PREDOMINANT EXPRESSION OF MONOAMINE-OXIDASE-B ISOFORM IN RABBIT RENALPROXIMAL TUBULE - REGULATION BY I-2 IMIDAZOLINE LIGANDS IN INTACT-CELLS

Previous studies have shown that a subpopulation of the catecholamine- degrading enzymes monoamine oxidase (MAO) A and B holds a previously u nknown regulatory site, the I-2-imidazoline binding site (I2BS). In th e present work, we characterized the isoforms of monoamine oxidases ex pressed in the rabbit renal proximal tubule, defined their relationshi p with I2BS, and investigated the ability of I2BS ligands to inhibit e nzyme activity in intact cells. Two findings indicate that MAO-B is th e predominant isoform expressed in the renal proximal tubule cells: 1) Western blot performed with an anti-MAO-A/MAO-B polyclonal antiserum revealed a single 55-kDa band corresponding to MAO-B; 2) enzyme assays showed an elevated MAO-B activity ([C-14]beta-phenylethylamine oxidat ion: V-max = 1.31 +/- 0.41 nmol/min/mg protein), whereas MAO-A activit y was only detectable ([C-14]5-HT oxidation: V-max = 80.3 +/- 19 pmol/ min/mg protein). Photoaffinity labeling with the I2BS ligand [I-125]2- (3-azido-4-iodophenoxy)-methylim revealed a single 55-kDa band, which indicates that MAO-B of the renal proximal tubule cells holds the I-2 imidazoline binding site. [H-3]Idazoxan binding studies and enzyme ass ays showed that, in intact cells, I2BS ligands bind to and inhibit MAO -B. Indeed, the increase in the accessibility of intracellular compart ment by cell permeabilization did not enhance [H-3]idazoxan binding, w hich indicates that, in intact cells, intracellular I2BS are fully occ upied by imidazoline ligands. In addition, enzyme assays showed that i ncubation of proximal tubule cells with imidazoline ligands leads to a complete, dose-dependent inhibition of MAO activity. These data show the predominant expression of MAO-B in rabbit renal proximal tubule an d its regulation by imidazoline ligands in intact cells.