Predominant expression of monoamine oxidase B isoform in rabbit renal proximal tubule: regulation by I2 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 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]beta-phenylethylamine oxidation: Vmax = 1.31 +/- 0.41 nmol/min/mg protein), whereas MAO-A activity was only detectable ([14C]5-HT oxidation: Vmax = 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.
Gargalidis-Moudanos
C
C
Institut National de la Santé et de la Recherche Médicale U388, Centre Hospitalier Universitaire Rangueil, Toulouse, France.
Remaury
A
A
Pizzinat
N
N
Parini
A
A
D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D019329:Idazoxan / Q000378:metabolism; D054755:Imidazoline Receptors; D007672:Kidney Cortex / Q000201:enzymology; D007687:Kidney Tubules, Proximal / Q000201:enzymology*; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008024:Ligands; D008297:Male; D008995:Monoamine Oxidase / Q000096:biosynthesis* / Q000378:metabolism; D008996:Monoamine Oxidase Inhibitors / Q000494:pharmacology; D011817:Rabbits; D011955:Receptors, Drug / Q000378:metabolism*; D014316:Tritium
D054755:Imidazoline Receptors; D008024:Ligands; D008996:Monoamine Oxidase Inhibitors; D011955:Receptors, Drug; D014316:Tritium; D007770:L-Lactate Dehydrogenase; D008995:Monoamine Oxidase; D019329:Idazoxan