Baled I. Khalefa

Relative contributions of peripheral versus supraspinal or spinal opioid receptors to the antinociception of systemic opioids

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 Freunds adjuvant hindpaw inflammation following different doses of intraplantar (i.pl.) as well as intravenous (i.v.) fentanyl (6.25–50 μg/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 fentanyls and morphines 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.

Rab7 Silencing Prevents μ-Opioid Receptor Lysosomal Targeting and Rescues Opioid Responsiveness to Strengthen Diabetic Neuropathic Pain Therapy

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.

Protein kinase C-mediated mu-opioid receptor phosphorylation and desensitization in rats, and its prevention during early diabetes.

Abstract 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.

Peripheral antinociceptive efficacy and potency of a novel opioid compound 14-O-MeM6SU in comparison to known peptide and non-peptide opioid agonists in a rat model of inflammatory pain

This study compared the peripheral analgesic effects of a novel opioid agonist 14-O-methylmorphine-6-O-sulfate (14-O-MeM6SU), to that of non-peptide (morphine, fentanyl) and peptide opioid agonists (Met-enkephalin; met-ENK and β-endorphin; β-END) in a model of localized inflammatory pain evoked by intraplantar (i.pl.) Freunds complete adjuvant (FCA). Nociceptive responses to local opioid agonists were measured by pressure paw-withdrawal procedures. In addition, the antinociceptive efficacy and potency of these test compounds in vivo was compared to that in vitro using the rat vas deferens (RVD) bioassay. Intraplantar 14-O-MeM6SU (0.32-2.53 nmol/rat), morphine (14.95-112.15 nmol/rat), fentanyl (0.19-2.36 nmol/rat), met-ENK (0.10-10 nmol/rat) and β-END (0.77-5.00 nmol/rat) dose dependently increased paw pressure thresholds exclusively in inflamed hindpaws. At higher doses analgesic effects were also seen in noninflamed paws for 14-O-MeM6SU, morphine and fentanyl but not for met-ENK or β-END. The maximal possible local analgesic effect (%) measured in inflamed paws was 50.6 ± 2.7, 18.23 ± 1.78, 37.44 ± 2.17, 36.00 ± 1.43, and 40.69 ± 0.91 for 14-O-MeM6SU, morphine, fentanyl, met-ENK and β-END, respectively. Interestingly, i.pl. administered opioid peptides met-ENK and β-END displayed a peripheral analgesic ceiling effect. This local antinociception was antagonized by co-administered opioid antagonist naloxone-methiodide (NAL-M). Similar to the analgesic testing, the RVD showed the following efficacy order of the test compounds: 14-O-MeM6SU>β-END>fentanyl>met-ENK≫morphine. Taken together, 14-O-MeM6SU was more potent than morphine, fentanyl and met-ENK and β-END and displayed superiority in the maximum antinociceptive effects. The superiority of local antinociceptive effects of 14-O-MeM6SU might be due to both pharmacodynamic and pharmacokinetic factors.

Accessibility of axonal G protein coupled mu-opioid receptors requires conceptual changes of axonal membrane targeting for pain modulation

&NA; The mechanisms of axonal trafficking and membrane targeting are well established for sodium channels, which are the principle targets for perineurally applied local anaesthetics. However, they have not been thoroughly investigated for G protein coupled receptors such as mu‐opioid receptors (MOR). Focusing on these axonal mechanisms, we found that axonal MOR functionality is quite distinct in two different pain states, i.e. hindpaw inflammation and nerve injury. We observed axonal membrane MOR binding and functional G protein coupling exclusively at sites of CCI nerve injury. Moreover at these axonal membrane sites, MOR exhibited extensive co‐localization with the membrane proteins SNAP and Na/K‐ATPase as well as NGF‐dependent enhanced lipid rafts and L1CAM anchoring proteins. Silencing endogenous L1CAM with intrathecal L1CAM specific siRNA, disrupting lipid rafts with the perineurial cholesterol‐sequestering agent M&bgr;CD, as well as suppressing NGF receptor activation with the perineurial NGF receptor inhibitor K252a abrogated MOR axonal membrane integration, functional coupling, and agonist‐elicited antinociception at sites of nerve injury. These findings suggest that local conceptual changes resulting from nerve injury are required for the establishment of functional axonal membrane MOR. Axonal integration and subsequent accessibility of functionally coupled MOR are of great relevance particularly for patients suffering from severe pain due to nerve injury or tumour infiltration. Graphical abstract Figure. No caption available.