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Comparative Study
. 2010 Nov 17;30(46):15400-8.
doi: 10.1523/JNEUROSCI.2391-10.2010.

Counter-regulation of opioid analgesia by glial-derived bioactive sphingolipids

Carolina Muscoli  1 , Tim Doyle, Concetta Dagostino, Leesa Bryant, Zhoumou Chen, Linda R Watkins, Jan Ryerse, Erhard Bieberich, William Neumman, Daniela Salvemini
Affiliations
Comparative Study

Counter-regulation of opioid analgesia by glial-derived bioactive sphingolipids

Carolina Muscoli et al. J Neurosci. .

Abstract

The clinical efficacy of opiates for pain control is severely limited by analgesic tolerance and hyperalgesia. Herein we show that chronic morphine upregulates both the sphingolipid ceramide in spinal astrocytes and microglia, but not neurons, and spinal sphingosine-1-phosphate (S1P), the end-product of ceramide metabolism. Coadministering morphine with intrathecal administration of pharmacological inhibitors of ceramide and S1P blocked formation of spinal S1P and development of hyperalgesia and tolerance in rats. Our results show that spinally formed S1P signals at least in part by (1) modulating glial function because inhibiting S1P formation blocked increased formation of glial-related proinflammatory cytokines, in particular tumor necrosis factor-α, interleukin-1βα, and interleukin-6, which are known modulators of neuronal excitability, and (2) peroxynitrite-mediated posttranslational nitration and inactivation of glial-related enzymes (glutamine synthetase and the glutamate transporter) known to play critical roles in glutamate neurotransmission. Inhibitors of the ceramide metabolic pathway may have therapeutic potential as adjuncts to opiates in relieving suffering from chronic pain.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1.
Figure 1.
Morphine-induced hyperalgesia and tolerance is associated with increased ceramide and S1P derived from activation of sphingosine kinase. When compared with rats that received a chronic subcutaneous infusion of saline (Veh–Sal, n = 5, ● or open bar) over 7 d, infusion of morphine over the same timeframe (Veh-Mor, n = 5, ■ or gray bar) led to the development of thermal hyperalgesia as evidenced by a significant reduction in paw-withdrawal latency (seconds) on days 3 and 6 when compared with paw-withdrawal latency from before implantation of the osmotic minipump (day 0) (A, C) and with the development of antinociceptive tolerance (B, D). Results shown in C and D are from behavioral measurements taken on day 6. These events were associated with increased formation of ceramide (red, E, F) and S1P (G, H) in dorsal horn tissues. Coadministration of morphine with DMS (0.3 μm/d for /5 d, n = 5, ● or black bar) or SK-I (0.3 μm/d for 5 d, n = 5, ♦ or hashed bar) blocked the development of hyperalgesia (A), tolerance (B), and S1P (G, H). Dose–response curves for DMS (0.03–0.3 μm/d for 5 d, n = 5, ● or black bar) or SK-I (0.03–0.3 μm/d for 5 d, n = 5, ○ or hashed bar) on day 6 are shown in C for hyperalgesia, D for tolerance, and H for S1P. Micrographs shown in E and F are representative of at least three images of the superficial layers of dorsal horn (L5–L6) harvested on day 6 from three different animals. Results are expressed as mean ± SEM for n = 5 animals and analyzed by ANOVA with Bonferroni's post hoc test: *p < 0.001 for Veh–Mor versus Veh–Sal; §p < 0.01 or §§p < 0.001 versus day 0; and †p < 0.01 or ††p < 0.001 for Drugs–Mor versus Veh–Mor.
Figure 2.
Figure 2.
Colocalization of ceramide with activated glial cells but not neurons. Fixed frozen spinal cord sections from Veh–Sal (A–C) and Veh–Mor (D–F) rats were stained for ceramide (red) and for astrocytes (GFAP+, green) (A, D), microglia (Iba1+, green) (B, E), or neurons (NeuN+, green) (C, F). In these merged images, ceramide was low or absent in spinal cords from Veh–Sal animals and substantially increased in spinal cords from Veh–Mor animals. Furthermore, ceramide levels colocalized (yellow) with astrocytes (D) and microglia (E) but not with neurons (F). Negative controls using normal rabbit serum (for ceramide) or normal mouse IgG (for GFAP, Iba1, and NeuN) at the same concentrations as for the immune serum and immune IgG exhibited only low levels of background fluorescence. Micrographs are representative of at least three images of the superficial layers of dorsal horn (L5–L6) from three different animals performed on different days.
Figure 3.
Figure 3.
Therapeutic manipulation with inhibitors of ceramide biosynthesis blocks hyperalgesia and antinociceptive tolerance. Intrathecal delivery of inhibitors of the de novo (Myr, 0.3 μm/d for 5 d; FB1, 1 μm/d for 5 d) and sphingomyelin (D609, 1 μm/d for 5 d) pathways blocked increased S1P levels in dorsal horn tissues (A) and subsequent development of morphine-induced thermal hyperalgesia (B) and antinociceptive tolerance (C). Results are expressed as mean ± SEM for n = 5 animals and analyzed by ANOVA with Dunnett's post hoc test: *p < 0.001 for Veh–Mor versus Veh–Sal and †p < 0.001 for drugs–Mor versus Veh–Mor.
Figure 4.
Figure 4.
DMS blocks glial cell activation and increased spinal production of cytokines. When compared with Veh–Sal, the development of tolerance (Veh–Mor) was associated with significant activation of astrocytes and microglial cells as evidenced, respectively, by increased GFAP (A, A1) and Iba1 (B, B1) protein expression and increased formation of TNF-α (C), IL-1β (D), and IL-6 (E) levels in dorsal horn tissues. Coadministration of morphine with DMS (0.3 μm/d for 5 d) prevented glial cell activation (A, A1, B, B1) and increased cytokine formation (C–E). Composite densitometric analyses for gels of five rats are expressed as mean ± SEM of percentage β-actin as shown in A1 and B1. Results for cytokines are expressed as mean ± SEM for n = 5 animals. Data were analyzed by ANOVA with Dunnett's post hoc test: *p < 0.001 for Veh–Mor versus Veh–Sal and †p < 0.01 or ††p < 0.001 for DMS–Mor versus Veh–Mor.
Figure 5.
Figure 5.
DMS blocks posttranslation protein nitration in spinal cord. When compared with Veh–Sal (A), the development of tolerance (Veh–Mor) was associated with significant protein nitration as detected by immunohistochemistry (B, see arrows), in particular significant nitration of the glutamate transporter (GLT-1; D, D1) and GS (E, E1). These events were attenuated by coadministration of morphine with DMS (0.3 μm/d for 5 d; C–E). Gels shown in D and E are representative of gels from n = 5 rats. Composite densitometric analyses for gels of nitrated proteins of five rats are expressed as mean ± SEM of percentage β-actin as shown in D1 and E1 and analyzed by ANOVA with Dunnett's post hoc test: *p < 0.001 for Veh–Mor versus Veh–Sal and †p < 0.001 for DMS–Mor versus Veh–Mor. Micrographs are representative of at least five from different animals performed on different days and are taken from the superficial layers of the dorsal horn (L5–L6), the anatomical site that stains for NT during tolerance (Muscoli et al., 2007).
Figure 6.
Figure 6.
Proposed working hypothesis. Chronic administration of morphine activates the ceramide metabolic pathway, resulting in increased formation of S1P in glial cells during activation of sphingosine kinase (SphK) 1 and/or 2. After its extracellular release, S1P binds S1P receptors (which remain to be identified) on glial cells, initiating a series of events culminating in enhanced production of proinflammatory cytokines and PN-mediated nitration of glutamate transporters (GTs) and GS. Activation of glial TLR4 may provide a link between chronic administration of morphine and activation of the ceramide metabolic pathway in the development of morphine-induced hyperalgesia and antinociceptive tolerance.
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