Human Endothelial Cells Bind and Activate Noncleavable Prorenin

Abstract

P15 Circulating prorenin (PR), following binding to endothelial mannose 6-phosphate (M6P) receptors, is activated proteolytically, and may thus play a role in vascular angiotensin (A) generation (Admiraal et al., J Hypertension 1999). In addition, in vivo studies with noncleavable PR (i.e., PR in which Lys in position 42 of the prosegment is mutated to Ala, thereby preventing cleavage by known proteases) in mice have revealed that PR may also generate AI following nonproteolytic activation (Methot et al., Circ Res 1999). Here, we studied the nature of the PR-activating enzyme in human umbilical vein endothelial cells (HUVECs), as well as the activation of noncleavable and nonglycosylated PR (i.e., PR mutated at its glycosylation sites) by these cells. HUVECs were incubated with 100 U/L recombinant human native, noncleavable, or nonglycosylated PR at 4°C or 37°C for 4 h with or without 10 mM M6P or inhibitors of serine (AEBSF, aprotinin), cysteine (E64, leupeptin), metallo (EDTA, phenanthrolin), or aspartic (pepstatin A) proteases. Intact and activated PR were measured in cell lysates by immunoradiometric assays with monoclonal antibodies (mAbs) directed against the prosegment (amino acids 20-43) or a renin-specific epitope. The prosegment-specific mAb recognizes intact PR only after nonproteolytic activation (either in the cells or after incubation of the lysates with the renin inhibitor remikiren for 48 h). At 37°C, HUVECs bound and internalized native and noncleavable, but not nonglycosylated, PR. Internalization did not occur at 4°C, and binding was blocked by M6P. More than 60% of the internalized native PR was activated. This was due to proteolytic cleavage, since the prosegment-specific mAb recognized