The expression and function of the OGF–OGFr axis – a tonically active negative regulator of growth – in COS cells

Abstract

This study was designed to examine the presence and role of the opioid growth factor (OGF, [Met 5]-enkephalin) and the OGF receptor (OGFr) in COS-7 cells; these cells lack classical opioid receptors. Preproenkephalin mRNA, which encodes OGF, was detected by Northern blot analysis, and OGFr mRNA was recorded by RT-PCR. Receptor binding analysis showed specific and saturable binding ( K d=3.5 nm, B max=44 fmol/mg protein) for OGFr in the nuclear fraction. Both OGF and OGFr were recorded in COS-7 cells by immunocytochemistry. Addition of OGF to log-phase COS-7 cultures depressed growth by 41.6% from control levels, whereas opioid-receptor blockade by the opioid antagonist, naltrexone, increased the number of cells by 29.8% from control values. The effect of OGF was receptor mediated. Exposure to a wide variety of synthetic and natural opioid peptides, including those selective for μ, δ, and κ opioid receptors, showed that only OGF had an effect. Treatment with antisense OGFr oligonucleotides increased the number of cells by over 2-fold compared to wild-type cultures of COS-7 cells and preparations receiving scrambled oligonucleotides. These results indicate that the OGF–OGFr axis is present and functions in COS-7 cells, and in the absence of classical opioid receptors.