Opioid peptide receptor studies. 17. Attenuation of chronic morphine effects after antisense oligodeoxynucleotide knock‐down of RGS9 protein in cells expressing the cloned Mu opioid receptor

RGS proteins are a recently described class of regulators that influence G‐protein‐mediated signaling pathways. We have shown previously that chronic morphine results in functional uncoupling of the μ opioid receptor from its G protein in CHO cells expressing cloned human μ opioid receptors. In the present study, we examined the effects of morphine treatment (1 μM, 20 h) on DAMGO‐stimulated high‐affinity [ 35 S]GTP‐γ‐S binding and DAMGO‐mediated inhibition of forskolin‐stimulated cAMP accumulation in HN9.10 cells stably expressing the cloned rat μ opioid receptor, in the absence and presence of the RGS9 protein knock‐down condition (confirmed by Western blot analysis). RGS9 protein expression was reduced by blocking its mRNA with an antisense oligodeoxynucleotide (AS‐114). Binding surface analysis resolved two [ 35 S]GTP‐γ‐S binding sites (high affinity and low affinity sites). In sense‐treated control cells, DAMGO‐stimulated [ 35 S]GTP‐γ‐S binding by increasing the B max of the high‐affinity site. In sense‐treated morphine‐treated cells, DAMGO‐stimulated [ 35 S]GTP‐γ‐S binding by decreasing the high‐affinity K d without changing the B max . AS‐114 significantly inhibited chronic morphine‐induced upregulation of adenylate cyclase activity and partially reversed chronic morphine effects as measured by DAMGO‐stimulated [ 35 S]GTP‐γ‐S binding. Morphine treatment increased the EC 50 (6.2‐fold) for DAMGO‐mediated inhibition of forskolin‐stimulated cAMP activity in control cells but not in cells treated with AS‐114 to knock‐down RGS9. These results provide additional evidence for involvement of RGS9 protein in modulating opioid signaling, which may contribute to the development of morphine tolerance and dependence. Synapse 52:209–217, 2004. Published 2004 Wiley‐Liss, Inc.