PHARMACOLOGY AND SUBCELLULAR-DISTRIBUTION OF [H-3]RILMENIDINE BINDING-SITES IN RAT-BRAIN

We have previously reported that in rat brain membranes, [H-3]rilmenid ine, in addition to labelling alpha(2)-adrenoceptors and the I-2B-subt ype of imidazoline receptor binding site (I-2B-RBS), may label an addi tional I-RES population, distinct from previously classified I-1-RBS a nd I-2-RBS. In this study, using crude or fractionated rat brain membr anes we examined the possible association of [H-3]rilmenidine-labelled I-RES with the A- and B-isoforms of monoamine oxidase (MAO) by studyi ng the inhibition of [H-3]rilmenidine binding by a number of MAO inhib itors; and comparing the maximal binding density (B-max) and subcellul ar distribution of [H-3]rilmenidine binding sites with that of MAO-A a nd MAO-B catalytic sites labelled by [H-3]RO41-1049 and [H-3]RO19-6327 and I-2-RBS labelled by [H-3]2-BFI. Inhibition of [H-3]rilmenidine bi nding by all MAO inhibitors tested produced very shallow curves (slope 0.29-0.56). Clorgyline and moclobemide (selective MAO-A inhibitors) d isplayed moderate affinities (60-140 nM), while pargyline (non-selecti ve MAO-inhibitor), RO41-1049 (selective MAO-A inhibitor) and RO19-6327 (selective MAO-B inhibitor) exhibited very low affinities (> 2 mu M) for 50-75% of [H-3]rilmenidine-labelled I-RES in crude brain membranes and even lower affinity for the remaining binding. Under identical bu ffer conditions, the B-max of [H-3]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 [H-3]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 fi ve fractions of rat cortex homogenates-nuclear (N), heavy (M) and ligh t (L) mitochondrial, microsomal non-mitochondrial (P), and soluble cyt osolic (S) fractions-revealed that 45% of total [3H]rilmenidine bindin g was present in the P fraction cf. 20 and 23% in the M and L fraction s, in contrast to [H-3]RO19-6327 and [H-3]2-BFI which bound 11-13% in the P fraction and 36-38% and 35-44% in the M and L fractions, respect ively. Binding of all ligands in the N fraction was 6-15% of total. Th ese studies reveal that [H-3]rilmenidine-labelled I-RES, unlike the I- 2-RBS, are not predominantly associated with mitochondrial fractions c ontaining the MAO enzymes (and cytochrome oxidase activity), but appea r to be distributed in both the mitochondrial and plasma membrane frac tions in rat cerebral cortex. (C) 1998 Elsevier Science B.V. All right s reserved.