Role of agonist-dependent receptor internalization in the regulation of μ opioid receptors

Abstract

Organotypic cultures and ileal neuromuscular preparations were used to determine (i) whether endogenous release of opioids by electrical stimulation induces μ receptor endocytosis, and (ii) whether and under which conditions ligand-induced μ receptor endocytosis influences the responsiveness of neurons expressing native μ receptors. In longitudinal muscle–myenteric plexus preparations, electrical stimulation at 20 Hz induced a prominent endocytosis of μ receptors in enteric neurons, indicating endogenous release of opioids. A similar massive endocytosis was triggered by exogenous application of the μ receptor agonist, [ d-Ala 2,MePhe 4,Gly-ol 5] enkephalin, whereas exogenous application of morphine was ineffective. [ d-Ala 2,MePhe 4,Gly-ol 5] enkephalin and morphine induced a concentration-dependent inhibition of neurogenic cholinergic twitch contractions to electrical stimulation at 0.1 Hz. β-Chlornaltrexamine shifted to the right the inhibitory curve of both agonists with a concentration-dependent reduction of the maximum agonist response, which is consistent with the existence of spare μ opioid receptors. Under these conditions, the induction of μ receptor endocytosis by exogenously applied [ d-Ala 2,MePhe 4,Gly-ol 5] enkephalin diminished the inhibitory effect of this agonist on twitch contractions and tritiated acetylcholine release. In contrast, there was no reduction of the inhibitory effect of morphine, which failed to induce μ receptor endocytosis, on neurogenic cholinergic response. These results provide the first evidence for the occurrence of μ receptor endocytosis in neurons by endogenously released opioids and show that agonist-dependent μ receptor endocytosis could serve as a mechanism to regulate μ opioid receptor responsiveness to ligand stimulation when the opioid receptor reserve is reduced.