Endosomal pH Determines Ligand-Specific Endocytic Sorting Of The Mu-Opioid Receptor By Morphine

David J. Lee and Peregrine B. Osborne, Pain Management Research Institute, University of Sydney at the Royal North Shore Hospital, Sydney

Purpose of Study

Continuous exposure to high concentrations of opioid agonists can cause mu-opioid receptors (MOPRs) to be trafficked away from the cell surface into endosomes (endocytosis). In the clinical situation, this cellular mechanism is thought to factor in the development of opioid tolerance in patients. Recent studies have established that morphine is very poor at eliciting endocytosis relative to other common opioid drugs. We have investigated if this ligand-specific property of morphine is determined by the low pH of early-endosomes. This hypothesis predicts the acid pH inside endosomes dissociates morphine from internalized MOPRs, and alllows ligand-free MOPRs to be rapidly recycled back to the cell surface. In contrast, ligand-MOPR complexes formed by pH insensitive opioid ligands are trafficked into deep endosomes, which delays MOPR recycling and prolongs cellular tolerance.

Method

Morphine binding to MOPRs expressed in an AtT20 cell line was measured directly by radioligand binding, and indirectly by electrophysiological patch clamp recording.

Result

Both methods provided evidence of morphine-MOPR interactions being reduced more strongly by a low pH environment (pH 5.0-7.0) than other opioid ligands. To examine this further, we measured the fade of opioid-induced currents, which is related to the rate of receptor internalization in AtT20 cells, and used bafilomycin to neutralize the internal endosomal pH. Bafilomycin significantly increased (P < 0.05) fading of morphine-induced currents, but had not effect on currents induced by the agonist DAMGO. This effect was prevented when cells were treated with concamycin A to block endocytic trafficking.

Conclusion

Our findings suggest the low pH of early endosomes limits MOPR endocytosis induced by morphine. This pH-sensitivity may identify a novel feature of the opioid pharmacophore, which could be exploited for rationale drug design aimed at reducing opioid analgesic tolerance. (supported by ANZCA project 05/019)