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

Abstract

The biotransformation of nociceptin/orphanin FQ (NOFQ) by enzyme activity isolated from U1690 human lung carcinoma and SH-SY5Y human neuroblastoma cell lines, and from rat brain cortex cells in primary culture was investigated. The identification and quantification of the cleavage products were performed using electrospray ionization mass spectrometry linked to size-exclusion 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 cell cultures. However, different proportions of the formed peptides were noted. The cleavage was inhibited by EDTA, PMSF, Hg 2+, Cu 2+ and Zn 2+. Dynorphin A2–13 inhibited NOFQ cleavage in a manner suggesting competition of the two peptides for the same enzyme. Chronic morphine treatment of the cell cultures resulted in a substantial increase in the enzyme activity, leading to higher levels of the major fragments and accumulation of N1–12 and the shorter peptides N1–5, N1–6. Since the effect of morphine treatment of the cells was blocked by naloxone, it is likely that it was receptor specific. Taken together, the findings suggest that a metallosensitive endopeptidase, the activity of which is increased by chronic morphine treatment of the cells, is responsible for the biotransformation of NOFQ with fragments N1–9 and N1–13 being the major products.