Psychostimulants including amphetamines and cocaine, opioids including morphine, and some recreational drugs share the ability to cause drug dependence and addiction. Although these drugs of abuse primarily act on distinct molecular targets, such as monoamine transporters or receptors, they finally converge to common neural pathways. Several lines of evidence suggest that their chronic treatment leads to the enhancement of the mesocorticolimbic dopaminergic neurons from the ventral tegmental area (VTA) to the nucleus accumbens (NAc) and the medial prefrontal cortex (mPFC) and leads to abnormal glutamatergic function from the mPFC to the NAc and VTA. The neural adaptation of dopaminergic-glutamatergic system is considered to be critically implicated in neuropsychotoxic effects of these drugs of abuse. In addition, recent studies suggest that the serotonergic neurons from the raphe nuclei to limbic areas modulate the mesocorticolimbic dopaminergic-glutamatergic system and participate in the neuropsychotoxicity. In this review, our recent in vitro studies on the molecular targets and neural adaptation of methamphetamine, 3,4-methylenedioxymethanphetamine (MDMA, “ecstasy”), and 5-methoxy-N,N-diisopropyltryptamine (5-MeO-DiPT, “foxy”) using Xenopus oocytes, organotypic slice cultures of the mesocorticolimbic dopaminergic-glutamatergic system, and the raphe serotonergic system are introduced.

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Matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) function to remodel the pericellular environment. We have investigated the role of the MMP/TIMP system in methamphetamine (METH) dependence in rodents, in which the remodeling of neural circuits may be crucial. Repeated METH treatment induced behavioral sensitization, which was accompanied by an increase in MMP-2/-9/TIMP-2 activity in the brain. An antisense TIMP-2 oligonucleotide enhanced the sensitization, which was associated with a potentiation of the METH-induced release of dopamine in the nucleus accumbens (NAc). MMP-2/-9 inhibitors blocked the METH-induced behavioral sensitization and conditioned place preference (CPP), a measure of the rewarding effect of a drug, and reduced the METH-increased dopamine release in the NAc. In MMP-2– and MMP-9–deficient mice, METH-induced behavioral sensitization and CPP as well as dopamine release were attenuated. The MMP/TIMP system may be involved in METH-induced sensitization and reward by regulating extracellular dopamine levels.

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Repeated administration of psychostimulants elicits a progressive enhancement of locomotor activity known as behavioral sensitization. Central dopamine (DA) neurons play key roles as the neural substrates mediating behavioral sensitization, but the role of the serotonin (5-HT) system in the sensitization is not fully elucidated. We have recently demonstrated that osemozotan, a specific 5-HT_1A–receptor agonist, and ritanserin, a 5-HT₂–receptor antagonist, inhibited the expression and development of both methamphetamine- and cocaine-induced behavioral sensitization in mice and that these drugs attenuated the maintenance of behavioral sensitization of methamphetamine, but not that of cocaine. We also found that azasetron, a 5-HT₃–receptor antagonist, inhibited the expression and development of the sensitization induced by methamphetamine and cocaine, respectively. Neurochemical studies using a microdialysis technique showed that repeated methamphetamine enhanced the methamphetamine-induced increase in 5-HT release in the prefrontal cortex. The sensitization of 5-HT release in methamphetamine-treated mice was attenuated by osemozotan and ritanserin. These findings suggest that the 5-HT system plays an important role in methamphetamine- and cocaine-induced behavioral sensitization in mice and imply that 5-HT_1A–receptor agonists and 5-HT₂–receptor antagonists may have a potential therapeutic value for the treatment of methamphetamine abuse or psychosis.

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Drug abuse involving dextromethorphan, an antitussive, has been a social problem in various geographic locations since the 1960s. Ironically, high doses of the drug confer neuroprotective activity with central nervous system and behavioral effects. Accumulating evidence suggests that metabolism to phencyclidine-like dextrorphan is not essential for the neuroprotective activity of dextromethorphan. Here, we review the neuroprotective properties of dextromethorphan and its potential for abuse and the potential neuroprotective effects of the drug’s analogs and 3-hydroxymorphinan, a metabolite of dextromethorphan. These compounds may provide a novel therapeutic direction for the treatment of neurodegenerative diseases such as convulsive or parkinsonian-like disorders.

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Brain-pancreas relative protein (BPRP) is a novel protein that we found in our laboratory. Previously we demonstrated that it is involved in ischemia and depression. In light of the putative association between diabetes and clinical depression, and the selective expression of BPRP in brain and pancreas, the present study examined whether BPRP levels are affected by induction of diabetes by alloxan injection in rats and exposure to high glucose levels in PC12 cells. Western blot and immunohistochemical analyses revealed that BPRP levels were decreased in the hippocampal CA1 neurons of diabetic rats 4 and 8 weeks post-alloxan injection and in PC12 cells 48 h after exposure to high concentrations of glucose. BPRP protein levels were not affected by osmolarity control treatments with mannitol. Follow-up pharmacological experiments in PC12 cells revealed that glucose-induced BPRP down-regulation was markedly attenuated by the calpain inhibitors N-acetyl-Leu-Leu-norleucinal (ALLN) or calpeptin, but not the proteasome-specific inhibitor carbobenzoxy-Leu-Leu-leucinal (MG132). The ability of calpain inhibitors to specifically counter the effects of high glucose exposure on BPRP levels further suggests that BPRP and calpain activity may contribute to diabetes complications in the central nervous system.

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The action of fluoxetine, a serotonin reuptake inhibitor, on the cloned neuronal rat Kv3.1 channels stably expressed in Chinese hamster ovary cells was investigated using the whole-cell patch-clamp technique. Fluoxetine reduced Kv3.1 whole-cell currents in a reversible, concentration-dependent manner, with an IC₅₀ value and a Hill coefficient of 13.4 μM and 1.4, respectively. Fluoxetine accelerated the decay rate of inactivation of Kv3.1 currents without modifying the kinetics of current activation. The inhibition increased steeply between 0 and +30 mV, which corresponded with the voltage range for channel opening. In the voltage range positive to +30 mV, inhibition displayed a weak voltage dependence, consistent with an electrical distance δ of 0.38. The binding (k₊₁) and dissociation (k₋₁) rate constants for fluoxetine-induced block of Kv3.1 were 5.7 μM⁻¹s⁻¹ and 53.5 s⁻¹, respectively. The theoretical K_D value derived by k₋₁/k₊₁ yielded 9.3 μM. Fluoxetine did not affect the ion selectivity of Kv3.1. Fluoxetine slowed the deactivation time course, resulting in a tail crossover phenomenon when the tail currents, recorded in the presence and absence of fluoxetine, were superimposed. Inhibition of Kv3.1 by fluoxetine was use-dependent. The present results suggest that fluoxetine acts on Kv3.1 currents as an open-channel blocker.

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We attempted to assess the prophylactic effect of human umbilical cord blood–derived CD34⁺ cells in experimental heatstroke. Anesthetized rats, 1 day before heat stress, were divided into 2 major groups and given CD34⁻ cells (defined by 1 × 10⁶ human cord blood lymphocytes and monocytes that contained <0.2% CD34⁺ cells) or CD34⁺ cells (defined by 1 × 10⁶ human cord blood lymphocytes and monocytes that contained >95% CD34⁺ cells). They were exposed to ambient temperature of 43°C for 70 min to induce heatstroke. When the CD34⁻ cells–treated or untreated rats underwent heat stress, their survival time values were found to be 20 – 24 min. Pretreatment with CD34⁺ cells significantly increased survival time (123 – 351 min). As compared with normothermic controls, all CD34⁻ cells–treated heatstroke animals displayed hypotension, hepatic and renal failure, hypercoagulable state, activated inflammation, and cerebral ischemia and injury. However, these heatstroke reactions all were significantly suppressed by CD34⁺ cells pretreatment. In addition, the levels of interlukin-10 in plasma and glial cell line–derived neurotrophic factors in brain were all significantly increased after CD34⁺ cell administration during heatstroke. Our data indicate that human umbilical cord–derived CD34⁺ cells can be used as a prophylactic agent for experimental heatstroke.

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To elucidate the molecular mechanism of glomerular events in lupus nephritis, we performed genome-wide mRNA expression analysis of glomeruli microdissected from lupus mice. MRL/lpr mice (12-week-old) were orally given vehicle or prednisolone (10 mg/kg per day) for 4 weeks. Renal histology of MRL/lpr mice revealed mesangial proliferative glomerulonephritis with cellular infiltration of macrophages, T cells, and neutrophils. We identified 567 up-regulated genes in MRL/lpr glomeruli compared to control congenic mice. Those included complement components, adhesion molecules, chemokines and their receptors, and molecules related to antigen presentation. Over 130 genes were considered preferentially or exclusively expressed in hematopoietic cell lineages possibly reflecting leukocytes accumulation. Of note is the finding that chemokines and chemokine receptors (CCL3, CCL4, CCL5, CXCL9, CXCL10, CXCL11, CXCL16, CCR5, CXCR3, and CXCR6) that are related to T helper 1 (Th1) cells accumulation were up-regulated concomitantly with increased expression of Ebi3, a subunit of IL-27 that plays a role in Th1 predominance. These changes were accompanied by increased mRNA expression of many genes that were inducible by Th1 cytokine interferon-γ. Prednisolone markedly attenuated glomerular lesion and leukocyte influx parallel with the reduction of enhanced gene expression. The present study shows additional evidence supporting glomerular Th1 cells accumulation and their role. Our data also provide an important resource in seeking new therapeutic targets to lupus nephritis.
Supplemental table: available only at http://dx.doi.org/10.1254/jphs.FP0071337

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Previous work has shown that the Smilacis chinae rhizome (SCR) markedly inhibits amyloid β protein (25-35)–induced neuronal cell damage in cultured rat cortical neurons. The present study was conducted to further verify the neuroprotective effect of SCR on excitotoxic and cerebral ischemic injury using both in vitro and in vivo studies. Exposure of cultured cortical neurons to 1 mM N-methyl-D-aspartate (NMDA) for 12 h induced neuronal cell death. SCR (10 and 50 μg/ml) inhibited NMDA-induced neuronal death, elevation of intracellular calcium ([Ca²⁺]_i), and generation of reactive oxygen species (ROS) in primary cultures of rat cortical neurons. In vivo, SCR prevented cerebral ischemic injury induced by 3-h middle cerebral artery occlusion (MCAO) and 24-h reperfusion. The ischemic infarct was significantly reduced in rats that received SCR (30 and 50 mg/kg, orally), with a corresponding improvement in neurological function. Moreover, SCR treatment significantly decreased the histological changes observed following ischemia. Oxyresveratrol and resveratrol isolated from SCR also inhibited NMDA-induced neuronal death, increase in [Ca²⁺]_i, and ROS generation in cultured cortical neurons, suggesting that the neuroprotective effect of SCR may be attributable to these compounds. Taken together, these results suggest that the neuroprotective effect of SCR against focal cerebral ischemic injury is due to its anti-excitotoxic effects and that SCR may have a therapeutic role in neurodegenerative diseases such as stroke.

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The changes of electroencephalogram induced by maximal electroshock were studied in comparison with behavioral seizures in mice. After electroshock, mice showed tonic flexor (TF) seizure, tonic extensor (TE) seizure, and clonic (CL) seizure, in this order of occurrence. At the same time, high frequency spike or spike and wave complex in the cortex was observed, and thereafter, the frequency of the spike or spike and wave complex gradually fell. The antiepileptics used in the present study, except for ethosuximide, caused a dose-dependent shortening of the TE seizure duration. In addition, phenobarbital and carbamazepine at relatively high doses also showed a significant shortening of the duration of CL seizure. Although the duration of total electroencephalographic (EEG) seizures was not influenced by all the antiepileptics used in the present study, the duration of high frequency spike or spike and wave complex observed for 7 – 8 s after electroshock was dose-dependently shortened by all the antiepileptics used. From these findings, it may be concluded that high frequency spike or spike and wave complex induced by maximal electroshock is a useful index to assess antiepileptics that are effective in not only tonic-clonic seizures but also absence seizures in humans.

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The purpose of the present study was to examine the effect of beta-amyloid (Aβ) oligomers, not the fibrils that make up Aβ plaques, on spatial memory and the cholinergic system in rats. Recently, several researchers have suggested that small assemblies of Aβ, Aβ oligomers, caused memory loss during the early stages of Alzheimer’s disease without showing cell death. In the present study, the combination of Aβ oligomers and cerebral ischemia, but not cerebral ischemia alone, significantly impaired spatial memory without apoptosis in the CA1 region of the hippocampus. Donepezil, an acetylcholinesterase inhibitor, ameliorated this memory impairment. Therefore we examined acetylcholine (ACh) release from the dorsal hippocampus. A microdialysis study showed that spontaneous release of ACh was not significantly decreased by the combination of Aβ oligomers and cerebral ischemia; however, high K⁺-evoked ACh release was decreased. These results suggest that a combination of Aβ oligomers and cerebral ischemia induces memory impairment by cholinergic synapse dysfunction without apoptosis. This model may be useful for developing new drugs for the treatment of early-phase Alzheimer’s disease.

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The development of non-human primate models of asthma requires a period of time (e.g., 0.5 – 1 year). To develop the models in a short period, male cynomolgus monkeys were sensitized with dinitrophenyl-Ascaris suum (DNP-As) allergen by intraperitoneal and intramuscular injection and by intratracheal inhalation. All sensitized animals developed positive intradermal skin reaction to DNP-As. Sensitization elevated allergen-specific IgE levels in serum, the number of CCR4-positive T helper lymphocytes in peripheral blood, and IL-4 and IL-5 releases from phorbol 12-myristate 13-acetate- and ionomycin-stimulated peripheral blood. In addition, allergen challenge induced increases in lung resistance, airway inflammation, and hyperresponsiveness to inhaled methacholine. Next, animals were sensitized with house dust mite extracts (HDM) under the similar procedure. In these animals sensitized with DNP-As or HDM, inhaled fluticasone propionate and oral prednisolone inhibited the allergen-induced airway hyperresponsiveness. Taken together, monkey asthma models were successfully developed by sensitization with DNP-As or HDM under a short-term protocol (within 7 weeks). These models should be useful for the evaluation of anti-inflammatory drugs for asthma treatment.

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We examined the influence of imipramine, a traditional tricyclic antidepressant, on the binding to serotonin (5-HT)₂ receptors and levels of 5-HT_2A–receptor mRNA in the frontal cortex of rats treated with adrenocorticotropic hormone (ACTH). Chronic treatment with ACTH significantly increased the binding of [³H]-ketanserin to 5-HT₂ receptors and the expression of 5-HT_2A–receptor mRNA in the frontal cortex. However, it did not alter the concentration of 5-HT or 5-hydroxyindole acetic acid. The effect of chronic ACTH treatment on 5-HT₂ receptor and 5-HT_2A–receptor mRNA levels was not altered by the chronic administration of imipramine. Also, imipramine did not affect the hyperfunction of 5-HT_2A receptors caused by chronic ACTH treatment. These findings suggest that chronic treatment with ACTH acts to increase 5-HT_2A–receptor synthesis through increased gene transcription, without modulating presynaptic serotonergic neurotransmission.

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As TrkA, a high-affinity receptor of nerve growth factor (NGF), is a potential target for relieving uncontrolled inflammatory pain, an effective inhibitor of TrkA has been required for pain management. To identify a specific inhibitor of TrkA activity, we designed cell-penetrating peptides combined with amino-acid sequences in the activation loop of TrkA to antagonize tyrosine kinase activity. To select a peptide inhibiting TrkA activity, we examined the effect of cell-penetrating peptides on tyrosine kinase activity of recombinant TrkA in vitro and studied their effects on NGF-stimulated neurite outgrowth and protein phosphorylation in PC12 cells. Thereafter we investigated the effect of the selected peptide on NGF-stimulated TrkA activity and the expression of transient receptor potential channel 1 in PC12 cells. The selected peptide inhibited TrkA activity, but did not inhibit tyrosine kinase activities of other receptor-type tyrosine kinases in vitro. It also suppressed NGF-stimulated responses in PC12 cells. The selected synthetic cell-penetrating peptide antagonizing TrkA function would be a candidate for inflammatory pain therapy.

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Computer simulations of the human α_1d-adrenergic receptor (α_1d-AR) based on the crystal structure of rhodopsin have been combined with experimental site-directed mutagenesis to investigate the role of residues in the transmembrane domains in antagonist binding. Our results indicate that the amino acids Asp176 in the third transmembrane domain (TMD), Glu237 in TMD IV, and Ser258 in TMD V of α_1d-AR were directly involved in prazosin and tamsulosin binding. The Asp176Ala mutant did not exhibit any affinity for [³H]prazosin and neither did it show agonist-stimulated inositol phosphates (IP) formation. On the other hand, the Glu237Ala and Ser258Ala mutant α_1d-AR showed increased binding affinity for [³H]prazosin. Competition binding experiments showed that prazosin affinity had increased to 5-fold and 3-fold in the Glu237Ala and Ser258Ala mutants, respectively, versus wild-type; and tamsulosin affinity only increased in the Ser258Ala mutant (2-fold vs wild-type). It seems that these two residues constrain the receptor by interaction with other residues and this disruption of the interaction increased the receptor’s binding affinity towards antagonists. However, the Glu237Ala and Ser258Ala mutant receptors retained the ability to stimulate the formation of myo-[³H]inositol but had activities lower than that of the wild-type receptor. The present results provide direct evidence that these amino acid residues are responsible for the interactions between α_1d-AR and the radioligand [³H]prazosin as well as tamsulosin.

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The pharmacological properties of T-2328 were evaluated as an antagonist of the tachykinin neurokinin 1 (NK₁) receptor. T-2328 inhibited the specific binding of [³H][Sar⁹,Met(O₂)¹¹]substance P to tachykinin NK₁ receptors in human lymphoblastic IM9 cells with K_i of 0.08 nM. In the same assay, K_i for aprepitant, a brain-penetrating NK₁ antagonist, was 1.3 nM. The antagonism of T-2328 is highly selective for the human NK₁ receptors since the affinities for human NK₂, NK₃ receptors, and 13 other kinds of receptors and ion channels were >1000-fold lower than for NK₁ receptors. Reduction in Bmax with no change in affinity suggests the non-competitive nature of T-2328 interaction with the NK₁ receptor. T-2328 (0.03 – 0.1 mg/kg, i.v.) and aprepitant (1 – 3 mg/kg, i.v.) significantly prevented the GR73632 (i.c.v.)-induced foot tapping response in gerbils. The potencies of T-2328 in both in vitro and in vivo studies were more than 10 times greater than those of aprepitant. I.v. administration of T-2328 (0.1 – 0.3 mg/kg) potently blocked both acute and delayed emetic responses induced by cisplatin (5 mg/kg, i.p.) in ferrets. It is concluded that T-2328 is a potent, centrally active NK₁ antagonist. T-2328 may have potential as a novel therapeutic agent for the treatment of chemotherapy-induced emesis.

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Phosphatidylinositol (PI), a phospholipid in component of cell membranes, is widely distributed in animals, plants, and microorganisms. Here, we examined in vitro whether PI inhibits the angiogenesis induced by vascular endothelial growth factor-A (VEGF-A). PI concentration-relatedly and significantly (at 10 and 30 μg/ml) inhibited VEGF-A–induced tube formation in a co-culture of human umbilical vein endothelial cells (HUVECs) and fibroblasts. PI also inhibited the migration, but not proliferation, induced in HUVECs by VEGF-A. Furthermore, PI at 30 μg/ml inhibited the VEGF-A–induced phosphorylation of serine/threonine protein kinase family protein kinase B (Akt) and p38 mitogen activate kinase (p38MAPK), key molecules in cell migration, but not phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), a key molecule in cell proliferation. These findings indicate that PI inhibits VEGF-induced angiogenesis by inhibiting HUVECs migration and that inhibition of phosphorylated-Akt and -p38MAPK may be involved in the mechanism. Therefore, PI may be expected to prevent some diseases caused by angiogenesis.

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This study examined the effects of baicalin, a bioactive flavonoid isolated from Scutellariae Radix, on carbon tetrachloride (CCl₄)-induced liver injury. Mice were treated intraperitoneally with 0.5 ml/kg CCl₄ and different groups of animals received 25, 50, 100, and 200 mg/kg baicalin. At 24 h after the CCl₄ treatment, the level of serum aminotransferases and lipid peroxidation was significantly elevated, whereas the hepatic glutathione content was decreased. These changes were attenuated by baicalin. The histological studies showed that baicalin inhibited the portal inflammation, centrizonal necrosis, and Kupffer cell hyperplasia, which are the three most common characteristics of CCl₄-induced liver damage. The serum level and mRNA expression of tumor necrosis factor-α were markedly increased by the CCl₄ treatment but suppressed by baicalin. The mRNA and protein expression levels of inducible nitric oxide synthase and heme oxygenase-1 increased significantly at 24 h after the CCl₄ treatment. Baicalin attenuated the increase in the protein and gene expression of inducible nitric oxide synthase but augmented the increase in those of heme oxygenase-1. These findings suggest that baicalin protects hepatocytes from the oxidative damage caused by CCl₄, and this protection is likely due to the induction of HO-1 expression and the inhibition of the proinflammatory mediators.

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The purpose of this study was to establish a novel imaging system for evaluation of cataract in small animals. Diabetic cataract was induced in male Wistar rats by a combination of a bolus injection of streptozotocin (65 mg/kg, i.v.) and 5% D-glucose given as drinking water. To assess cataract development, we designed a digital camera system equipped with a non-reflecting illuminator for capturing clear high-resolution full lens images in the horizontal plane. Cataract was evaluated from the resulting images using both an observer-based scoring system and quantitative digital image-analysis techniques. The onset of cataract was detected in the peripheral section of the lens in 57% of cases 2 weeks after induction of hyperglycemia. Central opacities were visible following 3 weeks in hyperglycemic conditions. The cataract increased in severity with time, so that by week 9 in hyperglycemic conditions, mature cataracts were developed in over 90% of lenses. Treatment of diabetic rats with GP-1447, an aldose reductase inhibitor, completely prevented the formation of diabetic cataracts. These results indicate that the digital imaging system established in the present study permits an assessment of all stages of cataract development and it is helpful for accurately evaluating the effects of therapeutic drugs on cataracts.

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To reveal the involvement of extracellular nucleotides in the ossification process in ossification of the posterior longitudinal ligament of the spine (OPLL), the mRNA expression profiles of P2 purinoceptors, mechanical stress-induced ATP release, and ATP-stimulated expression of osteogenic genes were analyzed in ligament cells derived from the spinal ligament of OPLL patients (OPLL cells) and non-OPLL cells derived from the spinal ligaments of cervical spondylotic myelopathy patients as a control. The extracellular ATP concentrations of OPLL cells in static culture were significantly higher than those of non-OPLL cells, and this difference was diminished in the presence of ARL67156, an ecto-nuclease inhibitor. Cyclic stretch markedly increased the extracellular ATP concentrations of both cell types to almost the same level. P2Y1 purinoceptor subtypes were intensively expressed in OPLL cells, but only weakly expressed in non-OPLL cells. Not only ATP addition but also cyclic stretch raised the mRNA levels of alkaline phosphatase and osteopontin in OPLL cells, which were blocked by MRS2179, a selective P2Y1 antagonist. These increases in the expression of osteogenic genes were not observed in non-OPLL cells. These results suggest an important role of P2Y1 and extracellular ATP in the progression of OPLL stimulated by mechanical stress.

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Bee venom (BV) has been used in patients with rheumatoid arthritis, a condition characterized by rheumatoid joint destruction mediated, in large part, by matrix metalloproteinases (MMPs). We investigated the effects of melittin, a major component of bee venom, on the production of MMPs in human rheumatoid arthritic fibroblast-like synoviocytes (FLS). Lipopolysaccharide (LPS)-stimulated MMP3 production was significantly inhibited by melittin, which also inhibited LPS-induced DNA binding by nuclear factor κB (NF-κB). Mellitin had no effect on IL-1β– or TNF-α–induced MMP1 or MMP3 production and did not decrease LPS-induced secretion of MMP1. Taken together, these findings suggest that melittin may exert its anti-rheumatoid effects, at least in part, by inhibiting MMP3 production, most likely through inhibition of NF-κB activity.

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