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-1ß, interleukin-6, and tumor necrosis factor-α and the increase in interleukin-1ß 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.

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 µg) 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.

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.

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 microg/h), morphine (15 microg/h), p38 mitogen-activated protein kinase inhibitor SB203580 (0.5 microg/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 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 microg in 5 microl saline). Four days after PTX injection, they were randomly given a different dose of naloxone (either 15 microg or 15 ng in 5 microl saline), followed by a morphine injection (10 microg in 5 microl 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.

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.

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 microg/10 microl, 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, PKCalpha, PKCbetaII, and PKCgamma. 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 and up-regulates glutamate transporters in morphine-tolerant rats.

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 microg/h) and/or amitriptyline (15 microg/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 microg/10 microl) 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 (TNFalpha, IL-1beta, 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.

Functional interaction of AT1 and AT2 receptors in fructose-induced insulin resistance and hypertension in rats.

The present study was performed to evaluate the potential role and functional interaction of angiotensin II AT1 and AT2 receptors (AT1R and AT2R) in the regulation of blood pressure and glucose homeostasis in fructose-induced insulin-resistant, hypertensive rats. Male Sprague-Dawley rats on fructose-enriched or regular diets for 4 weeks were subjected to 2-step euglycemic euinsulinemic (EEI) and euglycemic hyperinsulinemic (EHI) clamp studies with [3-3H]glucose infusion. After a 40-minute basal period, selective AT1R and AT2R antagonists, losartan (LOS, 10 mg/kg IV bolus) and PD123319 (PD, 50 microg/kg/min), alone or in combination were separately given to control and fructose-fed groups in the 2 clamp periods. The results showed that during the EEI period, LOS significantly reduced the elevated blood pressure in fructose-fed rats, whereas PD further increased fructose-induced high blood pressure. Coadministration of LOS and PD did not alter the elevated blood pressure in fructose-fed rats. Administration of LOS and/or PD failed to change the blood pressure in control rats. During the EHI period, blockade of both AT1R and AT2R eliminated the insulin-induced blood pressure elevation in control and fructose-fed rats. Hepatic glucose production (HGP) did not alter among groups in the basal and EEI periods. Insulin infusion (EHI period) markedly suppressed HGP in control rats, but this suppressive effect was significantly attenuated in fructose-fed rats. LOS administration further reduced the insulin-induced suppression of HGP in fructose-fed rats. The whole-body glucose uptakes (rates of glucose disappearance, Rd) during the basal and EEI periods were similar among groups. During the EHI period, Rd was markedly increased in all groups and the magnitude of increase was significantly greater in control rats than in fructose-fed rats except those with LOS treatment. LOS treatment also redirected Rd in favor of glycolysis in fructose rats, but not in control rats, during the EEI and EHI periods. The effects of LOS on glycolysis during the 2 clamp periods and on HGP during the EHI period were reversed when PD was concomitantly administered, but PD alone did not alter glucose metabolism throughout the experiment in fructose-fed rats. Administration of LOS and/or PD did not change the glucose metabolism in control rats. Our data suggest that AT2R can counterbalance the AT1R-mediated effects on blood pressure and glucose metabolism in fructose-induced insulin-resistant, hypertensive rats. Furthermore, AT1R- and AT2R-mediated effects on blood pressure are disassociated with their actions on glucose metabolism in this hypertensive model.

Aqueous extract of Monascus purpureus M9011 prevents and reverses fructose-induced hypertension in rats.

This study aimed to determine the antihypertensive and metabolic effects of an aqueous extract of Monascus purpureus M9011 on fructose-induced hypertensive rats. After dietary feeding of fructose for 2 weeks, the rats exhibited significantly higher systolic blood pressure (SBP), mean arterial pressure (MAP), and plasma insulin and triglyceride levels, but lower insulin sensitivity than those in control rats on regular diet. The intragastric loading of fructose-fed rats with M9011 containing gamma-aminobutyric acid (GABA, 1 mg.kg(-)(1).day(-)(1)) prevented the development of fructose-induced hypertension. After fructose-induced hypertension had been established, intragastric loading of M9011 reversed the elevated blood pressure to normal level. Administration of pure GABA at the same dose as that contained in M9011 failed to prevent or reverse hypertension due to fructose consumption. Chronic M9011 treatment significantly suppressed the fructose-induced elevation in total cholesterol levels and enhanced the recovery of high-density lipoprotein cholesterol/total cholesterol ratio. However, M9011 treatment did not alter insulin sensitivity or the plasma levels of insulin, glucose, and triglyceride in fructose-fed and control rats. The present results suggest that M9011 is a novel, potent, food-based antihypertensive agent with the capability to improve long-term control of cholesterol metabolism in rats and may be of importance in clinical application for the hypertensive diabetic population.