RBS are closely associated with MAO enzymes, these binding sites do not correspond to the active catalytic site of the enzyme at which the classical MAO inhibitors interact (Olmos et al., 1993; Renouard et al., 1993; Tesson et al., 1995).

In order to identify the I-RBS protein(s) and further investigate the possible relationships of these proteins with MAO enzymes, techniques such as photoaffinity labelling and immunoblotting have been employed to isolate I-RBS proteins from different tissues using [3H]idazoxan and other selective I2-RBS ligands (Limon et al., 1992; Lanier et al., 1995a). Comparison of partial amino acid sequences of I2-RBS proteins purified from rabbit kidney mitochondria revealed more than 70% sequence similarity with a portion of rat, rabbit and human MAO enzymes (Tesson et al., 1995). The pharmacological profile of this purified protein equated to that described for I2-RBS. It has even been suggested that I2A- and I2B-RBS subtypes correspond to MAO-A and MAO-B, respectively (Parini et al., 1996). There are, however, reports of other I-RBS that are not associated with MAO (Escriba et al., 1994; Greney et al., 1994; Garcı́a-Sevilla et al., 1995; Lanier et al., 1995a, Lanier et al., 1995b; Ernsberger and Shen, 1997).

There is evidence that [3H]rilmenidine labels both an I2B-like-RBS and an atypical I-RBS in brain and kidney (King et al., 1992, King et al., 1995a, King et al., 1995b) and so we examined the possible association of the [3H]rilmenidine-labelled I-RBS with MAO enzymes in rat brain by: (i) studying the inhibition of [3H]rilmenidine binding by a number of reversible and irreversible MAO inhibitors; (ii) comparing the maximal binding density (Bmax) of [3H]rilmenidine with those of [3H]RO41-1049 and [3H]RO19-6327, MAO-A and MAO-B selective ligands, in brain membranes; and (iii) comparing the subcellular distribution of [3H]rilmenidine binding with that of the I2-RBS selective ligand, [3H]2-BFI, and [3H]RO19-6327, in fractions of rat brain prepared by differential centrifugation.

Section snippets

Animals

Male Wistar-Kyoto rats, 240–260 g, were obtained from the Biological Research Laboratories at the Austin and Repatriation Medical Centre (Melbourne, Australia). Ethical approval was granted for all procedures associated with these studies by the Austin and Repatriation Medical Centre Animal Welfare Committee and experiments were carried out according to guidelines issued by the National Health and Medical Research Council of Australia.

Membrane preparation

For radioligand binding studies, rat forebrain membranes

Inhibition of [3H]rilmenidine binding by MAO inhibitors

Affinities of a number of selective and non-selective MAO inhibitors for [3H]rilmenidine binding sites in rat brain membranes were determined. Drug inhibition constants (Ki values) were derived using the EBDA program (McPherson, 1994) by constraining the radioligand Kd to a value previously determined in equilibrium saturation studies. All of the MAO inhibitors tested gave a pseudo-Hill coefficient (nH) significantly less than unity (0.29–0.56) using `Sigmoidal' curve analysis.

Clorgyline, an

Discussion

We have previously reported that in rat brain the oxazoline, [3H]rilmenidine, labelled an I2B-like RBS and an additional I-RBS which appeared to be distinct from either I1- or I2-RBS (King et al., 1992, King et al., 1995a). The present data generally support these conclusions.

Under identical buffer conditions, the Bmax of [3H]rilmenidine-labelled I-RBS (1.45±0.14 pmol/mg protein) was considerably lower than those of MAO-A (13.10±0.15 pmol/mg) and MAO-B (10.35±0.50 pmol/mg) sites. These results

Acknowledgements

This work was supported by the National Health and Medical Research Council of Australia and the Austin Hospital Medical Research Foundation. A.R.H. was the recipient of a scholarship from the Ministry of Health and Medical Education, Iran. The authors gratefully acknowledge Servier Laboratories, France for the gift of [3H]rilmenidine and Hoffmann-La Roche, Switzerland for providing [3H]RO41-1049 and [3H]RO19-6327.

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