PMA-ACTIVATED NEUTROPHILS DECREASE PULMONARY ENDOTHELIAL ECTOENZYME ACTIVITIES IN PERFUSED RABBIT LUNGS

We have studied the effects of phorbol 12-myristate 13-acetate (PMA, 15 mug) on pulmonary endothelial ectoenzyme [angiotensin-converting enzyme (ACE) and 5'-nucleotidase (NCT)] function in isolated rabbit lungs perfused in situ with platelet-poor (PPP) or platelet-rich (PRP) plasma in the presence or absence of neutrophils. Enzyme activities were estimated from the hydrolysis of the substrates [H-3]benzoyl-Phe-Ala-Pro ([H-3]BPAP) by ACE and C-14-labeled AMP by NCT during a single transpulmonary passage, using indicator-dilution techniques. In all treatment groups PMA produced a delayed increase in pulmonary vascular resistance to about three times the control value. PMA alone [in lungs perfused with PPP (n = 5 animals) or PRP (n = 6)] or neutrophils alone (in PPP-perfused lungs, n = 5) had no effect on enzyme activity. However, PMA-activated netrophils (n = 5) decreased percent metabolism (%M) of [H-3]BPAP from 87 +/- 3 to 77 +/- 4% (30 min after PMA), and the apparent first-order parameter [ratio of maximum activity to Michaelis constant (A(max)/K(m))] for ACE from 821 +/- 114 to 613 +/- 61 ml/min (30 min after PMA). At the same time, K(m) values of BPAP for ACE and AMP for NCT were elevated from 9.2 +/- 2.2 to 19.3 +/- 3 muM and 6.7 +/- 1.2 to 15.1 +/- 3.6 muM, respectively, whereas A(max) (product of enzyme mass and rate of product formation, thus an index of perfused microvascular surface area) did not change. Pretreatment of neutrophils with anti-CD18 monoclonal antibody 60.3, which inhibits neutrophil adherence to endothelial cells (n = 5), prevented the PMA-induced change in %M, A(max)/K(m), and K(m) for ACE and NCT. These results indicate that 1) PMA induces pulmonary vasoconstriction independently of blood cell elements, 2) PMA-induced decrease in endothelial ectoenzyme activities is platelet independent and neutrophil dependent, 3) neutrophil adherence to endothelial cells, via the leukocyte CD18 integrin, is required for the observed PMA-induced decrease in pulmonary endothelial ectoenzyme activities.