0026-895X/97/040637-07$3.00/0
Copyright © by The American Society for Pharmacology and Experimental Therapeutics
All rights of reproduction in any form reserved.
MOLECULAR PHARMACOLOGY 51:637-643 (1997).

Predominant Expression of Monoamine Oxidase B Isoform in Rabbit Renal Proximal Tubule: Regulation By I₂ Imidazoline Ligands in Intact Cells

C. Gargalidis-Moudanos, A. Remaury, N. Pizzinat, and A. Parini

Institut National de la Santé et de la Recherche Médicale U388, Institut Louis Bugnard, Centre Hospitalier Universitaire Rangueil, 31403 Toulouse Cedex 04, France

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]-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.