Characterization and Control of Expression of Cell Surface Aminopeptidase N Activity in Human Mesangial Glomerular Cells

Abstract

Intact human glomerular mesangial cells in culture hydrolyzed synthetic substrates of aminopeptidase N. Mesangial cell aminopeptidase N is an ectoenzyme as demonstrated by the extracellular location of the product, the inhibition of enzyme activity by the diazonium salt of sulfanilic acid, a nonpenetrating agent, the similar activities measured with intact and sonicated cells and the localization of the immunoreactive antigen by immunofluorescence on the cell surface. A broad substrate specificity was documented with various <i>p</i>-nitroanilide substrates in the following order of affinity: Ala, Leu > Lys, Arg > Pro, Gly and Val. Using Ala <i>p</i>-nitroanilide as substrate, apparent K_m and V_max values of 0.86 ± 0.70 m<i>M</i> and 3.54 ± 0.13 nmol min^–1 mg^–1, respectively were obtained. Enzyme activity was inhibited by bestatin and 1, 10-phenanthroline. Inhibition by 1 10-phenanthroline was noncompetitive. Cell surface aminopeptidase N activity increased in the presence of phorbolmyristate acetate (PMA). This effect was time-dependent, requiring a lag time of 12 h. It was also concentration-dependent with a threshold at 1 ng/ml and a maximum stimulation (2.2 times basal value) at 10 ng/ml. The effect of PMA was prevented by cycloheximide, an inhibitor of protein synthesis, and actinomycin D, an inhibitor of RNA synthesis. It was also suppressed by H7, an inhibitor of protein kinase C activity. The presence of aminopeptidase N in cultured glomerular mesangial cells and control of its expression by mitogens suggest that this enzyme may play a role in glomerular cell proliferation.