Down-regulation and Inactivation of Neutral Endopeptidase 24.11 (Enkephalinase) in Human Neutrophils

E G Erdös,B Wagner,C B Harbury,R G Painter,R A Skidgel,X G Fa

doi:10.1016/s0021-9258(18)71709-2

Abstract

Neutral endopeptidase (EC 3.4.24.11, NEP) is an integral membrane protein of human neutrophils. NEP is identical with the common acute lymphoblastic leukemia antigen (CALLA) of leukemic cells. The expression of NEP on the surface of neutrophils is down-regulated by endocytosis which can be induced by phorbol 12-myristate 13-acetate (PMA) at 37 degrees C. The activity of the enzyme on the surface of intact cells decreases by 76% within 5 min. The activity can be recovered, however, if the cells are lysed within 5 min of the endocytosis. After 30 min, only 32% of the NEP activity is present in the neutrophil lysates. The loss of activity is presumably due to proteolytic inactivation. Diacylglycerol and monoclonal antibody to CALLA/NEP also induce internalization of NEP. PMA induces endocytosis even at 4 degrees C, but NEP is not inactivated at that temperature. The disappearance of NEP activity after adding PMA was inhibited by various agents. Among the most active were the phospholipase inhibitor 4-bromophenacyl bromide and a combination of the serine protease and cathepsin inhibitors, diisopropylfluorophosphate and N-ethylmaleimide. The employment of fluorescent monoclonal antibody confirmed the down-regulation and internalization of NEP antigen on the neutrophils. Since NEP inactivates chemotactic peptides and thereby affects chemotaxis of neutrophils (Painter, R. G., Dukes, R., Sullivan, J., Carter, R., Erdös, E. G., and Johnson, A. R. (1988) J. Biol. Chem. 263, 9456-9461), the down-regulation of NEP activity on the cell membrane may modulate the function of these cells in inflammation.

Highlights

Neutral endopeptidase (EC 3.4.24.11,NEP) is an substance P (4, 5 ), the atrial natriuretic peptide, and others integral membrane proteinof human neutrophils
The expres- Phe4 intoa tripeptide and a dipeptide [1]. NEP was sion of NEP on the surface of neutrophils is down- originally discovered in the kidney (71, the human and animal regulated by endocytosiswhich can beinduced by enzymes are presenton the plasma membranein specific phorbol 12-myristate 13-acetate (PMA) at 37 “C. The structures in the centralnervous system (1, 8 ), lung [9], male activity of the enzyme on the surface of intact cells decreases by 76% within 5 min
The present experimentsshow that neutrophils stimulated recently that a chemotactic peptide is rapidly internalizebdy with PMA a t 37 "C rapidly internalize NEP by endocytosis. neutrophils via non-coated pits [43]

Summary

EXPERIMENTAL PROCEDURES

The following reagents,inhibitors, and enzymes were obtained from Sigma: Histopaque 1077, PMA,retinal,adenosine-5’-monophosphate, chymostatin, N-ethylmaleimide, 1-oleoyl 2-acetyl-racglycerol (analogue of diacylglycerol; DAG), superoxidedismutase, catalase, melittin, and diisopropylfluorophosphate (DFP). The elastase inhibitor methoxySuc-Ala-Ala-Pro-Val-CH2C(lCK20) and the NEP substragtelutarylAla-Ala-Phe-4-methoxy-2-naphthylami(MneNA)were from Enzyme Systems Products. Phosphoramidon was from Peninsula Laboratories and Ficoll from Pharmacia LKB Biotechnology Inc. Monoclonal antibody to NEP andfluorescein isothiocyanate (FITC) labeled antibody were purchased from Coulter as 55 antibody to CALLA. The other chemicals used were of reagent-grade obtained commercially

Methods

RESULTS

Findings

DISCUSSION