Neutrophils are Required for PMA-Induced Endothelial Ectoenzyme Dysfunction: Study in Cultured Endothelial Cells

Abstract

We have studied the role of neutrophils (PMN) in phorbol myristate acetate (PMA)-induced endothelial ectoenzyme (angiotensin converting enzyme, ACE and 5 ’ -nucleotidase, NCT) dysfunction in cultured rabbit aortic endothelial cells (EC), using [3H]-Benzoyl-Phe-Ala-Pro and [14C]-5’-AMP as substrates, respectively, under first order reaction conditions. PMA alone (1–1000 ng/ml) or PMN in the absence of PMA did not affect ACE activity. When PMA was incubated together with PMN (PMN:EC ratio=1.25:1) for 4 h in Earl’s salts, a dose dependent decrease in ACE activity was observed: threshold PMA concentration was 2 ng/ml and at 10 ng/ml, ACE activity was totally inhibited. This decrease in ACE activity was also dependent on PMN concentration and was detectable at as low as 1.25:10 PMN:EC ratio. The inhibition of ACE activity was also time-dependent, occurring as early as 1 h after incubation with PMN and PMA (10 ng/ml) and reaching maximum at 4 h. When incubated alone with EC for 4 h, PMA caused a small but significant increase in NCT activity (12–29%), which was PMA dose independent from 2–1,000 ng/ml. In the presence of PMN (1.25:1, PMN:EC ratio), PMA produced a significant decrease in NCT activity (20–26%) which, however, was dose-independent from 2–10 ng/ml. Pretreatment of EC with PMA (10 ng/ml) for 12 h, followed by incubation of EC with PMN in the absence of PMA for 4 h, did not affect ACE activity. Pretreatment of PMN (1:1, PMN:EC ratio), with PMA (10 ng/ml) for 1 h, followed by incubation of EC with activated PMN (after washing out the PMA) for 4 h, produced a significant decrease in ACE activity, whereas, the supernatant (containing PMA) from activated PMN had no effect on ACE activity.