Abstract

Previous studies have shown that a subpopulation of the catecholamine-degrading enzymes monoamine oxidase (MAO) A and B holds a previously unknown regulatory site, the I₂-imidazoline binding site (I₂BS). In the present work, we characterized the isoforms of monoamine oxidases expressed in the rabbit renal proximal tubule, defined their relationship with I₂BS, and investigated the ability of I₂BS ligands to inhibit enzyme activity in intact cells. Two findings indicate that MAO-B is the 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 ([¹⁴C]β-phenylethylamine oxidation:V _max = 1.31 ± 0.41 nmol/min/mg protein), whereas MAO-A activity was only detectable ([¹⁴C]5-HT oxidation: V _max = 80.3 ± 19 pmol/min/mg protein). Photoaffinity labeling with the I₂BS ligand [¹²⁵I]2-(3-azido-4-iodophenoxy)-methylimidazoline revealed a single 55-kDa band, which indicates that MAO-B of the renal proximal tubule cells holds the I₂ imidazoline binding site. [³H]Idazoxan binding studies and enzyme assays showed that, in intact cells, I₂BS ligands bind to and inhibit MAO-B. Indeed, the increase in the accessibility of intracellular compartment by cell permeabilization did not enhance [³H]idazoxan binding, which indicates that, in intact cells, intracellular I₂BS are fully occupied by imidazoline ligands. In addition, enzyme assays showed that incubation 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 and its regulation by imidazoline ligands in intact cells.