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CHRONIC MORPHINE TREATMENT DOES NOT AFFECT AMINOPEPTIDASE N (APN) ENZYME ACTIVITY ON 293 CELLS TRANSFECTED WITH mu OPIOID RECEPTOR

INTRODUCTION: Chronic exposure of cells to opioids leads to tolerance and adaptation, due to down-regulation of opioid receptors and their desensitisation. Aminopeptidase N (APN/CD13; E.C.3.4.11.2.) and neutral endopeptidase (NEP/CD10; E.C.3.4.24.11.) are key enzymes for degradation and inactivation of endogenous opioid peptides (enkaphalins, endomorphins and dynorphins). A correlation between the expression of mu opioid receptor on one side, and expression of APN (Malfroy et al, Nature, 276: 523-526,1978) and NEP (Fischer HS et al, Regulatory Peptides, 96:53-58, 2000) on the other side, were reported. We propose that in addition to opioid receptors, enzymes that inactivate opioid ligands, might also be involved in process of adaptation by up-regulating their expression and/or activity. AIM: In this study we tested the possibility that chronic treatment with mu selective, non-peptide opioid agonist, morphine, would result in up-regulation of APN enzyme activity. MATERIALS AND METHODS: Human embryonic kidney (HEK) 293 cells, stably transfected with mouse mu opioid receptor (293-MOR1) (Keith DE et al, J Biol Chem 271:19021-24,1996) were used. The cells were seeded in 96-well plates and treated with morphine (10-10 to 10-3 M) for 1, 2, 3, 4 or 5 days. APN enzyme activity was determined by using L-Ala-pNA as a substrate, and cell number by using WST-assay. RESULTS: Cells of 293-MOR1 exert strong APN activity (488 ą 83 nM/106 cells/30 min; n = 4) which could be effectively (over 90%) inhibited by specific inhibitor, probestin. Chronic treatment (3, 4 or 5 days) of 293-MOR1 cells with higher concentrations of morphine (10-4 M and 10-3 M) resulted in significant, dose-dependent decrease of APN enzyme activity. However, the observed decrease of APN was associated with similarly reduced cell number (maximal decrease obtained with 10-3 M morphine after 4-day treatment: 75% in APN, vs 71% in cell number), suggesting that no change of APN enzyme activity on per cell basis occurred. CONCLUSIONS: (1) 293-MOR1 cells exert strong APN enzyme activity. (2) Chronic treatment with morphine (10-10 to 10-3 M) for 1, 2, 3, 4 or 5 days did not affect APN activity of 293-MOR1 on a per cell basis. (3) Chronic treatment with morphine of higher concentration (10-4 and 10-3 M) for 3, 4 or 5 days strongly reduced cell number. (4) Thus, the data obtained do not support the idea that chronic exposure to morphine may induce up-regulation of APN enzyme activity as a part of adaptative mechanism. Opioid ligand of peptide structure, serving as an enzyme substrate, might be necessary in order to induce such adaptative changes.

morphine; opioid receptors; APN

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