Regulation of G Protein-Coupled Receptor Kinase 2 in Brains of Opiate-Treated Rats and Human Opiate Addicts

Abstract: The effects of opiate drugs (heroin, morphine, and methadone) on the levels of G protein-coupled receptor kinase 2 (GRK2) were studied in rat and human brain frontal cortices. The density of brain GRK2 was measured by immunoblot assays in acute and chronic opiate-treated rats as well as in opiate-dependent rats after spontaneous or naloxone-precipitated withdrawal and in human opiate addicts who had died of an opiate overdose. In postmortem brains from human addicts, total GRK2 immunoreactivity was not changed significantly, but the level of the membrane-associated kinase was modestly but significantly increased (12%) compared with matched controls. In rats treated chronically with morphine or methadone modest increases of the enzyme levels (only significant after methadone) were observed. Acute treatments with morphine and methadone induced dose- and time-dependent increases (8–22%) in total GRK2 concentrations [higher increases were observed for the membrane-associated enzyme (46%)]. Spontaneous and naloxone-precipitated withdrawal after chronic morphine or methadone induced a marked up-regulation in the levels of total GRK2 in the rat frontal cortex (18–25%). These results suggest that GRK2 is involved in the short-term regulation of μ-opioid receptors in vivo and that the activity of this regulatory kinase in brain could have a relevant role in opiate tolerance, dependence, and withdrawal.     

吗啡联合有创呼吸机辅助通气治疗重症急性左心衰竭的疗效

目的:探讨吗啡联合有创呼吸机辅助通气治疗重症急性左心衰竭的临床疗效。方法:将2012年5月-2014年5月我院收治的82例重症急性左心衰竭患者随机分为观察组和对照组,各41例。两组患者均因常规药物治疗无效再行经口气管插管机械通气及对症治疗,观察组在此基础上加用吗啡静脉注射。对比两组患者的体征、症状、动血气指标及临床疗效。结果:两组治疗后血压、心率(HR)、呼吸频率(BR)、血气指标明显优于治疗前,比较差异有统计学意义(P0.05),且观察组各指标改善明显优于对照组,比较差异有统计学意义(P0.05);治疗后观察组总有效率为92.7%(38/41),高于对照组的82.9%(34/41),比较差异有统计学意义(P0.05)。结论:吗啡联合有创呼吸机辅助通气治疗重症急性左心衰竭可有效缓解患者症状,改善临床体征,临床疗效显著,值得推广。     

Toll-like receptor 2 is required for opioids-induced neuronal apoptosis

Toll-like receptor 2 (TLR2), a key immune receptor in the TLR family, is widely expressed in various systems, including the immune and nervous systems and plays a critical role in controlling innate and adaptive immune responses. We previously reported that opioids inhibit cell growth and trigger apoptosis. However, the underlying mechanism by which TLR2 mediates apoptosis in response to opioids is not yet known. Here we show that chronic morphine treatment in primary neurons dramatically increased the expression of TLR2 at both the messenger RNA and protein levels. In addition, TLR2 deficiency significantly inhibited chronic morphine-induced apoptosis in primary neurons. Activation of caspase-3 after morphine treatment is impaired in TLR2 deficient primary neurons. Moreover, morphine treatment failed to induce an increased level of phosphorylated glycogen synthase kinase 3 beta (GSK3β) in TLR2 deficient primary neurons, suggesting an involvement of GSK3β in morphine-mediated TLR2 signaling. These results thus demonstrate that opioids prime neurons to undergo apoptosis by inducing TLR2 expression. Our data suggest that inhibition of TLR2 is capable of preventing opioids-induced damage to neurons.     

Morphine sulphate induced histopathological and histochemical changes in the rat liver

In this study, the histopathological and histochemical changes due to chronic usage of morphine sulphate in liver were assessed in rats with both light and electron microscopes. Twenty male albino rats (Rattus norvegicus) (130-150 g) were included and divided into four groups. Normal saline (5 ml) was given orally as placebo in the control group (N = 5). Morphine groups (N = 5) received morphine orally at a single dose of 5 ml/kg/day for 10, 20 and 30 days (groups II, III and IV), respectively. Liver specimens from all groups were evaluated for histopathological and histochemical changes. Light microscopy revealed severe centrilobular congestion, portal fibrosis with bile ductal proliferation and an increased inflammatory infiltration and focal parenchymal necrosis. Histochemical study revealed a progressive depletion of general carbohydrates and an increase in total protein contents. These changes were confirmed at ultrastructural level, including the presence of accumulated lipid in the hepatocytes; deposits of a collagen-like fibrous material were seen in the space of Disse and a reduction in the number of endothelial cell fenestrations. Our findings pointed out the risk of increased lipid fibrosis and hepatic damage due to long-term use of morphine. Although opioids are reported to be effective in pain management, their toxic effects should be kept in mind during chronic usage.     

RGS14 prevents morphine from internalizing Mu-opioid receptors in periaqueductal gray neurons

Opioid agonists display different capacities to stimulate mu-opioid receptor (MOR) endocytosis, which is related to their ability to provoke the phosphorylation of specific cytosolic residues in the MORs. Generally, opioids that efficiently promote MOR endocytosis and recycling produce little tolerance, as is the case for [d-Ala², N-MePhe⁴,Gly-ol⁵] encephalin (DAMGO). However, morphine produces rapid and profound antinociceptive desensitization in the adult mouse brain associated with little MOR internalization. The regulator of G-protein signaling, the RGS14 protein, associates with MORs in periaqueductal gray matter (PAG) neurons, and when RGS14 is silenced morphine increased the serine 375 phosphorylation in the C terminus of the MOR, a GRK substrate. Subsequently, these receptors were internalized and recycled back to the membrane where they accumulated on cessation of antinociception. These mice now exhibited a resensitized response to morphine and little tolerance developed. Thus, in morphine-activated MORs the RGS14 prevents GRKs from phosphorylating those residues required for β-arresting-mediated endocytosis. Moreover morphine but not DAMGO triggered a process involving calcium/calmodulin-dependent kinase II (CaMKII) in naïve mice, which contributes to MOR desensitization in the plasma membrane. In RGS14 knockdown mice morphine failed to activate this kinase. It therefore appears that phosphorylation and internalization of MORs disrupts the CaMKII-mediated negative regulation of these opioid receptors.     

Sex differences in morphine-induced behavioral sensitization and social behaviors in ICR mice

Gender and genetic strain are two prominent variants that influence drug abuse. Although certain sexrelated behavioral responses have been previously characterized in ICR mice, little is known about the effects of sex on morphine-induced behavioral responses in this outbred strain. Therefore, in this study, we investigated the sex differences of morphine-induced locomotion, anxiety-like and social behaviors in ICR mice. After morphine or saline exposure for four consecutive days(twice daily), increased locomotion, more time spent in the central area, as well as attenuated rearing and self-grooming behaviors were found in morphine-treated females in an open field; no differences were found in locomotion and the time spent in the central area between male and female controls. When interacting with the samesex individuals, female controls were engaged in more social investigation, following, body contacting and self-grooming behaviors than controls; morphine exposure reduced contacting and self-grooming behaviors in females; in contrast, these effects were not found in males. These results indicate that female ICR mice are more prosocial and are more susceptible to morphine exposure than males.     

Protective effect of pioglitazone on morphine-induced neuroinflammation in the rat lumbar spinal cord

Background

Morphine-induced tolerance is associated with the spinal neuroinflammation. The aim of this study was to explore the effects of oral administration of the pioglitazone, the peroxisome proliferator activated receptor gamma (PPAR-γ) agonist, on the morphine-induced neuroinflammation in the lumbar region of the male Wistar rat spinal cord.

Results

Co-administration of the pioglitazone with morphine not only attenuated morphine-induced tolerance, but also prevented the up-regulation of pro-inflammatory cytokines (tumor necrosis factor alpha, interleukin-1beta, and interleukin 6) and nuclear factor-kappa B activity. Administration of the GW-9662 antagonized the above mentioned effects of the pioglitazone.

Conclusions

It is concluded that oral administration of the pioglitazone attenuates morphine-induced tolerance and the neuroinflammation in the lumbar region of the rat spinal cord. This action of the pioglitazone may be, at least in part, due to an interaction with the spinal pro-inflammatory cytokine expression and the nuclear factor-kappa B activity.     

Methylnaltrexone prevents morphine-induced kaolin intake in the rat

Opioids are frequently used analgesics, and emesis is a common opioid-induced adverse effect. Methylnaltrexone, a peripheral opioid antagonist, has the potential to block the undesired effects of opioids that are mediated by peripheral receptors while sparing the analgesic effect. We used a rat model of simulated emesis or pica to study if methylnaltrexone decreases morphine induced-kaolin consumption. We observed that after morphine administration, kaolin intake increased significantly compared to intake in the vehicle group, and the increase could be attenuated by ondansetron administration. Methylnaltrexone dose-dependently reduced kaolin ingestion induced by morphine. Morphine and methylnaltrexone did not significantly affect food intake and body weight in the experimental animals. Our data suggest that methylnaltrexone has therapeutic value in treating opioid-induced nausea and vomiting.     

Antinociceptive effect of 7-hydroxymitragynine in mice: Discovery of an orally active opioid analgesic from the Thai medicinal herb Mitragyna speciosa

Mitragynine is an indole alkaloid isolated from the Thai medicinal plant Mitragyna speciosa. We previously reported the morphine-like action of mitragynine and its related compounds in the in vitro assays. In the present study, we investigated the opioid effects of 7-hydroxymitragynine, which is isolated as its novel constituent, on contraction of isolated ileum, binding of the specific ligands to opioid receptors and nociceptive stimuli in mice. In guinea-pig ileum, 7-hydroxymitragynine inhibited electrically induced contraction through the opioid receptors. Receptor-binding assays revealed that 7-hydroxymitragynine has a higher affinity for micro-opioid receptors relative to the other opioid receptors. Administration of 7-hydroxymitragynine (2.5-10 mg/kg, s.c.) induced dose-dependent antinociceptive effects in tail-flick and hot-plate tests in mice. Its effect was more potent than that of morphine in both tests. When orally administered, 7-hydroxymitragynine (5-10 mg/kg) showed potent antinociceptive activities in tail-flick and hot-plate tests. In contrast, only weak antinociception was observed in the case of oral administration of morphine at a dose of 20 mg/kg. It was found that 7-hydroxymitragynine is a novel opioid agonist that is structurally different from the other opioid agonists, and has potent analgesic activity when orally administered.     

Molecular mechanism of changes in the morphine-induced pharmacological actions under chronic pain-like state: suppression of dopaminergic transmission in the brain

In the present study, we demonstrated whether a neuropathic pain-like state induced by sciatic nerve ligation in rodents could cause a long-lasting change in intracellular signaling in both supraspinal and spinal cord related to the suppression of morphine's effect. Mice with sciatic nerve ligation exhibited a significant suppression of the morphine-induced antinociception. Under this condition, phosphorylated-conventional protein kinase C-like immunoreactivity (p-cPKC-IR) and phosphorylated-micro-opioid receptor (p-MOR)-IR were clearly increased on the ipsilateral side in the dorsal horn of the spinal cord of nerve-ligated mice. It is of interest to note that astroglial hypertrophy as well as its proliferation was also noted in this area of sciatic nerve-ligated mice. Like nerve injury, the increase in cPKC activities and astroglial hypertrophy/proliferation in this region was observed by repeated morphine treatment. These findings suggest that the phosphorylation of both cPKC and MOR in the dorsal horn of the spinal cord by sciatic nerve ligation may play a substantial role in the suppression of morphine-induced antinociception under a neuropathic pain-like state. Sciatic nerve injury also caused a significant inhibition of MOR-mediated G-protein activation onto GABAergic neurons and a dramatic reduction in ERK activities onto dopaminergic neurons in the ventral tegmental area (VTA) regulating the rewarding effect of opioids. Furthermore, we found that the inhibition of ERK cascade in the VTA by treatment with specific inhibitors suppressed the morphine-induced rewarding effect in normal mice. These findings provide evidence that the direct reduction in MOR function and the persistent decrease in ERK activity of dopaminergic neurons in the VTA may contribute to the suppression of the morphine-induced rewarding effect under a neuropathic pain-like state. Conclusively, our recent findings provide novel evidences for the mechanism underlying the less sensitivity to opioids under a neuropathic pain-like state.