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

Autori
Gabrilovac, Jelka ; Breljak, Davorka

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
2001 Annual Meeting of the Croatian Immunological Society, Book of Abstracts / - Zagreb, 2001

Skup
2001 Annual Meeting of the Croatian Immunological Society

Mjesto i datum
Zagreb, Hrvatska, 07.12.2001

Vrsta sudjelovanja
Poster

Vrsta recenzije
Domaća recenzija

Ključne riječi
morphine; opioid receptors; APN

Sažetak
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.

Izvorni jezik
Engleski

Znanstvena područja
Kliničke medicinske znanosti

POVEZANOST RADA