Regulation by chronic clonidine of adenylate cyclase and cyclic AMP-dependent protein kinase in the rat locus coeruleus

Clonidine and morphine are known to produce tolerance and dependence in rat locus coeruleus (LC) neurons after chronic administration based on electrophysiological criteria. Previous studies have shown that morphine tolerance and dependence is associated with increases in levels of adenylate cyclase, pertussis toxin-mediated ADP-ribosylation of G-proteins, and cyclic AMP-dependent protein kinase in this brain region. The present study was aimed at investigating whether clonidine tolerance and dependence is also associated with alterations in these intracellular messengers. It was found that, similar to chronic morphine, chronic (2 weeks) clonidine administration, under conditions that produce electrophysiological evidence of tolerance and dependence in LC neurons, increased levels of adenylate cyclase activity and cyclic AMP-dependent protein kinase activity in this brain region, but not in several other regions studied, which included the frontal cortex, neostriatum, and dorsal raphe. However, the changes induced by chronic clonidine in the LC, at maximal doses and duration of treatment, were only approximately 50% in magnitude of those observed in response to morphine. Unlike chronic morphine, chronic clonidine produced no change in G-protein ADP-ribosylation levels in the LC. Chronic administration of a number of other drugs, namely diazepam, chloral hydrate, and dextromethorphan, which produce electrophysiological actions distinct from those of clonidine and morphine in the LC, failed to alter adenylate cyclase and cyclic AMP-dependent protein kinase in this brain region. The results indicate that increased levels of adenylate cyclase and cyclic AMP-dependent protein kinase represent common adaptations by LC neurons to chronic clonidine and morphine, and raise the possibility that such changes contribute to the development of clonidine and morphine tolerance and dependence in these neurons.     

BiP,an endoplasmic reticulum chaperone,modulates the development of morphine antinociceptive tolerance

Morphine is a potent analgesic, but the molecular mechanism for tolerance formation after repeated use is not fully understood. Binding immunoglobulin protein (BiP) is an endoplasmic reticulum (ER) chaperone that is central to ER function. We examined knock‐in mice expressing a mutant BiP with the retrieval sequence deleted in order to elucidate physiological processes that are sensitive to BiP functions. We tested the thermal antinociceptive effect of morphine in heterozygous mutant BiP mice in a hot plate test. Paw withdrawal latencies before and after a single administration of morphine were not significantly different between the wild‐type and mutant BiP mice. Repeated morphine administration caused the development of morphine tolerance in the wild‐type mice. The activation of glycogen synthase kinase 3b (GSK‐3b) was associated with morphine tolerance, because an inhibitor of GSK‐3β prevented it. On the other hand, the mutant BiP mice showed less morphine tolerance, and the activation of GSK‐3b was suppressed in their brain. These results suggest that BiP may play an important role in the development of morphine tolerance. Furthermore, we found that a chemical chaperone which improves ER protein folding capacity also attenuated the development of morphine tolerance in wild‐type mice, suggesting a possible clinical application of chemical chaperones in preventing morphine tolerance.     

Treatment for psychological dependence on morphine: usefulness of inhibiting NMDA receptor and its associated protein kinase in the nucleus accumbens

A growing body of evidence indicates that the mesolimbic dopaminergic (DAergic) pathway projecting from the ventral tegmental area (VTA) to the nucleus accumbens (N.Acc.) play a critical role in the initiation of psychological dependence on morphine. As well as DAergic system, the involvement of non-DAergic neurotransmitter and neuromodulator systems in rewarding effects induced by morphine has been recently documented. We previously demonstrated that the morphine-induced rewarding effect was dramatically suppressed by co-treatment with NMDA receptor antagonists, such as dizocilpine (MK-801), ketamine and ifenprodil. Therefore, we propose here that inhibiting the N-methyl-D-aspartate (NMDA) receptor and its associated protein kinase in the N.Acc. is useful for the treatment for psychological dependence on morphine. The following review provides a summary of recent our findings regarding the role of NMDA receptor and its associated protein kinase in the development of psychological dependence on morphine.     

Accelerated communciation: Constitutive μ opioid receptor activation as a regulatory mechanism underlying narcotic tolerance and dependence

Chronic administration of narcotic μ opioid agonists results in tolerance and dependence. We propose that agonist stimulation causes a gradual conversion of μ receptors to a constitutively active state μ¹ as a key step in tolerance and physical dependence. We provide evidence in support of the existence of μ¹ in human neuroblastoma cells, SH-SY5Y, and μ¹ upregulation during morphine treatment. Naloxone blocked μ¹ activity, acting as an antagonist with negative intrinsic activity which accounts for its high potency in eliciting withdrawal. In contrast, the μ selective antagonist CTAP did not affect μ¹ activity but inhibited naloxone's effect. The protein kinase inhibitor H7 was found to suppress μ¹ formation, suggesting that μ¹ is phosphorylated. In a model of acute morphine tolerance/dependence in mice, H7 prevented naloxone induced withdrawal jumping and reversed morphine (antinociceptive) tolerance. CTAP cause only mild withdrawal and attenuated naloxone induced withdrawal, as predicted for an antagonist without negative activity. These results support a role for constitutive μ receptor activation in narcotic tolerance and dependence, affording potential separation of acute and chronic narcotic effects.     

New approaches to study the development of morphine tolerance and dependence

Morphine is now believed not to cause tolerance and dependence when it is appropriately used in clinic. However, in terminal cancer pain, patients' analgesic tolerance to morphine is developed due to the use of high doses of morphine for complete blockade of pain. At higher doses, morphine has more opportunity to show serious side effects, which worsens quality of life (QOL), and leads to the use of potent analgesic adjuvants to reduce the morphine dosage. Here we attempt to summarize recent studies of the molecular basis of morphine tolerance and dependence, and to discuss whether these mechanisms could provide new molecular targets as analgesic adjuvants. They include protein kinase C inhibitor, opioid agonist with low RAVE value, and antagonists of antiopioid receptors (GluRepsilon1 or nociceptin/OFQ receptor). In addition, we demonstrate new approaches to find further candidates of such molecular targets. These approaches include the visualization of neuronal networks in the downstream of opioid neurons by use of the WGA transgene technique and the single cell dissection technique to get new genes involved in plasticity during morphine tolerance and dependence.     

Differential Regulation of MAPK Phosphorylation in the Dorsal Hippocampus in Response to Prolonged Morphine Withdrawal-Induced Depressive-Like Symptoms in Mice

Depression is one of the most frequent neuropsychiatric comorbidities associated with opiate addiction. Mitogen activated protein kinase (MAPK) and MAPK phosphatase (MKP) are involved in drug addiction and depression. However, the potential role of MAPK and MKP in depression caused by morphine withdrawal remains unclear. We utilized a mouse model of repeated morphine administration to examine the molecular mechanisms that contribute to prolonged withdrawal induced depressive-like behaviors. Depressive-like behaviors were significant at 1 week after withdrawal and worsened over time. Phospho-ERK (extracellular signal-regulated protein kinase) was decreased and MKP-1 was elevated in the hippocampus, and JNK (c-Jun N-terminal protein kinase), p38 (p38 protein kinase) and MKP-3 were unaffected. A pharmacological blockade of MKP-1 by intra-hippocampal sanguinarine (SA) infusion prevented the development of depressive-like behaviors and resulted in relatively normal levels of MKP-1 and phospho-ERK after withdrawal. Our findings support the association between hippocampal MAPK phosphorylation and prolonged morphine withdrawal-induced depression, and emphasize the MKP-1 as an negative regulator of the ERK phosphorylation that contributes to depression.     

Activation of B-Raf and regulation of the mitogen-activated protein kinase pathway by the G(o) alpha chain

Many receptors coupled to the pertussis toxin-sensitive G(i/o) proteins stimulate the mitogen-activated protein kinase (MAPK) pathway. The role of the alpha chains of these G proteins in MAPK activation is poorly understood. We investigated the ability of Galpha(o) to regulate MAPK activity by transient expression of the activated mutant Galpha(o)-Q205L in Chinese hamster ovary cells. Galpha(o)-Q205L was not sufficient to activate MAPK but greatly enhanced the response to the epidermal growth factor (EGF) receptor. This effect was not associated with changes in the state of tyrosine phosphorylation of the EGF receptor. Galpha(o)-Q205L also potentiated MAPK stimulation by activated Ras. In Chinese hamster ovary cells, EGF receptors activate B-Raf but not Raf-1 or A-Raf. We found that expression of activated Galpha(o) stimulated B-Raf activity independently of the activation of the EGF receptor or Ras. Inactivation of protein kinase C and inhibition of phosphatidylinositol-3 kinase abolished both B-Raf activation and EGF receptor-dependent MAPK stimulation by Galpha(o). Moreover, Galpha(o)-Q205L failed to affect MAPK activation by fibroblast growth factor receptors, which stimulate Raf-1 and A-Raf but not B-Raf activity. These results suggest that Galpha(o) can regulate the MAPK pathway by activating B-Raf through a mechanism that requires a concomitant signal from tyrosine kinase receptors or Ras to efficiently stimulate MAPK activity. Further experiments showed that receptor-mediated activation of Galpha(o) caused a B-Raf response similar to that observed after expression of the mutant subunit. The finding that Galpha(o) induces Ras-independent and protein kinase C- and phosphatidylinositol-3 kinase-dependent activation of B-Raf and conditionally stimulates MAPK activity provides direct evidence for intracellular signals connecting this G protein subunit to the MAPK pathway.     

Thrombin activates p38 mitogen-activated protein kinase in vascular smooth muscle cells

Thrombin is a potent mitogen for vascular smooth muscle cells. However, the signaling pathways by which thrombin mediates its mitogenic response are not fully understood. The ERK (extracellular signal-regulated protein kinase) and JNK (c-Jun N-terminal kinase) members of the mitogen-activated protein kinase (MAPK) family are reported to be activated by thrombin. We have investigated the response to thrombin of another member of the MAPK family, p38 MAPK, which has been suggested to be activated by both stress and inflammatory stimuli in vascular smooth muscle cells. We found that thrombin induced time- and dose-dependent activation of p38 MAPK. Maximal stimulation of p38 MAPK was observed after a 10-min incubation with 1 unit ml(-1) thrombin. GF109203X, a protein kinase C inhibitor, and prolonged treatment with phorbol 12-myristate 13-acetate partially inhibited p38 MAPK activation. A tyrosine kinase inhibitor, genistein, also inhibited p38 MAPK activation in a dose-dependent manner. p38 MAPK activation was inhibited by overexpression of betaARK1ct (beta-adrenergic receptor kinase I C-terminal peptide). p38 MAPK activation was also inhibited by expression of dominant-negative Ras, not by dominant-negative Rac. We next examined the effect of a p38 MAPK inhibitor, SB203580, on thrombin-induced proliferation. SB203580 inhibited thrombin-induced DNA synthesis in a dose-dependent manner. These results suggest that thrombin activates p38 MAPK in a manner dependent on Gbetagamma, protein kinase C, a tyrosine kinase, and Ras, that p38 MAPK has a role in thrombin-induced mitogenic response in the cells.     

Toxoplasma gondii interferes with lipopolysaccharide-induced mitogen-activated protein kinase activation by mechanisms distinct from endotoxin tolerance

We show in this study that Toxoplasma gondii infection induces rapid activation of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase 1/2, and stress-activated protein kinase/c-Jun N-terminal kinase MAPK, followed promptly by their deactivation in mouse macrophages. Nevertheless, when infected cells were subsequently subjected to LPS triggering, MAPK activation was severely defective, in particular in the case of p38 MAPK, which is required for LPS-triggered TNF-alpha and IL-12 production. Similar effects occurred during endotoxin tolerance, but the phenomena were distinct. LPS pretriggering failed to activate the major p38 MAPK kinase, MAPK kinase 3/6. Toxoplasma infection, in contrast, resulted in sustained activation of this kinase. Furthermore, endotoxin pre-exposure blocked IkappaBalpha degradation upon subsequent LPS triggering, but this was not the case for Toxoplasma preinfection. Endotoxin-mediated down-regulation of the LPS receptor, Toll-like receptor 4, has been suggested as one possible mechanism contributing to tolerance, and we found in this study that LPS down-modulated Toll-like receptor 4 expression. In contrast, Toxoplasma infection induced up-regulation of this pattern recognition receptor. Our results show that T. gondii blocks LPS-triggered cytokine production in part through MAPK inactivation, and that this occurs through pathways distinct from endotoxin-induced tolerance.     

脊髓水平细胞外信号调节激酶激活参与吗啡依赖的形成及戒断反应的表达

在大鼠吗啡依赖和戒断模型上,采用行为学、免疫组织化学和Western blot方法观察吗啡依赖及戒断大鼠脊髓神经元磷酸化细胞外信号调节激酶（phospho-extracellular signal-regulated kinase,pERK）表达的变化,及鞘内注射促分裂原活化蛋白激酶激酶（mitogen-activated protein kinase kinase,MEK）抑制剂U0126或ERK反义寡核苷酸对吗啡依赖大鼠纳洛酮催促戒断反应、触诱发痛及脊髓神经元pERK表达的影响,探讨脊髓水平pERK在介导吗啡依赖和戒断过程中的作用。结果显示：（1）在吗啡依赖形成过程中,大鼠脊髓胞浆与胞核非磷酸化ERK表达没有改变,但pERK表达逐渐增加,纳洛酮催促戒断后,仍有进一步增加的趋势,戒断1h后,其表达量明显下降,但仍高于对照组。（2）鞘内预先注射MEK抑制剂U0126或ERK反义寡核苷酸能明显抑制吗啡戒断反应和戒断引起的痛觉异常;与行为学结果一致,脊髓背角pERK阳性神经元表达与脊髓胞浆和胞核pERK表达也明显降低。上述结果提示,脊髓水平ERK激活和核转位参与吗啡依赖的形成及戒断反应的表达。     

The mechanism of μ-opioid receptor (MOR)-TRPV1 crosstalk in TRPV1 activation involves morphine anti-nociception,tolerance and dependence

Initiated by the activation of various nociceptors, pain is a reaction to specific stimulus modalities. The μ-opioid receptor (MOR) agonists, including morphine, remain the most potent analgesics to treat patients with moderate to severe pain. However, the utility of MOR agonists is limited by the adverse effects associated with the use of these drugs, including analgesic tolerance and physical dependence. A strong connection has been suggested between the expression of the transient receptor potential vanilloid type 1 (TRPV1) ion channel and the development of inflammatory hyperalgesia. TRPV1 is important for thermal nociception induction, and is mainly expressed on sensory neurons. Recent reports suggest that opioid or TRPV1 receptor agonist exposure has contrasting consequences for anti-nociception, tolerance and dependence. Chronic morphine exposure modulates TRPV1 activation and induces the anti-nociception effects of morphine. The regulation of many downstream targets of TRPV1 plays a critical role in this process, including calcitonin gene-related peptide (CGRP) and substance P (SP). Additional factors also include capsaicin treatment blocking the anti-nociception effects of morphine in rats, as well as opioid modulation of TRPV1 responses through the cAMP-dependent PKA pathway and MAPK signaling pathways. Here, we review new insights concerning the mechanism underlying MOR-TRPV1 crosstalk and signaling pathways and discuss the potential mechanisms of morphine-induced anti-nociception, tolerance and dependence associated with the TRPV1 signaling pathway and highlight how understanding these mechanisms might help find therapeutic targets for the treatment of morphine induced antinociception, tolerance and dependence.     

The mechanism of μ-opioid receptor (MOR)-TRPV1 crosstalk in TRPV1 activation involves morphine anti-nociception,tolerance and dependence

Initiated by the activation of various nociceptors, pain is a reaction to specific stimulus modalities. The μ-opioid receptor (MOR) agonists, including morphine, remain the most potent analgesics to treat patients with moderate to severe pain. However, the utility of MOR agonists is limited by the adverse effects associated with the use of these drugs, including analgesic tolerance and physical dependence. A strong connection has been suggested between the expression of the transient receptor potential vanilloid type 1 (TRPV1) ion channel and the development of inflammatory hyperalgesia. TRPV1 is important for thermal nociception induction, and is mainly expressed on sensory neurons. Recent reports suggest that opioid or TRPV1 receptor agonist exposure has contrasting consequences for anti-nociception, tolerance and dependence. Chronic morphine exposure modulates TRPV1 activation and induces the anti-nociception effects of morphine. The regulation of many downstream targets of TRPV1 plays a critical role in this process, including calcitonin gene-related peptide (CGRP) and substance P (SP). Additional factors also include capsaicin treatment blocking the anti-nociception effects of morphine in rats, as well as opioid modulation of TRPV1 responses through the cAMP-dependent PKA pathway and MAPK signaling pathways. Here, we review new insights concerning the mechanism underlying MOR-TRPV1 crosstalk and signaling pathways and discuss the potential mechanisms of morphine-induced anti-nociception, tolerance and dependence associated with the TRPV1 signaling pathway and highlight how understanding these mechanisms might help find therapeutic targets for the treatment of morphine induced antinociception, tolerance and dependence.     

蛋白激酶C与吗啡耐受

蛋白激酶C(protein kinase C,PKC)属于AGC蛋白激酶家族(即PKA/PKG/PKC激酶家族),在吗啡介导的μ-阿片受体脱敏及吗啡耐受中具有重要作用,因此研究PKC的细胞信号传导机制对吗啡耐受的治疗具有重要的临床意义。本文综述了PKC在吗啡耐受中的作用。     

Inability of cyclo (Leu-Gly) to facilitate the development of tolerance to and physical dependence on morphine in the rat

The effect of cyclo (Leu-Gly), an analog of melanotropin release inhibition factor on the development of tolerance to and physical dependence on morphine in the rat was investigated. Administration of cyclo (Leu-Gly) (1 μg/rat/day) prior to and during morphine pellet implantation failed to facilitate the development of tolerance to the analgesic and hypothermic effects of morphine. Similarly the development of dependence on morphine was not facilitated by cyclo (Leu-Gly) as evidenced by changes in body weight and body temperature observed during abrupt withdrawal of morphine. These studies do not lend support to the previous observations that cyclo (Leu-Gly) and other related peptides facilitate the development of tolerance to and physical dependence on morphine.     

Morphine-induced receptor endocytosis in a novel knockin mouse reduces tolerance and dependence

Opioid drugs, such as morphine, are among the most effective analgesics available. However, their utility for the treatment of chronic pain is limited by side effects including tolerance and dependence. Morphine acts primarily through the mu-opioid receptor (MOP-R) , which is also a target of endogenous opioids. However, unlike endogenous ligands, morphine fails to promote substantial receptor endocytosis both in vitro, and in vivo. Receptor endocytosis serves at least two important functions in signal transduction. First, desensitization and endocytosis act as an "off" switch by uncoupling receptors from G protein. Second, endocytosis functions as an "on" switch, resensitizing receptors by recycling them to the plasma membrane. Thus, both the off and on function of the MOP-R are altered in response to morphine compared to endogenous ligands. To examine whether the low degree of endocytosis induced by morphine contributes to tolerance and dependence, we generated a knockin mouse that expresses a mutant MOP-R that undergoes morphine-induced endocytosis. Morphine remains an excellent antinociceptive agent in these mice. Importantly, these mice display substantially reduced antinociceptive tolerance and physical dependence. These data suggest that opioid drugs with a pharmacological profile similar to morphine but the ability to promote endocytosis could provide analgesia while having a reduced liability for promoting tolerance and dependence.     

Functionally Selective Signaling for Morphine and Fentanyl Antinociception and Tolerance Mediated by the Rat Periaqueductal Gray

Functionally selective signaling appears to contribute to the variability in mechanisms that underlie tolerance to the antinociceptive effects of opioids. The present study tested this hypothesis by examining the contribution of G protein-coupled receptor kinase (GRK)/Protein kinase C (PKC) and C-Jun N-terminal kinase (JNK) activation on both the expression and development of tolerance to morphine and fentanyl microinjected into the ventrolateral periaqueductal gray of the rat. Microinjection of morphine or fentanyl into the periaqueductal gray produced a dose-dependent increase in hot plate latency. Microinjection of the non-specific GRK/PKC inhibitor Ro 32-0432 into the periaqueductal gray to block mu-opioid receptor phosphorylation enhanced the antinociceptive effect of morphine but had no effect on fentanyl antinociception. Microinjection of the JNK inhibitor SP600125 had no effect on morphine or fentanyl antinociception, but blocked the expression of tolerance to repeated morphine microinjections. In contrast, a microinjection of Ro 32-0432 blocked the expression of fentanyl, but not morphine tolerance. Repeated microinjections of Ro 32-0432 blocked the development of morphine tolerance and inhibited fentanyl antinociception whether rats were tolerant or not. Repeated microinjections of SP600125 into the periaqueductal gray blocked the development of tolerance to both morphine and fentanyl microinjections. These data demonstrate that the signaling molecules that contribute to tolerance vary depending on the opioid and methodology used to assess tolerance (expression vs. development of tolerance). This signaling difference is especially clear for the expression of tolerance in which JNK contributes to morphine tolerance and GRK/PKC contributes to fentanyl tolerance.     

Comparison of the effects of specific and nonspecific inhibition of nitric oxide synthase on morphine analgesia,tolerance and dependence in mice

The effects of L-Canavanine, a selective inducible nitric oxide synthase (NOS) inhibitor and N(G)-nitro-L-arginine methyl ester (L-NAME), a nonselective NOS inhibitor, on pain threshold and morphine induced analgesia, tolerance and dependence in mice were investigated and compared. Morphine was administered by subcutaneous implantation of a pellet containing 40 mg free base and withdrawal was precipitated by intraperitoneal (i.p.) injection of naloxone (2 mg/kg). L-Canavanine (200 mg/kg, i.p.) did not affect the pain threshold, morphine-induced analgesia and the induction and expression phases of morphine tolerance and dependence. L-NAME (20 mg/kg, i.p.) significantly (p < 0.05) enhanced the pain threshold, potentiated morphine-induced analgesia and attenuated the expression phase of morphine dependence which has been characterized by withdrawal signs and body weight loss, but did not modify the induction phase of morphine tolerance and dependence. It is concluded that constitutive NOS isoforms which were inhibited by L-NAME may be involved specifically in the mechanisms of morphine induced analgesia, tolerance and dependence.     

The Rho family GTPase Cdc42 regulates the activation of Ras/MAP kinase by the exchange factor Ras-GRF

The Ras guanine-nucleotide exchange factor Ras-GRF/Cdc25(Mn) harbors a complex array of structural motifs that include a Dbl-homology (DH) domain, usually found in proteins that interact functionally with the Rho family GTPases, and the role of which is not yet fully understood. Here, we present evidence that Ras-GRF requires its DH domain to translocate to the membrane, to stimulate exchange on Ras, and to activate mitogen-activated protein kinase (MAPK). In an unprecedented fashion, we have found that these processes are regulated by the Rho family GTPase Cdc42. We show that GDP- but not GTP-bound Cdc42 prevents Ras-GRF recruitment to the membrane and activation of Ras/MAPK, although no direct association of Ras-GRF with Cdc42 was detected. We also demonstrate that catalyzing GDP/GTP exchange on Cdc42 facilitates Ras-GRF-induced MAPK activation. Moreover, we show that the potentiating effect of ionomycin on Ras-GRF-mediated MAPK stimulation is also regulated by Cdc42. These results provide the first evidence for the involvement of a Rho family G protein in the control of the activity of a Ras exchange factor.