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, 124 (2), 592-603

Opioid Receptor-Triggered Spinal mTORC1 Activation Contributes to Morphine Tolerance and Hyperalgesia

Opioid Receptor-Triggered Spinal mTORC1 Activation Contributes to Morphine Tolerance and Hyperalgesia

Ji-Tian Xu et al. J Clin Invest.

Abstract

The development of opioid-induced analgesic tolerance and hyperalgesia is a clinical challenge for managing chronic pain. Adaptive changes in protein translation in the nervous system are thought to promote opioid tolerance and hyperalgesia; however, how opioids drive such changes remains elusive. Here, we report that mammalian target of rapamycin (mTOR), which governs most protein translation, was activated in rat spinal dorsal horn neurons after repeated intrathecal morphine injections. Activation was triggered through μ opioid receptor and mediated by intracellular PI3K/Akt. Spinal mTOR inhibition blocked both induction and maintenance of morphine tolerance and hyperalgesia, without affecting basal pain perception or locomotor functions. These effects were attributed to the attenuation of morphine-induced increases in translation initiation activity, nascent protein synthesis, and expression of some known key tolerance-associated proteins, including neuronal NOS (nNOS), in dorsal horn. Moreover, elevating spinal mTOR activity by knocking down the mTOR-negative regulator TSC2 reduced morphine analgesia, produced pain hypersensitivity, and increased spinal nNOS expression. Our findings implicate the μ opioid receptor-triggered PI3K/Akt/mTOR pathway in promoting morphine-induced spinal protein translation changes and associated morphine tolerance and hyperalgesia. These data suggest that mTOR inhibitors could be explored for prevention and/or reduction of opioid tolerance in chronic pain management.

Figures

Figure 1
Figure 1. Intrathecal rapamycin attenuates the development and maintenance of morphine tolerance and hyperalgesia. n = 5–7 rats per group.
(AC) Morphine (M) was administered intrathecally twice daily for 6 days. (A) Rapamycin (R), but not ascomycin (A), attenuated the reduction in morphine’s maximal potential analgesic effect (MPAE) on days 5 and 7. S, saline; V, vehicle. (B) Rapamycin, but not ascomycin, blocked a rightward shift in the cumulative dose-response curve of morphine on day 7. (C) Rapamycin dose-dependently attenuated the reduction in morphine’s MPAE on days 5 and 7. (D) Coinjection of rapamycin did not affect analgesia induced by a submaximal dose of intrathecal morphine. (E and F) Morphine was administered intrathecally twice daily for 6 days. Coadministration of rapamycin, but not ascomycin, blocked morphine-induced decreases in left hind paw withdrawal threshold (E) and latency (F) on day 7. (GI) Morphine was administered intrathecally twice daily for 11 days. (G) Coadministration of rapamycin beginning on day 7 reversed morphine-induced reductions in MPAE on days 9 and 12. n = 5–6 rats per group. (H and I) Coadministration of rapamycin beginning on day 7 reversed the reductions in left hind paw withdrawal threshold (H) and latency (I) on day 12. (J) Concanavalin A–stimulated (ConA) proliferation of splenocytes and effect of rapamycin pretreatment. *P < 0.05, **P < 0.01 vs. baseline; #P < 0.05, ##P < 0.01 vs. morphine plus vehicle.
Figure 2
Figure 2. Spinal mTOR knockdown attenuates the development of morphine-induced tolerance and hyperalgesia.
(A) Intrathecal mTOR siRNA (Si, 5 εg), but not mTOR scrambled siRNA (Sc, 5 εg), significantly reduced expression of mTOR without affecting expression of PSD-95, S6K1, or 4E-BP1 in dorsal horn. Representative Western blots and summary of densitometric analysis (normalized to β-actin loading control) are shown. *P < 0.05 vs. saline plus vehicle. n = 5 rats per group. (B) Intrathecal mTOR siRNA, but not mTOR scrambled siRNA, dramatically blocked the morphine-induced decrease in MPAE on days 5 and 7 of twice-daily intrathecal morphine injections. **P < 0.01 vs. morphine plus vehicle. n = 5 rats per group. (CF) Intrathecal mTOR siRNA, but not mTOR scrambled siRNA, markedly blocked morphine-induced decreases in left (C and E) and right (D and F) hindpaw withdrawal thresholds in response to mechanical stimuli (C and D) and withdrawal latencies in response to thermal stimuli (E and F) after 7 days of twice-daily morphine injections. *P < 0.05, **P < 0.01 vs. baseline; #P < 0.05, ##P < 0.01 vs. morphine plus saline. n = 5 rats per group.
Figure 3
Figure 3. Repeated intrathecal morphine injections activate the mTOR pathway in dorsal horn neurons.
(A and B) Repeated intrathecal morphine injections caused time-dependent increases in p-mTOR, p-S6K1, and p–4E-BP1 (A), but did not change the levels of total mTOR, S6K1, or 4E-BP1 (B), in dorsal horn. Representative Western blots and summary of densitometric analysis (normalized to β-actin loading control) are shown. *P < 0.05 vs. day 0. n = 5 rats per group. (C) Distribution of neurons labeled by p-mTOR, p-S6K1, or p–4E-BP1 in dorsal horn 7 days after saline or morphine injection. Scale bar: 100 εm. (D) Exclusive localization of p-mTOR, p-S6K1, and p–4E-BP1 in dorsal horn neurons on day 7 of twice-daily intrathecal morphine injections. Scale bars: 50 εm.
Figure 4
Figure 4. Morphine-induced activation of the mTOR pathway is ε opioid receptor dependent in dorsal horn neurons during chronic morphine exposure.
(A and B) Naltrexone (Nal, 100 εM; A) and CTOP (CT, 10 εM; B) blocked morphine-induced (20 εM) increases in p-mTOR, p-S6K1, and p–4E-BP1 in cultured dorsal horn neurons. *P < 0.05, **P < 0.01 vs. control (C); ##P < 0.01 vs. morphine. n = 4–5 repeats per group. (C) CTOP (10 εM) blocked DAMGO-induced (DA, 20 εM) increases in p-mTOR, p-S6K1, and p–4E-BP1 in cultured dorsal horn neurons. **P < 0.01 vs. control; ##P < 0.01 vs. DAMGO. n = 4–5 repeats per group. (D) Coinjection of CTOP (1 ng) blocked morphine-induced (10 εg) increases in p-mTOR, p-S6K1, and p–4E-BP1 in dorsal horn on day 7 of twice-daily intrathecal morphine injections. **P < 0.01 vs. saline plus vehicle; #P < 0.05, ##P < 0.01 vs. morphine plus vehicle. n = 5 rats per group. (E) Subcutaneous injection of morphine (20 mg/kg, twice daily for 5 days) increased the level of dorsal horn p-mTOR in wild-type mice, but not in ε opioid receptor knockout mice. *P < 0.05 vs. saline-treated wild-type. n = 4 mice per group. (F) Colocalization (arrows) of ε opioid receptor (MOR) mRNA with mTOR and p-mTOR in dorsal horn neurons on day 7 of twice-daily intrathecal morphine injections. Scale bar: 50 εm.
Figure 5
Figure 5. PI3K/Akt mediates ε opioid receptor–dependent activation of the mTOR pathway in dorsal horn neurons during chronic morphine exposure.
(A and B) Coadministration of LY294002 (Ly, 10 εM; A) or Akt inhibitor IV (Akt, 5 εM; B) blocked morphine-induced (20 εM) increases in p-mTOR and p-S6K1 in cultured dorsal horn neurons. **P < 0.01 vs. saline; ##P < 0.01 vs. morphine. n = 4 repeats per group. (C and D) Coadministration of LY294002 (10 εM; C) or Akt inhibitor IV (5 εM; D) blocked DAMGO-induced (20 εM) increases in p-mTOR, p-S6K1, and p–4E-BP1 in cultured dorsal horn neurons. **P < 0.01 vs. saline; #P < 0.05, ##P < 0.01 vs. DAMGO. n = 4 repeats per group. (EH) Coinjection of LY294002 (10 εg; E and F) or Akt inhibitor IV (10 εg; G and H) blocked morphine-induced (10 εg) decrease in MPAE (E and G) and increases in p-mTOR, p-S6K1, and p–4E-BP1 in dorsal horn (F and H) on day 7 of twice-daily intrathecal morphine injections. *P < 0.05, **P < 0.01 vs. saline plus vehicle; ##P < 0.01 vs. morphine plus vehicle. n = 5 rats per group. (I) Coexpression of ε opioid receptor mRNA with PI3Kγ, Akt, and mTOR mRNA in individual dorsal horn neurons. Con, control. n = 3 rats.
Figure 6
Figure 6. Twice-daily intrathecal morphine injections enhance mTOR-dependent protein translation in dorsal horn on day 7.
(A) Coinjection of rapamycin (10 εg) blocked morphine-induced (10 εg) increases in p-mTOR, p-S6K1, and p–4E-BP1 in dorsal horn. (B) Coinjection of rapamycin blocked the morphine-induced increase in association between eIF4A and eIF4E and reversed the morphine-induced decrease in association between 4E-BP1 and eIF4E in dorsal horn. (C and D) Coinjection of rapamycin blocked the morphine-induced increase in AHA incorporation into newly synthesized proteins in dorsal horn. (E and F) Coinjection of rapamycin blocked morphine-induced increases in CaMKIIα, nNOS, and PKCγ, but did not affect basal levels of PKCβ or PSD-95, in dorsal horn. (AF) **P < 0.01 vs. saline plus vehicle; ##P < 0.01 vs. morphine plus vehicle. n = 4 rats per group. (G) Coexpression of ε opioid receptor and mTOR mRNA with nNOS, PKCγ, or CaMKIIα mRNA in individual dorsal horn neurons. n = 3 rats.
Figure 7
Figure 7. Elevating mTOR activity by knocking down TSC2 reduces morphine analgesia and induces pain hypersensitivity.
Saline, vehicle, TSC2 siRNA, or scrambled TSC2 siRNA was injected intrathecally once daily for 3 days. Protein expression and MPAE were measured on day 7. (A) TSC2 siRNA (4 εg), but not scrambled TSC2 siRNA (4 εg), reduced TSC2 expression, increased p-mTOR, p-S6K1, and nNOS levels, and had no effect on total mTOR or S6K1 protein expression. (B and C) TSC2 siRNA, but not scrambled TSC2 siRNA, reduced morphine MPAE (B) and produced a significant rightward shift in the cumulative dose-response curve of morphine (C). (D and E) TSC2 siRNA, but not scrambled TSC2 siRNA, reduced left and right hindpaw withdrawal thresholds in response to mechanical stimuli (D) and withdrawal latencies in response to thermal stimuli (E). *P < 0.05, **P < 0.01 vs. saline. n = 4 (A) or 5 (BE) rats per group.

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