Altered amounts of G-protein mRNA and cAMP accumulation after long-term opioid receptor stimulation of neurons in primary culture from the rat cerebral cortex

Abstract

Primary neuronal enriched cultures were incubated with μ (morphine, 10 −5 M), δ (DPDPE, 10 −6 M) and κ (U-50,488H, 10 −5 M) receptor agonists for 5 days, respectively. Thereafter the acute inhibitory actions of μ, δ or κ receptor agonists on forskolin stimulated cAMP accumulation was assayed. The effect of long term opioid treatment on the steady-state level of G-protein mRNA (G αs, G αi-1 and G αi-2) was analyzed using an RNAase protection hybridization assay. Incubation for 5 days with κ receptor agonist resulted in an attenuated ability to decrease the accumulation of cAMP by κ receptors, as well as μ and δ receptors, which was also observed after 5 days of incubation with the δ receptor agonist. Furthermore, the adenylate cyclase responsiveness to forskolin stimulation was markedly reduced in cultures treated with either δ or κ receptor agonists. Five days of incubation with κ receptor agonist resulted in an increase in the levels of G αs and G αi-2 mRNAs. No effects on the amounts of G αs mRNA, G αi-1 mRNA or G αi-2 mRNA were detected after 5 days of δ receptor stimulation. On the other hand, 5 days of μ receptor stimulation decreased the amounts of G αs, G αi-1 and G αi-2 mRNA. Incubation with κ receptor agonist for 24 h resulted in a significant decrease in the forskolin-stimulated accumulation of cAMP. The stimulatory effect of forskolin was further decreased after 3 days incubation with κ receptor agonist. A decreased sensitivity to the acute inhibitory action of μ, δ or κ receptor agonists was seen after 3 days incubation with the κ receptor agonist. The basal accumulation of cAMP was decreased after 24 h and 3 days of incubation with the κ agonist in the neuronal enriched cultures. The present data demonstrate that long-term activation of κ and δ receptors in neuronal enriched cultures from the rat cerebral cortex reduced the stimulated formation of cAMP, induced decreased sensitivity of μ, δ or κ receptors. Furthermore, the present data indicate that long term activation of μ and κ opioid receptors induces alterations in the expression of G-protein mRNA.