PMID- 1661804 OWN - NLM STAT- MEDLINE DCOM- 19920206 LR - 20191021 IS - 0341-2040 (Print) IS - 0341-2040 (Linking) VI - 169 IP - 6 DP - 1991 TI - HOCl exposure of a human airway epithelial cell line decreases its plasma membrane neutral endopeptidase. PG - 311-23 AB - It has recently been demonstrated that luminal exposure of airway segments in vitro to HOCl produces airway muscle hyperresponsiveness to substance P and a decrease in neutral endopeptidase (NEP) activity of tissue segment homogenates, suggesting that HOCl may decrease airway epithelial cell NEP activity. To confirm that this effect occurs in humans and to investigate possible subcellular mechanisms for it, we assessed HOCl exposure of the human airway epithelial cell line Calu-1. These cells, grown to confluency in Dulbecco's modified Eagle medium with 10% fetal bovine serum and penicillin-streptomycin, were exposed in situ for 5 min to 100 microM HOCl in a phosphate-buffered saline solution (PBS; pH 7.0 at 37 degrees C) or to PBS alone. Thereafter, cells were rinsed and assayed for NEP activity employing reverse-phase high-pressure liquid chromatography. This activity was characterized by the generation of phosphoramidon-inhibitable product (ANA) cleaved from the synthetic substrate succinyl-(ala)3-p-nitroaniline during a 30 min incubation at 37 degrees C. Cell viability was assessed by changes in LDH release, trypan blue exclusion, and cell volume. In some experiments, crude plasma membrane and soluble components of exposed cells were isolated and differential NEP activity was assayed. We found that a 5 min exposure to HOCl decreased whole cell NEP activity from 74.1 +/- 4.4 (mean +/- SE) to 54.3 +/- 6.0 pmoles of ANA/min/10(6) cells (p less than 0.05), while no parameter of cell viability was affected. NEP activity in the crude membrane fraction decreased 36.3 +/- 3.1% after exposure (p less than 0.01), whereas NEP activity in the soluble fraction increased 4.0 +/- 0.6%. Isolated membrane NEP exposed by itself was not affected. Subsequent experiments with reducing agents demonstrated that NEP activity of cell cultures pretreated with 100 mM of either beta-mercaptoethanol or dithiothrietol before HOCl exposure was not significantly different from control values. We conclude that whole cell HOCl exposure decreases Calu-1 plasma membrane NEP. This loss appears to occur by internalization of cell membrane NEP. FAU - Lang, Z H AU - Lang ZH AD - Department of Medicine (Pulmonary), Rush University Chicago, Il 60612. FAU - Murlas, C G AU - Murlas CG LA - eng GR - HL-01965/HL/NHLBI NIH HHS/United States GR - HL-34228-04/HL/NHLBI NIH HHS/United States GR - OH-00060/OH/NIOSH CDC HHS/United States PT - Journal Article PT - Research Support, Non-U.S. Gov't PT - Research Support, U.S. Gov't, P.H.S. PL - United States TA - Lung JT - Lung JID - 7701875 RN - 712K4CDC10 (Hypochlorous Acid) RN - EC 3.4.24.11 (Neprilysin) SB - IM MH - Cell Line MH - Cell Membrane/enzymology MH - Cell Survival MH - Chromatography, High Pressure Liquid MH - Endocytosis/drug effects MH - Epithelial Cells MH - Humans MH - Hypochlorous Acid/*pharmacology MH - In Vitro Techniques MH - Neprilysin/drug effects/*metabolism MH - Respiratory System/cytology/*drug effects EDAT- 1991/01/01 00:00 MHDA- 1991/01/01 00:01 CRDT- 1991/01/01 00:00 PHST- 1991/01/01 00:00 [pubmed] PHST- 1991/01/01 00:01 [medline] PHST- 1991/01/01 00:00 [entrez] AID - 10.1007/BF02714168 [doi] PST - ppublish SO - Lung. 1991;169(6):311-23. doi: 10.1007/BF02714168.