Small G-Protein Rheb Gates Mammalian Target of Rapamycin Signaling to Regulate Morphine Tolerance in Mice.

BACKGROUND: Analgesic tolerance due to long-term use of morphine remains a challenge for pain management. Morphine acts on µ-opioid receptors and downstream of the phosphatidylinositol 3-kinase signaling pathway to activate the mammalian target of rapamycin (mTOR) pathway. Rheb is an important regulator of growth and cell-cycle progression in the central nervous system owing to its critical role in the activation of mTOR. The hypothesis was that signaling via the GTP-binding protein Rheb in the dorsal horn of the spinal cord is involved in morphine-induced tolerance. METHODS: Male and female wild-type C57BL/6J mice or transgenic mice (6 to 8 weeks old) were injected intrathecally with saline or morphine twice daily at 12-h intervals for 5 consecutive days to establish a tolerance model. Analgesia was assessed 60 min later using the tail-flick assay. After 5 days, the spine was harvested for Western blot or immunofluorescence analysis. RESULTS: Chronic morphine administration resulted in the upregulation of spinal Rheb by 4.27 ± 0.195-fold (P = 0.0036, n = 6), in turn activating mTOR by targeting rapamycin complex 1 (mTORC1). Genetic overexpression of Rheb impaired morphine analgesia, resulting in a tail-flick latency of 4.65 ± 1.10 s (P < 0.0001, n = 7) in Rheb knock-in mice compared to 10 s in control mice (10 ± 0 s). Additionally, Rheb overexpression in spinal excitatory neurons led to mTORC1 signaling overactivation. Genetic knockout of Rheb or inhibition of mTORC1 signaling by rapamycin potentiated morphine-induced tolerance (maximum possible effect, 52.60 ± 9.56% in the morphine + rapamycin group vs. 16.60 ± 8.54% in the morphine group; P < 0.0001). Moreover, activation of endogenous adenosine 5'-monophosphate-activated protein kinase inhibited Rheb upregulation and retarded the development of morphine-dependent tolerance (maximum possible effect, 39.51 ± 7.40% in morphine + metformin group vs. 15.58 ± 5.79% in morphine group; P < 0.0001). CONCLUSIONS: This study suggests spinal Rheb as a key molecular factor for regulating mammalian target of rapamycin signaling.

Chronic morphine induces cyclic adenosine monophosphate formation and hyperpolarization-activated cyclic nucleotide-gated channel expression in the spinal cord of mice.

Chronic morphine exposure persistently activates Gαi/o protein-coupled receptors and enhances adenylyl cyclase (AC) activity, which can increase cyclic adenosine monophosphate (cAMP) production. Direct binding of cAMP to the cytoplasmic site on hyperpolarization-activated cyclic nucleotide-gated (HCN) channels increases the probability of channel opening. HCN channels play a prominent role in chronic pain the disease that shares some common mechanisms with opioid tolerance. This compensatory AC activation may be responsible for the induction of morphine-induced analgesic tolerance. We investigated spinal cAMP formation and expression of HCN2 in the spinal cord, and observed the effect of AC inhibition on the induction of morphine analgesic tolerance. We found that chronic morphine-induced antinociceptive tolerance increased spinal cAMP formation and the expression of spinal HCN2. Inhibition of spinal AC partially blocked chronic morphine-induced cAMP formation and prevented the induction of morphine-induced analgesic tolerance. Inhibition of HCN2 also showed a partial preventive effect on morphine-induced tolerance, hypothermia tolerance and also the right-shift of the dose-response curve. We conclude that repeated morphine treatment increases AC activity and cAMP formation, and also spinal HCN2 expression, blockade of AC or HCN2 can prevent the development of morphine-induced analgesic tolerance.

Exchange factor directly activated by cAMP-PKCε signalling mediates chronic morphine-induced expression of purine P2X3 receptor in rat dorsal root ganglia.

BACKGROUND AND PURPOSE: The P2X3 receptor is a major receptor in the processing of nociceptive information in dorsal root ganglia. We investigated the role of the P2X3 receptor and the detailed mechanisms underlying chronic morphine-induced analgesic tolerance in rats. EXPERIMENTAL APPROACH: Repeated i.t. morphine treatment was used to induce anti-nociceptive tolerance. The expression of spinal P2X3 receptor, phosphorylated PKCε and exchange factor directly activated by cAMP (Epac) were evaluated. Effects of A-317491 (P2X3 antagonist), ε-V1-2 (PKCε inhibitor) and ESI-09 (Epac inhibitor) on mechanical pain thresholds and tail-flick latency after chronic morphine treatment were determined. Co-localization of P2X3 receptor with NeuNs (marker of neuron), IB4 (marker of small DRG neurons), peripherin, PKCε and Epac were performed by double immunofluorescence staining. KEY RESULTS: Chronic morphine time-dependently increased the expression of P2X3 receptor, phosphorylated PKCε and Epac in DRGs. ε-V1-2 prevented chronic morphine-induced expression of P2X3 receptor. ESI-09 decreased the phosphorylation of PKCε and up-regulated expression of Epac after chronic morphine exposure. Mechanical pain thresholds and tail-flick latency showed that A317491, ε-V1-2 and ESI-09 significantly attenuated the loss of morphine's analgesic potency. Morphine-induced P2X3 receptor expression mainly occurred in neurons staining for IB4 and peripherin. Co-localization of P2X3 receptor with PKCε and Epac was demonstrated in the same neurons. CONCLUSIONS AND IMPLICATIONS: Chronic morphine exposure increased the expression of P2X3 receptor, and i.t. P2X3 receptor antagonists attenuated the loss of morphine's analgesic effect. Inhibiting Epac/PKCε signalling was shown to play a significant inhibitory role in chronic morphine-induced P2X3 receptor expression and attenuate morphine-induced tolerance.