Effect of neuronal nitric oxide synthase inhibitors and antioxidants on the development of tolerance by different opioid agonists in the rat locus coeruleus

Nitric oxide (NO) is involved in acute μ-opioid receptor (MOR) desensitization in the locus coeruleus (LC) and in the neuroadaptations following chronic morphine administration. However, the role of NO and NO-derived reactive oxygen species (ROS) in the development of cellular tolerance to different opioids remains unclear. Herein, we examined the effect of the selective neuronal nitric oxide synthase (nNOS) inhibitor 7-nitroindazole (7-NI; 30 mg/kg/12 h, i.p.) and the antioxidants Trolox + ascorbic acid (TX+AA; 40 and 100 mg/kg/day, respectively, i.p.) and U-74389G (10 mg/kg/day, i.p.) on the development of cellular tolerance induced by morphine, methadone and fentanyl. For induction of morphine tolerance, rats were treated subcutaneously with a slow release emulsion containing free base morphine (200 mg/kg, 3 days). For methadone (60 mg/kg/day, 6 days) and fentanyl (0.2 mg/kg/day, 7 days), tolerance was induced by s.c. implantation of osmotic pumps. Concentration-effect curves for the inhibitory effect of Met⁵-enkephalin (ME; 0.05-12.8 μM, 2x, 1 min) on the firing rate were performed by single-unit extracellular recordings of LC neurons from rat brain slices. Morphine, methadone and fentanyl treatments shifted to the right concentration-effect curves for ME and increased the EC₅₀ by 2-4 folds. Co-administration of TX+AA or U-74389G in morphine-treated animals prevented the development of tolerance in LC neurons. Conversely, co-treatments with U-74389G or 7-NI failed to affect the induction of cellular tolerance after methadone or fentanyl treatments. Our results suggest that MOR agonists with different intrinsic efficacies cause variable degrees of cellular tolerance in LC cells. Moreover, NO/ROS pathways are differentially involved in opioid tolerance after prolonged treatments with morphine, methadone and fentanyl.