Chronic morphine induces downregulation of spinal glutamate transporters: implications in morphine tolerance and abnormal pain sensitivity

Abstract

Tolerance to the analgesic effects of an opioid occurs after its chronic administration, a pharmacological phenomenon that has been associated with the development of abnormal pain sensitivity such as hyperalgesia. In the present study, we examined the role of spinal glutamate transporters (GTs) in the development of both morphine tolerance and associated thermal hyperalgesia. Chronic morphine administered through either intrathecal boluses or continuous infusion induced a dose-dependent downregulation of GTs (EAAC1 and GLAST) in the rat's superficial spinal cord dorsal horn. This GT downregulation was mediated through opioid receptors because naloxone blocked such GT changes. Morphine-induced GT downregulation reduced the ability to maintain in vivo glutamate homeostasis at the spinal level, because the hyperalgesic response to exogenous glutamate was enhanced, including an increased magnitude and a prolonged time course, in morphine-treated rats with reduced spinal GTs. Moreover, the downregulation of spinal GTs exhibited a temporal correlation with the development of morphine tolerance and thermal hyperalgesia. Consistently, the GT inhibitor l-trans-pyrrolidine-2-4-dicarboxylate (PDC) potentiated, whereas the positive GT regulator riluzole reduced, the development of both morphine tolerance and thermal hyperalgesia. The effects from regulating spinal GT activity by PDC were at least in part mediated through activation of the NMDA receptor (NMDAR), because the noncompetitive NMDAR antagonist MK-801 blocked both morphine tolerance and thermal hyperalgesia that were potentiated by PDC. These results indicate that spinal GTs may contribute to the neural mechanisms of morphine tolerance and associated abnormal pain sensitivity by means of regulating regional glutamate homeostasis.

Fig. 1.

Downregulation of spinal GTs after chronic morphine. Both EAAC1-ir (B) and GLAST-ir (E) were reduced in rats receiving a 7 d intrathecal, twice daily treatment with 20 μg of morphine as compared with the corresponding saline control (A, D). Coadministration of morphine (20 μg) with naloxone (10 μg), twice daily for 7 d, blocked the reduction of both EAAC1-ir (C) and GLAST-ir (F). Scale bar, 50 μm.

Fig. 2.

Quantification of the EAAC1-ir and GLAST-ir reduction. The relative density of immunostaining in laminas I–II was measured by subtracting the background density in each image. The percentage reduction of density from the corresponding saline group was calculated as described in Materials and Methods. A,B10, B20, 10 or 20 μg of morphine boluses; B, C10, C20, 10 or 20 nm · μl⁻¹ · hr⁻¹morphine infusion; C, B20, 20 μg of morphine bolus alone; B20+NX, 20 μg of morphine plus 10 μg of naloxone boluses; NX, 10 μg of naloxone bolus alone. **p < 0.01 as compared with the saline group and ⁺ p < 0.01 as compared with the corresponding low morphine dose or saline groups.

Fig. 3.

Morphine-induced EAAC1 and GLAST reduction in Western blotting. Both EAAC1 and GLAST protein contents were reduced in rats receiving a 7 d intrathecal infusion with 20 nm · μl⁻¹ · hr⁻¹morphine as compared with the saline control. *p < 0.05; two-tailed Student's t test. ERK2 is a loading control.

Fig. 4.

Development of morphine tolerance and its blockade by naloxone. A, Morphine antinociception was dose-dependently reduced on day 8 in rats receiving a 7 d intrathecal morphine treatment of either twice daily boluses or continuous infusion. B, Coadministration of morphine (20 μg) with naloxone (10 μg) for 7 d blocked the development of morphine tolerance (B20+NX), and a 7 d naloxone (10 μg; NX) treatment alone did not affect the morphine antinociception. See Figure 2 for the details of each group. **p < 0.01 as compared with the saline group, and ⁺ p < 0.05 as compared with the corresponding low morphine dose group.

Fig. 5.

Development of thermal hyperalgesia and its reversal by riluzole. A, The paw-withdrawal latency (PWL) was reduced on day 8 in the absence of exogenous glutamate as compared with that on day 1 in rats receiving either 10 (B10) or 20 μg (B20) of morphine boluses or continuous infusion of 10 (C10) or 20 nm · μl⁻¹ · hr⁻¹(C20) morphine for 7 d. B, Coadministration of morphine (20 μg) with naloxone (10 μg) for 7 d blocked the development of thermal hyperalgesia (B20+NX), and a 7 d naloxone (10 μg;NX) treatment alone did not affect baseline paw-withdrawal latency. C, The response to intrathecal 5 nm glutamate was exacerbated in morphine-infused rats (B20+GLU) as compared with saline-treated rats (SAL+GLU). A single intrathecal pretreatment with 20 μg of riluzole at 30 min before the glutamate injection attenuated the hyperalgesia (B20+GLU+R20). Riluzole alone (R20 alone) transiently increased the baseline paw-withdrawal latency. The data were presented as the percentage change of the paw-withdrawal latency from that of before the glutamate treatment on day 8 in each group. *p < 0.05, **p < 0.01, as compared with the correspondingSAL+GLU group. The paw-withdrawal latency in theR20 alone group was compared before and after riluzole treatment, and its change did not reach statistical significance.

Fig. 6.

Time course of EAAC1 and GLAST changes after chronic morphine. Both EAAC1 and GLAST protein contents were progressively reduced after twice daily intrathecal treatment with 20 μg of morphine. The M2 to M8 groups stand for rats receiving 20 μg of morphine, and their spinal cords were harvested at day 2, 4, 6, or 8 of the treatment period. *p < 0.05, **p < 0.01, as compared with the corresponding saline group. ERK2 is a loading control.

Fig. 7.

Time course of the development of morphine tolerance and thermal hyperalgesia after chronic morphine. Both morphine tolerance and thermal hyperalgesia developed on days 6 (D6) and 8 (D8) after twice daily intrathecal treatment with 20 μg of morphine. Note that the time course of behavioral changes correlated with that of EAAC1 and GLAST changes in Western blot analysis (Fig. 6). **p < 0.01, as compared with the corresponding saline group.

Fig. 8.

Regulation of morphine tolerance by the GT inhibitor PDC and activator riluzole. A, The onset for the development of morphine tolerance was shortened by coadministration of 10 μg of morphine (B10) with 20 μg of PDC (B10+P20) but prolonged by coadministration of 10 μg of morphine (B10) with 20 μg of riluzole (B10+R20). **p < 0.01, as compared with the saline group, and ⁺ p < 0.05, as compared with the morphine alone group. B,C, The cumulative dose–response curves were shifted dose dependently to the right in rats treated with 10 μg of morphine (B10) with 5, 10, or 20 μg of PDC (B10+P5, B10+P10,B10+P20), whereas the dose–response curves were shifted dose dependently to the left in rats treated with 10 μg of morphine with 5, 10, or 20 μg of riluzole (B10+R5,B10+R10, B10+R20), as compared with the rats receiving either saline alone or 10 μg of morphine plus saline (B10+SAL).

Fig. 9.

Inhibition by MK-801 of morphine tolerance and thermal hyperalgesia potentiated by PDC. A, The development of thermal hyperalgesia was potentiated in rats treated with 10 μg of morphine plus 20 μg of PDC (B10+P20) but prevented in rats receiving 10 μg of morphine plus 20 μg of riluzole (B10+R20). PDC or riluzole alone changed baseline paw-withdrawal latencies (PWL) on day 8 but did not reach the statistical significance at the current dose.B, The morphine antinociception was dose-dependently reduced on day 8 in rats receiving 7 d intrathecal 20 μg of morphine boluses (B20). The GT activator riluzole (20 μg), given intrathecally at 30 min before the morphine antinociceptive test on day 8 (B20+R20; Day 8), did not reverse the behavioral manifestation of morphine tolerance. C, The development of morphine tolerance was potentiated by intrathecal coadministration of 10 μg of morphine with 20 μg of PDC (B10+P) twice daily for 7 d. This potentiation was blocked by adding 10 nm MK-801 into this combination (B+P+M). Treatment with 10 nm MK-801 alone (MK) for 7 d did not change the antinociceptive effects of morphine. *p < 0.05, **p < 0.01, as compared with the corresponding saline group, and⁺ p < 0.05,⁺⁺ p < 0.01, as compared with the corresponding morphine alone group.