The Expression and Function of the OGF-OGFr Axis - a Tonically Active Negative Regulator of Growth - in COS Cells

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)=3.5nm, B(max)=44fmol/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 micro, delta, and kappa 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.