FATE OF A FLUORESCENT INHIBITOR OF ENDOPEPTIDASE-24.11 USING ENZYME-EXPRESSING MDCK CELLS - MODIFICATION OF ITS CELLULAR PROCESSING WITH A MONOCLONAL-ANTIBODY

Neutral endopeptidase-24,11 (NEP) is a membrane bound zinc metallopept idase which cleaves biologically active peptides such as the enkephali ns and atrial natriuretic peptide. Using the specific and fluorescent thiol inhibitor of the enzyme, N-[fluoresceinyl]N'-[1-(6(3-mercapto 2- benzyl-1-oxopropyl)-amino-1-hexyl]-thiocarbamide (FTI), the fate of th e inhibitor-enzyme complex was investigated by videomicrofluorimetry u sing MDCK epithelial cells expressing the rabbit peptidase thanks to a retroviral expression vector. N-[3-(R,S)-[(hydroxyamino) carbonyl]-2- benzyl-1-oxopropyl]-glycine (HACBOGly) and the corresponding tritiated molecule were also used to measure the cellular pathway of inhibitor- NEP complexes. In the present paper, we demonstrate that, for short in cubation times, the fluorescent probe preferentially labeled brush bor der membranes of the apical side of the MDCK cells. After more than 1 h incubation, a honeycomb pattern of fluorescence was observed in vide omicrofluorimetry suggesting that part of the inhibitor was bound or l ocalized close to the basolateral plasma membrane. Confocal experiment s confirmed the transcytosis of FTI/NEP complex, from the apical to th e basolateral domain. Using [H-3]HACBOGly on filter-grown cells, after 2 and 4 h incubation at 37 degrees C, the percentage of basolateral m embrane-bound molecules was estimated to be about 12 and 23%, respecti vely. The coincubation of the cells with FTI and 2B12, a monoclonal an tibody raised against the rabbit enzyme, greatly modified the fluoresc ence pattern. A patchy fluorescence was observed for short incubation times, corresponding to cluster formation induced by antigen-antibody binding. For longer incubation times (>1 h), in addition to the basola teral labeling, some intracellular fluorescent vesicles were observed essentially Localized in the vicinity of the nucleus. The colocalizati on of FTI with Texas Red isothiocyanate-labeled Concanavalin A (TRITC- Con A) strongly suggests an endosomal/lysosomal internalization pathwa y when FTI was incubated in the presence of 2B12 mAb.