Biotransformation of nociceptin orphanin FQ by enzyme activity from morphine-naive and morphine-treated cell cultures

The biotransformation of nociceptin/orphanin FQ (NOFQ) by enzyme activity i solated from U1690 human lung carcinoma and SH-SY5Y human neuroblastoma cel l lines, and from rat brain cortex cells in primary culture was investigate d. The identification and quantification of the cleavage products were perf ormed using electrospray ionization mass spectrometry linked to size-exclus ion chromatography. The effect of chronic morphine treatment of the cells ( 5 days) on NOFQ biotransformation was also studied. It was found that major products generated from NOFQ were the amino-terminal peptides N1-9 and N1- 13. The pattern of NOFQ biotransformation was quite similar for all three c ell cultures. However, different proportions of the formed peptides were no ted. The cleavage was inhibited by EDTA, PMSF, Hg2+, Cu2+ and Zn2+. Dynorph in A2-13 inhibited NOFQ cleavage in a manner suggesting competition of the two peptides for the same enzyme. Chronic morphine treatment of the cell cu ltures resulted in a substantial increase in the enzyme activity, leading t o higher levels of the major fragments and accumulation of N1-12 and the sh orter peptides N1-5, N1-6. Since the effect of morphine treatment of the ce lls was blocked by naloxone, it is likely that it was receptor specific. Ta ken together, the findings suggest that a metallosensitive endopeptidase, t he activity of which is increased by chronic morphine treatment of the cell s, is responsible for the biotransformation of NOFQ with fragments N1-9 and N1-13 being the major products. (C) 1999 Elsevier Science B.V. All rights reserved.