ANTISENSE PROENKEPHALIN CDNA TRANSFECTION DECREASES OPIOID BINDING IN NG 108-15 CELLS

The proenkephalin A (PENK) gene codes for several opioid peptides, including Met-enkephalin, an endogenous ligand of opioid receptors. These peptides are thought to play an important role in a variety of neural processes. To study the role of the PENK gene in opioid related processes, an antisense sequence of PENK gene was subcloned into pSVL SV 40 late promoter expression vector and stably transfected into NG 108-15 cells, which contain opioid receptors. The sense orientation of the same fragment was also cloned and transfected, serving as a control. The presence and expression of transfected recombinant plasmids in NG 108 cells were confirmed by DNA and RNA PCR and by subsequent sequencing. Surprisingly, the endogeneous PENK message level was found to be 3 times higher in antisense cells than in sense or NG 108 cells. This high steady-state mRNA level seemed to be due to the increased stability of PENK mRNA rather than to an elevated transcription rate. Nevertheless, the level of total Met-enkephalin was found to be reduced in antisense-transfected cells, though free Met-enkephalin content did not differ from sense-transfected or non-transfected cells. We suggest that both the increased PENK message and the unchanged levels of free Met-enkephalin may be the result of compensatory mechanisms induced by translational inhibition by antisense, although the underlying processes remain to be determined. Binding of the opioid ligand [H-3]diprenorphine was significantly reduced by 50-80% in the antisense-transfected cell lines. but not in the sense cells. In contrast, binding of the muscarinic ligand [H-3]scopolamine, or alpha2-adrenergic ligand [H-3]rauwolscine were unchanged either in antisense or sense cells. Thus the presence of antisense PENK in NG 108-15 cells appeared to have a specific effect on opioid receptors, though this effect was not mediated through alterations in the receptor's endogenous ligand. While our results did not enable us to determine the effect of altering endogenous opioid ligand levels on opioid receptor disposition in NG 108-15 cells, we speculate that opioid receptors may be regulated by the level of PENK mRNA.