The contribution of supraspinal, spinal or peripheral mu-opioid receptors (MORs) to the overall antinociception of systemic centrally penetrating versus peripherally restricted opioids has not been thoroughly investigated. Therefore, we examined paw pressure thresholds in Wistar rats with complete Freund's adjuvant hindpaw inflammation following different doses of intraplantar (i.pl.) as well as intravenous (i.v.) fentanyl (6.25-50 mg/kg), morphine (1-7.5 mg/kg) or loperamide (1-7.5 mg/kg). Antagonism of the i.v. mu-opioid agonists by intracerebroventricular (i.c.v.), intrathecal (i.t.) or i.pl. naloxone-methiodide (NLXM) revealed the relative contributions of supraspinal, spinal and peripheral MOR to the overall antinociceptive effects. In parallel, the MOR density at these three levels of pain transmission was assessed by radioligand binding. Antinociceptive effects of i.v. fentanyl and morphine, but not of the peripherally restricted loperamide were two-to threefold greater and longer lasting compared with their i.pl. administration. I.c.v. but not i.pl. NLXM significantly antagonized fentanyl's and morphine's antinociception by 70-80%, whereas i.t. NLXM reduced it by 20-30%. In contrast, antinociception of i.v. loperamide was abolished by i.pl. but not by i.c.v. or i.t. NLXM. In parallel, a respective 32-and sixfold higher MOR density in supraspinal and spinal versus peripheral sensory neurons was detected. In conclusion, in comparison with supraspinal and spinal opioid receptors, peripheral opioid receptors do not significantly contribute to the antinociception of systemic fentanyl and morphine during inflammatory pain. Antinociception of their i.v. administration was superior over both i.v and i.pl. loperamide, acting exclusively via peripheral MOR. These findings may guide the future development of novel peripherally restricted opioids.

Painful diabetic neuropathy is poorly controlled by analgesics and requires high doses of opioids, triggering side effects and reducing patient quality of life. This study investigated whether enhanced Rab7-mediated lysosomal targeting of peripheral sensory neuron μ-opioid receptors (MORs) is responsible for diminished opioid responsiveness in rats with streptozotocin-induced diabetes. In diabetic animals, significantly impaired peripheral opioid analgesia was associated with a loss in sensory neuron MOR and a reduction in functional MOR G-protein-coupling. In control animals, MORs were retained mainly on the neuronal cell membrane. In contrast, in diabetic rats, they were colocalized with upregulated Rab7 in LampI-positive perinuclear lysosome compartments. Silencing endogenous Rab7 with intrathecal Rab7-siRNA or, indirectly, by reversing nerve growth factor deprivation in peripheral sensory neurons not only prevented MOR targeting to lysosomes, restoring their plasma membrane density, but also rescued opioid responsiveness toward better pain relief. These findings elucidate in vivo the mechanisms by which enhanced Rab7 lysosomal targeting of MORs leads to a loss in opioid antinociception in diabetic neuropathic pain. This is in contrast to peripheral sensory neuron MOR upregulation and antinociception in inflammatory pain, and provides intriguing evidence that regulation of opioid responsiveness varies as a function of pain pathogenesis.

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Painful diabetic neuropathy is associated with impaired opioid analgesia; however, the precise mechanism in sensory neurons remains unclear. This study aimed to identify putative mechanisms involved in modified opioid responsiveness during early streptozotocin-induced diabetes in rats. In this study, we demonstrate that in diabetic animals, impaired peripheral opioid analgesia is associated with a reduction in functional mu-opioid receptor (MOR) G protein coupling. Mu-opioid receptor immunoreactive neurons colocalized with activated forms of protein kinase C (PKC) and with the receptor for advanced glycation end products (RAGE) during streptozotocin-induced diabetes. Moreover, MOR phosphorylation at Thr370 in sensory neurons of diabetic rats, and thus desensitization, was due to RAGE-dependent PKC activation. Importantly, blocking PKC activation using PKC selective inhibitor, silencing RAGE with intrathecal RAGE siRNA, or inhibiting advanced glycation end product (AGE) formation prevented sensory neuron MOR phosphorylation and, consequently, restored MOR G protein coupling and analgesic efficacy. Thus, our findings give the first in vivo evidence of a RAGE-dependent PKC-mediated heterologous MOR phosphorylation and desensitization in sensory neurons under pathological conditions such as diabetic neuropathy. This may unravel putative mechanisms and suggest possible prevention strategies of impaired opioid responsiveness.

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