Characterization of functional µ opioid receptor turnover in rat locus coeruleus: an electrophysiological and immunocytochemical study.

BACKGROUND AND PURPOSE: Regulation of µ receptor dynamics such as its trafficking is a possible mechanism underlying opioid tolerance that contributes to inefficient recycling of opioid responses. We aimed to characterize the functional turnover of µ receptors in the noradrenergic nucleus locus coeruleus (LC). EXPERIMENTAL APPROACH: We measured opioid effect by single-unit extracellular recordings of LC neurons from rat brain slices. Immunocytochemical techniques were used to evaluate µ receptor trafficking. KEY RESULTS: After near-complete, irreversible µ receptor inactivation with ß-funaltrexamine (ß-FNA), opioid effect spontaneously recovered in a rapid and efficacious manner. In contrast, α2 -adrenoceptor-mediated effect hardly recovered after receptor inactivation with the irreversible antagonist EEDQ. When the recovery of opioid effect was tested after various inactivating time schedules, we found that the longer the ß-FNA pre-exposure, the less efficient and slower the functional µ receptor turnover became. Interestingly, µ receptor turnover was slower when ß-FNA challenge was repeated in the same cell, indicating constitutive µ receptor recycling by trafficking from a depletable pool. Double immunocytochemistry confirmed the constitutive nature of µ receptor trafficking from a cytoplasmic compartment. The µ receptor turnover was slowed down when LC neuron calcium- or firing-dependent processes were prevented or vesicular protein trafficking was blocked by a low temperature or transport inhibitor. CONCLUSIONS AND IMPLICATIONS: Constitutive trafficking of µ receptors from a depletable intracellular pool (endosome) may account for its rapid and efficient functional turnover in the LC. A finely-tuned regulation of µ receptor trafficking and endosomes could explain neuroadaptive plasticity to opioids in the LC.

Contribution of nitric oxide-dependent guanylate cyclase and reactive oxygen species signaling pathways to desensitization of µ-opioid receptors in the rat locus coeruleus.

Nitric oxide (NO) is involved in desensitization of µ-opioid receptors (MOR). We used extracellular recordings in vitro to unmask the NO-dependent pathways involved in MOR desensitization in the rat locus coeruleus (LC). Perfusion with ME (3 and 10 µM) concentration-dependently reduced subsequent ME effect, indicative of MOR desensitization. ME (3 µM)-induced desensitization was enhanced by a NO donor (DEA/NO 100 µM), two soluble guanylate cyclase (sGC) activators (A 350619 30 µM and BAY 418543 1 µM) or a cGMP-dependent protein kinase (PKG) activator (8-pCPT-cGMP 30 µM). DEA/NO-induced enhancement was blocked by the sGC inhibitor NS 2028 (10 µM). A 350619 effect was also blocked by NS 2028, but not by the antioxidant Trolox. ME (10 µM)-induced desensitization was blocked by the neuronal NO synthase inhibitor 7-NI (100 µM) and restored by the PKG activator 8-Br-cGMP (100-300 µM). Paradoxically, ME (10 µM)-induced desensitization was not modified by sGC inhibitors (NS 2028 and ODQ), PKG inhibitors (H8 and Rp-8-Br-PET-cGMP) or antioxidant agents (Trolox, U-74389G and melatonin), but it was attenuated by a combination of NS 2028 and Trolox. In conclusion, MOR desensitization in the LC may be mediated or regulated by NO through sGC and reactive oxygen species signaling pathways.