Uptake and proteolytic activation of prorenin by cultured human endothelial cells

Objective To investigate the mechanisms of vascular uptake of prorenin and renin and to explore the possibility of vascular activation of prorenin, Design and methods Human umbilical vein endothelial cells (HUVECs) cultured in a chemically defined medium were incubated with recombinant human prore nin or renin in the presence or absence of putative inhibitors of renin int ernalization. Cell surface-bound and internalized prorenin or renin were se parated by the acid-wash method and were quantified by enzyme-kinetic assay s. The activation of prorenin was also monitored by a direct immunoradiomet ric assay (IRMA) with use of a monoclonal antibody directed against the -p2 4-Arg to -1p-Arg C-terminal propeptide sequence of prorenin. Results Prorenin and renin were internalized at 37 degrees C in a dose-depe ndent manner; with 1000 mu U prorenin/ml medium, the quantity of cell-assoc iated prorenin after 3 h of incubation was 9.3 +/- 1.0 mu U/4 x 10(5) cells , and with 75 000 mu U/ml medium it was 670 +/- 75 mu U/4 x 10(5) cells (me an +/- SD; n = 5), Results for renin were similar. Prorenin that had been t reated with endoglycosidase H to remove N-linked oligosaccharides was not i nternalized. Addition of mannose 6-phosphate (M-6-P) to the medium caused a dose-dependent inhibition of renin and prorenin internalization. Fifty per cent inhibition was observed at 70 mu mol/M-6-P, whereas mannose 1-phospha te, glucose 6-phosphate and alpha-methylmannoside at this concentration had no effect. Ammonium chloride (50 mmol/l) and monensin (10 mu mol/l) also i nhibited internalization. Prorenin was activated by HUVECs, and cell-activa ted prorenin was only found in the internalized fraction, whereas the surfa ce-bound prorenin remained inactive, Thus, it appears that the activation o f prorenin took place at the time of its internalization or thereafter. The results of the prorenin IRMA indicated that activation was associated with proteolytic cleavage of the propeptide, Conclusions Our findings provide evidence for M-6-P receptor-dependent endo cytosis of (pro)renin and proteolytic prorenin activation by vascular endot helial cells. J Hypertens 1999, 17:621-629 (C) Lippincott Williams & Wilkin s.