MODULATION OF ANGIOTENSIN-CONVERTING ENZYME IN CULTURED HUMAN VASCULAR ENDOTHELIAL-CELLS

Previous work has suggested that not all immunoreactive angiotensin-co nverting enzyme (ACE) in tissues or cells is in a biologically active state. We have explored this possibility in cultured human umbilical v ein endothelial cells (HUVEC), one of the most widely studied in vitro endothelial cell systems. Our approach included characterization of t he effect of increasing passage number on ACE activity and expression of immunoreactive ACE at the single cell level, the subcellular compar tmentalization of active ACE, and the effect of phorbol ester (PMA) tr eatment. We found that both ACE activity and expression of ACE antigen were downregulated by cultivation (30% of ACE-positive cells at seven th passage vs. 90% in primary culture). ACE downregulation is specific (number of CD31-positive cells did not change with cultivation) and c orrelated with do downregulation of factor VIII-antigen. The percentag e of ACE-positive cells in permeabilized HUVEC at third passage was al most twice that in nonpermeabilized HUVEC (90% vs. 50%), indicating th at HUVEC contain intracellular immunoreactive ACE. ACE activity, howev er, was similar when measured in intact cells and in cell lysates. Mor eover; diazonium salt of sulfanilic acid (DASA), a membrane-impermeabl e ACE inhibitor, inhibited ACE activity in intact cells and in cell ly sates at the same extent, thus implying that intracellular ACE is inac tive. PMA (100 nM) treatment increased the percentage of ACE-positive cells at third passage from 57 to 96%. ACE activity was increased 3-fo ld in cell and 1.5-fold in the culture medium of PMA-treated cells. An alysis of ACE activity in intact monolayers and cell. lysates of contr ol and PMA-treated cells revealed that all enzymatically active ACE in PMA-treated cells is localized on the plasma membrane and acts as an ectoenzyme. We conclude that expression of ACE hi HUVEC is downregulat ed by repeated passage in culture but can be restored by PMA treatment . In addition, ACE expression is heterogeneous between neighboring cel ls, and total immunoreactive ACE protein associated with HUVEC include s an inactive pool of the enzyme.