Ligand-directed signaling has been suggested as a basis for the differences in responses evoked by otherwise receptor-selective agonists. The underlying mechanisms are not understood, yet clearer ...definition of this concept may be helpful in the development of novel, pathway-selective therapeutic agents. We previously showed that κ-opioid receptor activation of JNK by one class of ligand, but not another, caused persistent receptor inactivation. In the current study, we found that the μ-opioid receptor (MOR) could be similarly inactivated by a specific ligand class including the prototypical opioid, morphine. Acute analgesic tolerance to morphine and related opioids (morphine-6-glucuronide and buprenorphine) was blocked by JNK inhibition, but not by G protein receptor kinase 3 knockout. In contrast, a second class of μ-opioids including fentanyl, methadone, and oxycodone produced acute analgesic tolerance that was blocked by G protein receptor kinase 3 knockout, but not by JNK inhibition. Acute MOR desensitization, demonstrated by reduced D-Ala²-Met⁵-Glyol-enkephalin—stimulated ³⁵SGTPγS binding to spinal cord membranes from morphine-pretreated mice, was also blocked by JNK inhibition; however, desensitization of D-Ala²-Met⁵-Glyol-enkephalin—stimulated ³⁵SGTPγS binding following fentanyl pretreatment was not blocked by JNK inhibition. JNK-mediated receptor inactivation of the κ-opioid receptor was evident in both agonist-stimulated ³⁵SGTPγS binding and opioid analgesic assays; however, gene knockout of JNK 1 selectively blocked κ-receptor inactivation, whereas deletion of JNK 2 selectively blocked MOR inactivation. These findings suggest that ligand-directed activation of JNK kinases may generally provides an alternate mode of G protein—coupled receptor regulation.
Long-term exposure to pyschostimulants and opioids induced neuronal plasticity. Accumulating evidence suggests that astrocytes actively participate in synaptic plasticity. We show here that a glial ...modulator propentofylline (PPF) dramatically diminished the activation of astrocytes induced by drugs of abuse, such as methamphetamine (METH) and morphine (MRP). In vivo treatment with PPF also suppressed both METH- and MRP-induced rewarding effects. On the other hand, intra-nucleus accumbens (N.Acc.) administration of astrocyte-conditioned medium (ACM) aggravated the development of rewarding effects induced by METH and MRP via the Janus kinase/signal transducers and activators of transcription (Jak/STAT) pathway, which modulates astrogliosis and/or astrogliogenesis. Furthermore, ACM, but not METH itself, clearly induced the differentiation of multipotent neuronal stem cells into glial fibrillary acidic protein-positive astrocytes, and this effect was reversed by cotreatment with the Jak/STAT inhibitor AG490. Intra-cingulate cortex (CG) administration of ACM also enhanced the rewarding effect induced by METH and MRP. In contrast to ACM, intra-N.Acc. administration of microglia-conditioned medium failed to affect the rewarding effects of METH and MRP in mice. These findings suggest that astrocyte-, but not microglia-, related soluble factors could amplify the development of rewarding effect of METH and MRP in the N.Acc. and CG. The present study provides direct evidence that astrocytes may, at least in part, contribute to the synaptic plasticity induced by drugs of abuse during the development of rewarding effects induced by psychostimulants and opioids.
The present study was undertaken to further investigate the role of glial cells in the development of the neuropathic pain‐like state induced by sciatic nerve ligation in mice. At 7 days after ...sciatic nerve ligation, the immunoreactivities (IRs) of the specific astrocyte marker glial fibrillary acidic protein (GFAP) and the specific microglial marker OX‐42, but not the specific oligodendrocyte marker O4, were increased on the ipsilateral side of the spinal cord dorsal horn in nerve‐ligated mice compared with that on the contralateral side. Furthermore, a single intrathecal injection of activated spinal cord microglia, but not astrocytes, caused thermal hyperalgesia in naive mice. Furthermore, 5‐bromo‐2′‐deoxyuridine (BrdU)‐positive cells on the ipsilateral dorsal horn of the spinal cord were significantly increased at 7 days after nerve ligation and were highly co‐localized with another microglia marker, ionized calcium‐binding adaptor molecule 1 (Iba1), but neither with GFAP nor a specific neural nuclei marker, NeuN, in the spinal dorsal horn of nerve‐ligated mice. The present data strongly support the idea that spinal cord astrocytes and microglia are activated under the neuropathic pain‐like state, and that the proliferated and activated microglia directly contribute to the development of a neuropathic pain‐like state in mice.
Thermal hyperalgesia and tactile allodynia induced by sciatic nerve ligation were completely suppressed by repeated intrathecal (i.t.) injection of a TrkB/Fc chimera protein, which sequesters ...endogenous brain‐derived neurotrophic factor (BDNF). In addition, BDNF heterozygous (+/–) knockout mice exhibited a significant suppression of nerve ligation‐induced thermal hyperalgesia and tactile allodynia compared with wild‐type mice. After nerve ligation, BDNF‐like immunoreactivity on the superficial laminae of the ipsilateral side of the spinal dorsal horn was clearly increased compared with that of the contralateral side. It should be noted that a single i.t. injection of BDNF produced a long‐lasting thermal hyperalgesia and tactile allodynia in normal mice, and these responses were abolished by i.t. pre‐treatment with either a Trk‐dependent tyrosine kinase inhibitor K‐252a or a selective protein kinase C (PKC) inhibitor Ro‐32‐0432. Supporting these findings, we demonstrated here for the first time that the increase in intracellular Ca2+ concentration by application of BDNF in cultured mouse spinal neurons was abolished by pre‐treatment with either K‐252a or Ro‐32‐0432. Taken together, these findings suggest that the binding of spinally released BDNF to TrkB by nerve ligation may activate PKC within the spinal cord, resulting in the development of a neuropathic pain‐like state in mice.
In the present study, we investigated the role of orexinergic systems in the activation of midbrain dopamine neurons. In an in vitro study, exposure to either orexin A or orexin B under superfusion ...conditions produced a transient increase in the intracellular Ca2+ concentration through the phospholipase C (PLC)/protein kinase C (PKC) pathway via Gq11α or Gβγ subunits in midbrain cultured neurons, which were shown to be tyrosine hydroxylase (TH)‐positive cells, but not in purified midbrain astrocytes. Here we show that in vivo injection with a selective PKC inhibitor chelerythrine chloride or 2‐{8‐(dimethylamino)methyl‐6,7,8,9‐tetrahydropyrido1,2‐aindol‐3‐yl}‐3‐1‐methyl‐1H‐indol‐3‐ylmaleimide HCl (Ro‐32–0432) into the ventral tegmental area (VTA) significantly suppressed the place preference and increased levels of dopamine in the nucleus accumbens (NAcc) induced by intra‐VTA injection of orexins. These results strongly support the idea that activation of the orexin‐containing neuron in the VTA leads to the direct activation of mesolimbic dopamine neurons through the activation of the PLC/PKC pathway via Gq11α or Gβγ‐subunit activation, which could be associated with the development of its rewarding effect.
Methamphetamine (METH) is a strongly addictive psychostimulant that dramatically affects the central nervous system (CNS). On the other hand, protein kinase C (PKC) plays a major role in cellular ...regulatory and signalling processes that involve protein phosphorylation. The purpose of this study was to investigate the role of neuronal and astrocytic PKC in changes in the central glutamatergic system induced by METH. We show here that in vitro treatment with METH caused the phosphorylation of both neuronal and astrocytic PKC and the activation of astrocytes in cortical neuron/glia co‐cultures. Treatment of cortical neuron/glia co‐cultures with either the PKC activator phorbol 12,13‐dibutyrate (PDBu) or glutamate also caused the PKC‐dependent activation of astrocytes. The PKC inhibitor chelerythrine suppressed the Ca2+ responses to glutamate in both cortical neurons and astrocytes. Moreover, a low concentration of PDBu significantly enhanced the Ca2+ responses to glutamate, but not to dopamine, in both cortical neurons and astrocytes. Notably, treatment with METH also enhanced the Ca2+ responses to glutamate in cortical neurons. The activation of astrocytes induced by METH was also reversed by co‐treatment with glutamate receptor antagonists (ifenprodil, DNQX or MPEP) in cortical neuron/glia co‐cultures. In the conditioned place preference paradigm, intracerebroventricular administration of glutamate receptor antagonists (ifenprodil, DNQX or MPEP) attenuated the METH‐induced rewarding effect. These findings provide evidence that the changes in PKC‐dependent neuronal and astrocytic glutamatergic transmission induced by METH may, at least in part, contribute to the development of psychological dependence on METH.
It is well known that long‐term exposure to psychostimulants induces neuronal plasticity. Recently, accumulating evidence suggests that astrocytes may actively participate in synaptic plasticity. In ...this study, we found that in vitro treatment of cortical neuron/glia co‐cultures with either methamphetamine (METH) or morphine (MRP) caused the activation of astrocytes via protein kinase C (PKC). Purified astrocytes were markedly activated by METH, whereas MRP had no such effect. METH, but not MRP, caused a long‐lasting astrocytic activation in cortical neuron/glia co‐cultures. Furthermore, MRP‐induced behavioral sensitization to hyper‐locomotion was reversed by 2 months of withdrawal following intermitted MRP administration, whereas behavioral sensitization to METH‐induced hyper‐locomotion was maintained even after 2 months of withdrawal. Consistent with this cell culture study, in vivo treatment with METH, which was associated with behavioral sensitization, caused a PKC‐dependent astrocytic activation in the cingulate cortex and nucleus accumbens of mice. These findings provide direct evidence that METH induces a long‐lasting astrocytic activation and behavioral sensitization through the stimulation of PKC in the rodent brain. In contrast, MRP produced a reversible activation of astrocytes via neuronal PKC and a reversibility of behavioral sensitization. This information can break through the definition of drugs of abuse and the misleading of concept that morphine produces a long‐lasting neurotoxicity.
Astrocytes are a subpopulation of glial cells that directly affect neuronal function. This review focuses on the potential functional roles of astrocytes in the development of behavioral ...sensitization and rewarding effects induced by chronic treatment with drugs of abuse. In vitro treatment of cortical neuron/glia cocultures with either methamphetamine or morphine caused activation of astrocytes via protein kinase C (PKC). Purified cortical astrocytes were markedly activated by methamphetamine, whereas morphine had no such effect. Methamphetamine, but not morphine, caused a long‐lasting astrocytic activation in cortical neuron/glia cocultures. Morphine‐induced behavioral sensitization, assessed as hyperlocomotion, was reversed by 2 months of withdrawal from intermittent morphine administration, whereas behavioral sensitization to methamphetamine‐induced hyperlocomotion was maintained even after 2 months of withdrawal. In vivo treatment with methamphetamine, which was associated with behavioral sensitization, caused PKC‐dependent astrocytic activation in the mouse cingulate cortex and nucleus accumbens. Furthermore, the glial modulator propentofylline dramatically diminished the activation of astrocytes and the rewarding effect induced by methamphetamine and morphine. On the other hand, intra–nucleus accumbens and intra–cingulate cortex administration of astrocyte‐conditioned medium aggravated the development of rewarding effects induced by methamphetamine and morphine. Furthermore, astrocyte‐conditioned medium, but not methamphetamine itself, clearly induced differentiation of neural stem cells into astrocytes. These findings provide direct evidence that astrocytes may, at least in part, contribute to the development of the rewarding effects induced by drugs of abuse in the nucleus accumbens and cingulate cortex.
It is well known that prolonged exposure to morphine results in tolerance to morphine‐induced antinociception. In the present study, we found that either intrathecal (i.t.) or subcutaneous (s.c.) ...injection of the selective metabotropic glutamate receptor 5 (mGluR5) antagonist, methyl‐6‐(phenylethynyl)‐pyridine hydrochloride (MPEP), attenuated the development of tolerance to morphine‐induced antinociception. Using the receptor binding assay, we found here that the number of mGluR5 in the mouse spinal cord was significantly increased by repeated treatment with morphine. Furthermore, repeated treatment with morphine produced a significant increase in the level of mGluR5 immunoreactivity in the dorsal horn of the mouse spinal cord. Double‐labeling experiments showed that the increased mGluR5 was predominantly expressed in the neurons and sparsely expressed in the processes of astrocytes following repeated treatment with morphine. Consistent with these results, the response of Ca2+ to the selective group I mGluR agonist, 3,5‐dihydroxyphenylglycine (DHPG), in cultured spinal cord neurons was potently enhanced by 3 days of in vitro treatment with morphine. These findings support the idea that the increased mGluR5 following repeated treatment with morphine leads to enhanced neuronal excitability and synaptic transmission in the dorsal horn of the spinal cord and, in turn, suppresses the morphine‐induced antinociception in mice.
α 1D -Adrenergic receptors (ARs) are key regulators of cardiovascular system function that increase blood pressure and promote vascular remodeling. Unfortunately, little information exists about the ...signaling pathways used by this important G protein-coupled receptor (GPCR). We recently discovered that α 1D -ARs form a “signalosome” with multiple members of the dystrophin-associated protein complex (DAPC) to become functionally expressed at the plasma membrane and bind ligands. However, the molecular mechanism by which the DAPC imparts functionality to the α 1D -AR signalosome remains a mystery. To test the hypothesis that previously unidentified molecules are recruited to the α 1D -AR signalosome, we performed an extensive proteomic analysis on each member of the DAPC. Bioinformatic analysis of our proteomic data sets detected a common interacting protein of relatively unknown function, α-catulin. Coimmunoprecipitation and blot overlay assays indicate that α-catulin is directly recruited to the α 1D -AR signalosome by the C-terminal domain of α-dystrobrevin-1 and not the closely related splice variant α-dystrobrevin-2. Proteomic and biochemical analysis revealed that α-catulin supersensitizes α 1D -AR functional responses by recruiting effector molecules to the signalosome. Taken together, our study implicates α-catulin as a unique regulator of GPCR signaling and represents a unique expansion of the intricate and continually evolving array of GPCR signaling networks.
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