Yueh-Hua Tai

Amitriptyline suppresses neuroinflammation and up-regulates glutamate transporters in morphine-tolerant rats.

Abstract The present study was performed to evaluate the effects of the tricyclic antidepressant amitriptyline on morphine tolerance in rats. Male Wistar rats were implanted with two intrathecal (i.t.) catheters with or without a microdialysis probe, then received a continuous i.t. infusion of saline (control) or morphine (15 μg/h) and/or amitriptyline (15 μg/h) for 5 days. The results showed that amitriptyline alone did not produce an antinociceptive effect, while morphine alone induced antinociceptive tolerance and down‐regulation of spinal glutamate transporters (GLAST, GLT‐1, and EAAC1) in the rat spinal cord dorsal horn. Co‐administration of amitriptyline with morphine attenuated morphine tolerance and up‐regulated GLAST and GLT‐1 expression. On day 5, morphine challenge (10 μg/10 μl) resulted in a significant increase in levels of the excitatory amino acids (EAAs), aspartate and glutamate, in CSF dialysates in morphine‐tolerant rats. Amitriptyline co‐infusion not only markedly suppressed this morphine‐evoked EAA release, but also preserved the antinociceptive effect of acute morphine challenge at the end of infusion. Glial cells activation and increased cytokine expression (TNFα, IL‐1β, and IL‐6) in the rat spinal cord were induced by the 5‐day morphine infusion and these neuroimmune responses were also prevented by amitriptyline co‐infusion. These results show that amitriptyline not only attenuates morphine tolerance, but also preserves its antinociceptive effect. The mechanisms involved may include: (a) inhibition of pro‐inflammatory cytokine expression, (b) prevention of glutamate transporter down‐regulation, and even up‐regulation of glial GTs GLAST and GLT‐1 expression, with (c) attenuation of morphine‐evoked EAA release following continuous long‐term morphine infusion.

Amitriptyline preserves morphine’s antinociceptive effect by regulating the glutamate transporter GLAST and GLT-1 trafficking and excitatory amino acids concentration in morphine-tolerant rats

Abstract The present study was undertaken to examine the effect of amitriptyline on the antinociceptive effect of morphine and its underlying mechanisms in regulating glutamate transporters trafficking in morphine‐tolerant rats. Long‐term morphine infusion induced antinociceptive tolerance and down‐regulation of glutamate transporters (GTs), GLAST, GLT‐1, and EAAC1, expression in the rat spinal cord dorsal horn. Acute amitriptyline treatment potentiated morphine’s antinociceptive effect, with a 5.3‐fold leftward shift of morphine’s dose–response curve in morphine‐tolerant rats, and this was associated with GLAST and GLT‐1 trafficking onto the cell surface. Similar to our previous studies, morphine challenge (10 μg/10 μl, i.t.) significant by increased the excitatory amino acids (EAAs) aspartate and glutamate level in the CSF dialysates of morphine‐tolerant rats. Acute amitriptyline treatment not only suppressed this morphine‐evoked EAA release, but further reduced the EAA concentration than baseline level. Furthermore, long‐term morphine infusion up‐regulated PKA and PKC protein expression in the spinal cord dorsal horn, while amitriptyline inhibited the increase in expression of phospho‐PKA, PKCα, PKCβII, and PKCγ. In morphine‐tolerant rats, acute treatment with PKA inhibitor H89 and PKC inhibitor Gö6805 attenuated morphine tolerance and the morphine‐induced CSF glutamate and aspartate elevation, and induced trafficking of GLAST and GLT‐1 from cytosol onto the cell surface. These results show that acute amitriptyline treatment preserved morphine’s antinociceptive effect in morphine‐tolerant rats; the mechanisms may be involved in inhibition of phospho‐PKA and PKC expression, and thus inducing the GLAST and GLT‐1 trafficking onto glial cell surface which enhances the EAA uptake from the synaptic cleft and reduces EAA concentration in the spinal CSF.

Amitriptyline suppresses neuroinflammation-dependent interleukin-10-p38 mitogen-activated protein kinase-heme oxygenase-1 signaling pathway in chronic morphine-infused rats.

Background:This study explores the underlying mechanism of the antiinflammatory effect of amitriptyline in chronic morphine-infused rats. Methods:Male Wistar rats were implanted with two intrathecal catheters. One catheter was for the continuous infusion of saline, amitriptyline (15 &mgr;g/h), morphine (15 &mgr;g/h), p38 mitogen-activated protein kinase inhibitor SB203580 (0.5 &mgr;g/h), morphine plus amitriptyline, or morphine plus amitriptyline plus SB203580 for 5 days. The other catheter was used for daily intrathecal injection of anti–interleukin-10 (IL-10) antibody or heme oxygenase-1 inhibitor zinc protoporphyrin for 5 days. Results:Amitriptyline/morphine coinfusion upregulated IL-10 protein expression in microglia; this was not observed in morphine-infused rats. Anti–IL-10 antibody effectively neutralized the amitriptyline-induced IL-10 expression in chronic morphine-infused rats. In addition, coinfusion of amitriptyline restored the antinociceptive effect of morphine (a 4.8-fold right-shift of the morphine dose-response curve compared to a 77.8-fold right-shift in its absence), and the injection of anti–IL-10 antibody or coinfusion of SB203580 partially reversed the effect of amitriptyline on the antinociceptive effect of morphine in morphine-infused rats (a 17.9-fold and 15.1-fold right-shift in morphine dose-response curves). Anti–IL-10 antibody and SB203580 significantly inhibited the amitriptyline-induced p38 mitogen-activated protein kinase and heme oxygenase-1 expression and the associated antiinflammatory effect of amitriptyline. Daily injection of zinc protoporphyrin also demonstrated that it reverses the effect of amitriptyline in morphine’s antinociception and antiinflammation in chronic morphine-infused rats. Conclusions:These results suggest that the antiinflammatory effect of amitriptyline on morphine tolerance, probably acting by increasing IL-10 expression, is mediated by p38 mitogen-activated protein kinase heme oxygenase-1 signal transduction cascade.

Ultra-low dose naloxone restores the antinociceptive effect of morphine in pertussis toxin-treated rats by reversing the coupling of μ-opioid receptors from Gs-protein to coupling to Gi-protein

Pertussis toxin (PTX) treatment results in ADP-ribosylation of Gi-protein and thus in disruption of mu-opioid receptor signal transduction and loss of the antinociceptive effect of morphine. We have previously demonstrated that pretreatment with ultra-low dose naloxone preserves the antinociceptive effect of morphine in PTX-treated rats. The present study further examined the effect of ultra-low dose naloxone on mu-opioid receptor signaling in PTX-treated rats and the underlying mechanism. Male Wistar rats implanted with an intrathecal catheter received an intrathecal injection of saline or PTX (1 microg in 5 microl of saline), then, 4 days later, were pretreated by intrathecal injection with either saline or ultra-low dose naloxone (15 ng in 5 microl of saline), followed, 30 min later, by saline or morphine (10 microg in 5 microl of saline). Four days after PTX injection, thermal hyperalgesia was observed, together with increased coupling of excitatory Gs-protein to mu-opioid receptors in the spinal cord. Ultra-low dose naloxone pretreatment preserved the antinociceptive effect of morphine, and this effect was completely blocked by the mu-opioid receptor antagonist CTOP, but not by the kappa-opioid receptor antagonist nor-BNI or the delta-opioid receptor antagonist naltrindole. Moreover, a co-immunoprecipitation study showed that ultra-low dose naloxone restored mu-opioid receptor/Gi-protein coupling and inhibited the PTX-induced mu-opioid receptor/Gs-protein coupling. In addition to the anti-neuroinflammatory effect and glutamate transporter modulation previously observed in PTX-treated rats, the re-establishment of mu-opioid receptor Gi/Go-protein coupling is involved in the restoration of the antinociceptive effect of morphine by ultra-low dose naloxone pretreatment by normalizing the balance between the excitatory and inhibitory signaling pathways. These results show that ultra-low dose naloxone preserves the antinociceptive effect of morphine, suppresses spinal neuroinflammation, and reduces PTX-elevated excitatory Gs-coupled opioid receptors in PTX-treated rats. We suggest that ultra-low dose naloxone might be clinically valuable in pain management.

Ultra-Low-Dose Naloxone Restores the Antinociceptive Effect of Morphine and Suppresses Spinal Neuroinflammation in PTX-Treated Rats

The aim of the present study was to examine the effect of ultra-low-dose naloxone on pertussis toxin (PTX)-induced thermal hyperalgesia in rats and its underlying mechanisms. Male Wistar rats, implanted with an intrathecal catheter with or without a microdialysis probe, received a single intrathecal injection of PTX (1 μg in 5 μl saline). Four days after PTX injection, they were randomly given a different dose of naloxone (either 15 μg or 15 ng in 5 μl saline), followed by a morphine injection (10 μg in 5 μl saline) after 30 min. The results found that PTX injection induced thermal hyperalgesia and increasing excitatory amino acid (EAA; L-glutamate and L-aspartate) concentration in the spinal CSF dialysates. Ultra-low-dose naloxone not only preserved the antinociceptive effect of morphine but also suppressed the PTX-evoked EAA release as well. Moreover, ultra-low-dose naloxone plus morphine administration inhibited the downregulation of L-glutamate transporters (GTs) and the L-glutamate-metabolizing enzyme glutamine synthetase (GS), and, moreover, inhibited microglial activation and suppressed cytokine expression in PTX-treated rat spinal cords. These results show that ultra-low-dose naloxone preserves the antinociceptive effect of morphine in PTX-treated rats. The mechanisms include (a) inhibition of pro-inflammatory cytokine expression, (b) attenuation of PTX-evoked EAA release, and (c) reversion of the downregulation of GT expression.

Ultra-low dose naloxone upregulates interleukin-10 expression and suppresses neuroinflammation in morphine-tolerant rat spinal cords

Co-infusion of ultra-low dose naloxone and morphine attenuates morphine tolerance through the prevention of mu opioid receptor-Gs protein coupling. We previously demonstrated that chronic intrathecal infusion of morphine leads to tolerance and spinal neuroinflammation. The aim of present study was to examine the possible mechanisms by which ultra-low dose naloxone modulates spinal neuroinflammation, particularly the role of anti-inflammatory cytokine interleukin 10 (IL-10). Morphine tolerance was induced in male Wistar rats by intrathecal infusion of morphine (15 microg/h) for 5 days, and co-infusion of naloxone (15 pg/h) was used to evaluate the impact on spinal cytokine expression. Recombinant rat IL-10 (rrIL-10) or anti-rat IL-10 antibody was injected to elucidate the effect of IL-10 on morphine tolerance. Our results showed that co-infusion of naloxone (15 pg/h) with morphine not only attenuated tolerance, shifting the AD(50) from 89.2 to 11.7 microg but also inhibited the increased expression of pro-inflammatory cytokine (TNF-alpha, IL-1beta, and IL-6) caused by chronic intrathecal morphine infusion. The increase of IL-10 protein and mRNA were 1.5- and 3-fold, respectively, compared to that in morphine-infused rat spinal cords. A combination of daily rrIL-10 (1 microg) injection with morphine infusion produced, in a less potent, preservative antinociception and inhibited pro-inflammatory cytokine production compared to ultra-low dose naloxone co-infusion, and the effect of ultra-low dose naloxone co-infusion was inhibited by daily intrathecal anti-rat IL-10 antibody injection. These results demonstrate that IL-10 contributes to the attenuation of pro-inflammatory cytokine expression caused by ultra-low dose naloxone/morphine co-infusion and thus the attenuation of morphine tolerance.

N-Methyl-d-aspartate receptor antagonist MK-801 suppresses glial pro-inflammatory cytokine expression in morphine-tolerant rats

Chronic opioid therapy induces tolerance and hyperalgesia, which hinders the efficacy of opioid treatment. Previous studies have shown that inhibition of neuroinflammation and glutamatergic receptor activation prevents the development of morphine tolerance. The aim of the present study was to examine whether N-Methyl-D-aspartate receptors are involved in the regulation of chronic morphine-induced neuroinflammation in morphine-tolerant rats. Morphine tolerance was induced in male Wistar rats by intrathecal infusion of morphine (15 μg/h) for 5 days. Tail-flick latency was measured to estimate the antinociceptive effect of morphine. Morphine challenge (15 μg, intrathecally) on day 5 at 3h after discontinuation of morphine infusion produced a significant antinociceptive effect in saline-infused rats, but not in morphine-tolerant rats. Pretreatment with MK-801 (20 μg, intrathecally) 30 min before morphine challenge preserved its antinociceptive effect in morphine-tolerant rats. Morphine-tolerant rats expressed high levels of the pro-inflammatory cytokines interleukin-1β, interleukin-6, and tumor necrosis factor-α and the increase in interleukin-1β and interleukin-6, and tumor necrosis factor-α levels was prevented by MK-801 pre-treatment at both the protein and mRNA levels. The results show that a single dose of MK-801 reduces the increase in pro-inflammatory cytokines in the spinal cord, thus re-sensitizing neurons to the antinociceptive effect of morphine in morphine-tolerant rats. This study provides a piece of theoretical evidence that NMDA antagonist can be a therapeutic adjuvant in treating morphine tolerant patients for pain relief.

Ultra-low dose naloxone restores the antinociceptive effect of morphine in pertussis toxin–treated rats and prevents glutamate transporter downregulation by suppressing the p38 mitogen-activated protein kinase signaling pathway

We previously demonstrated that ultra-low dose naloxone restores the antinociceptive effect of morphine in rats with pertussis toxin (PTX)-induced thermal hyperalgesia by reversing the downregulation of glutamate transporter (GT) expression and suppressing spinal neuroinflammation. In the present study, we examined the underlying mechanisms of this anti-inflammatory effect in PTX-treated rats, particularly on the expression of GTs. Male Wistar rats were implanted with an intrathecal catheter and, in some cases, with a microdialysis probe. All rats were injected intrathecally with saline (5 microl) or PTX (1 microg), then, 4 days later, were randomly assigned to receive a single injection of saline, ultra-low dose naloxone (15 ng), or the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 (5 microg), followed by morphine injection (10 microg) 30 min later. Our results showed that PTX injection induced activation of microglia and a significant increase in P-p38 MAPK expression in the spinal cord. Ultra-low dose naloxone plus morphine significantly inhibited the effect of PTX on P-p38 MAPK expression in the spinal cord, while the p38 MAPK inhibitor SB203580 attenuated the PTX-induced mechanical allodynia, thermal hyperalgesia, increase in spinal cerebrospinal fluid excitatory amino acids, and downregulation of GTs. These results show that the restoration of the antinociceptive effect of morphine and GT expression in PTX-treated rats by ultra-low dose naloxone involves suppression of the p38 MAPK signal transduction cascade.

Purinergic P2X receptor regulates N-methyl-D-aspartate receptor expression and synaptic excitatory amino acid concentration in morphine-tolerant rats.

Background:The present study examined the effect of P2X receptor antagonist 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate (TNP-ATP) on morphine tolerance in rats. Methods:Male Wistar rats were implanted with two intrathecal catheters with or without a microdialysis probe, then received a continuous intrathecal infusion of saline (control) or morphine (tolerance induction) for 5 days. Results:Long-term morphine infusion induced antinociceptive tolerance and up-regulated N-methyl-d-aspartate receptor subunits NR1 and NR2B expression in both total lysate and synaptosome fraction of the spinal cord dorsal horn. TNP-ATP (50 &mgr;g) treatment potentiated the antinociceptive effect of morphine, with a 5.5-fold leftward shift of the morphine dose–response curve in morphine-tolerant rats, and this was associated with reversal of the up-regulated NR1 and NR2B subunits in the synaptosome fraction. NR1/NR2B–specific antagonist ifenprodil treatment produced a similar effect as TNP-ATP; it also potentiated the antinociceptive effect of morphine. On day 5, morphine challenge resulted in a significant increase in aspartate and glutamate concentration in the cerebrospinal fluid dialysates of morphine-tolerant rats, and this effect was reversed by TNP-ATP treatment. Moreover, the amount of immunoprecipitated postsynaptic density-95/NR1/NR2B complex was increased in morphine-tolerant rats, and this was prevented by the TNP-ATP treatment. Conclusions:The findings suggest that attenuation of morphine tolerance by TNP-ATP is attributed to down-regulation of N-methyl-d-aspartate receptor subunits NR1 and NR2B expression in the synaptosomal membrane and inhibition of excitatory amino acids release in morphine-tolerant rats. The TNP-ATP regulation on the N-methyl-d-aspartate receptor expression may be involved in a loss of scaffolding proteins postsynaptic density-95.