Monocyte and Lymphocyte Activation in Bipolar Disorder: A New Piece in the Puzzle of Immune Dysfunction in Mood Disorders

Background:

This study tested the hypothesis that the low-grade inflammation presented in patients with bipolar disorder (BD) is associated with expansion of activated T cells, and this activated state may be due to a lack of peripheral regulatory cells.

Methods:

Specifically, we investigated the distribution of monocytes and lymphocyte subsets, and investigated Th1/Th2/Th17 cytokines in plasma by flow cytometry. Twenty-one BD type I patients and 21 age- and sex-matched controls were recruited for this study.

Results:

BD patients had increased proportions of monocytes (CD14+). Regarding lymphocyte populations, BD patients presented reduced proportions of T cells (CD3+) and cytotoxic T cells (CD3+CD8+). BD patients also exhibited a higher percentage of activated T CD4+CD25+ cells, and a lower percentage of IL-10 expressing Treg cells.

Conclusions:

Our data shed some light into the underlying mechanisms involved with the chronic low-grade inflammatory profile described in BD patients.     

A pilot study of plasma metabolomic patterns from patients treated with ketamine for bipolar depression: evidence for a response-related difference in mitochondrial networks

Background and Purpose

(R,S)-ketamine produces rapid and significant antidepressant effects in approximately 65% of patients suffering from treatment-resistant bipolar depression (BD). The genetic, pharmacological and biochemical differences between ketamine responders and non-responders have not been identified. The purpose of this study was to employ a metabolomics approach, a global, non-targeted determination of endogenous metabolic patterns, to identify potential markers of ketamine response and non-response.

Experimental Approach

Plasma samples from 22 BD patients were analyzed to produce metabolomic patterns. The patients had received ketamine in a placebo-controlled crossover study and the samples were obtained 230 min post-administration at which time the patients were categorized as responders or non-responders. Matching plasma samples from the placebo arm of the study were also analysed. During the study, the patients were maintained on either lithium or valproate.

Key Results

The metabolomic patterns were significantly different between the patients maintained on lithium and those maintained on valproate, irrespective of response to ketamine. In the patients maintained on lithium, 18 biomarkers were identified. In responders, lysophosphatidylethanolamines (4) and lysophosphatidylcholines (9) were increased relative to non-responders.

Conclusions and Implications

The results indicate that the differences between patients who respond to ketamine and those who do not are due to alterations in the mitochondrial β-oxidation of fatty acids. These differences were not produced by ketamine administration. The data indicate that pretreatment metabolomics screening may be a guide to the prediction of response and a potential approach to the individualization of ketamine therapy.

Linked Articles

This article is part of a themed issue on Mitochondrial Pharmacology: Energy, Injury & Beyond. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2014.171.issue-8     

Protein Kinase C Inhibition Rescues Manic-Like Behaviors and Hippocampal Cell Proliferation Deficits in the Sleep Deprivation Model of Mania

Background:

Recent studies revealed that bipolar disorder may be associated with deficits of neuroplasticity. Additionally, accumulating evidence has implicated alterations of the intracellular signaling molecule protein kinase C (PKC) in mania.

Methods:

Using sleep deprivation (SD) as an animal model of mania, this study aimed to examine the possible relationship between PKC and neuroplasticity in mania. Rats were subjected to SD for 72h and tested behaviorally. In parallel, SD-induced changes in hippocampal cell proliferation were evaluated with bromodeoxyuridine (BrdU) labeling. We then examined the effects of the mood stabilizer lithium, the antipsychotic agent aripiprazole, and the PKC inhibitors chelerythrine and tamoxifen on both behavioral and cell proliferation impairments induced by SD. The antidepressant fluoxetine was used as a negative control.

Results:

We found that SD triggered the manic-like behaviors such as hyperlocomotion and increased sleep latency, and reduced hippocampal cell proliferation. These alterations were counteracted by an acute administration of lithium and aripiprazole but not of fluoxetine, and only a single administration of aripiprazole increased cell proliferation on its own. Importantly, SD rats exhibited increased levels of phosphorylated synaptosomal-associated protein 25 (SNAP-25) in the hippocampus and prefrontal cortex, suggesting PKC overactivity. Moreover, PKC inhibitors attenuated manic-like behaviors and rescued cell proliferation deficits induced by SD.

Conclusions:

Our findings confirm the relevance of SD as a model of mania, and provide evidence that antimanic agents are also able to prevent SD-induced decrease of hippocampal cell proliferation. Furthermore, they emphasize the therapeutic potential of PKC inhibitors, as revealed by their antimanic-like and pro-proliferative properties.     

Atomoxetine acts as an NMDA receptor blocker in clinically relevant concentrations

Background and purpose:

There is increasing evidence that not only the monoaminergic but also the glutamatergic system is involved in the pathophysiology of attention-deficit hyperactivity disorder (ADHD). Hyperactivity of glutamate metabolism might be causally related to a hypoactive state in the dopaminergic system. Atomoxetine, a selective noradrenaline reuptake inhibitor, is the first non-stimulant approved for the treatment of this disorder. Here we have evaluated the effects of atomoxetine on glutamate receptors in vitro.

Experimental approach:

The whole-cell configuration of the patch-clamp technique was used to analyse the effect of atomoxetine on N-methyl-d-aspartate (NMDA) receptors in cultured rodent cortical and hippocampal neurons as well as on NMDA receptors heterologously expressed in human TsA cells.

Key results:

Atomoxetine blocked NMDA-induced membrane currents. Half-maximal inhibition emerged at about 3 µM which is in the range of clinically relevant concentrations found in plasma of patients treated with this drug. The inhibition was voltage-dependent, indicating an open-channel blocking mechanism. Furthermore, the inhibitory potency of atomoxetine did not vary when measured on NMDA receptors from different brain regions or with different subunit compositions.

Conclusions and implications:

The effective NMDA receptor antagonism by atomoxetine at low micromolar concentrations may be relevant to its clinical effects in the treatment of ADHD. Our data provide further evidence that altered glutamatergic transmission might play a role in ADHD pathophysiology.     

Hypothalamic-Pituitary-Adrenal Axis Dysfunction and Illness Progression in Bipolar Disorder

Background:

Impaired stress resilience and a dysfunctional hypothalamic-pituitary-adrenal (HPA) axis are suggested to play key roles in the pathophysiology of illness progression in bipolar disorder (BD), but the mechanisms leading to this dysfunction have never been elucidated. This study aimed to examine HPA axis activity and underlying molecular mechanisms in patients with BD and unaffected siblings of BD patients.

Methods:

Twenty-four euthymic patients with BD, 18 siblings of BD patients, and 26 healthy controls were recruited for this study. All subjects underwent a dexamethasone suppression test followed by analyses associated with the HPA axis and the glucocorticoid receptor (GR).

Results:

Patients with BD, particularly those at a late stage of illness, presented increased salivary post-dexamethasone cortisol levels when compared to controls (p = 0.015). Accordingly, these patients presented reduced ex vivo GR responsiveness (p = 0.008) and increased basal protein levels of FK506-binding protein 51 (FKBP51, p = 0.012), a co-chaperone known to desensitize GR, in peripheral blood mononuclear cells. Moreover, BD patients presented increased methylation at the FK506-binding protein 5 (FKBP5) gene. BD siblings presented significantly lower FKBP51 protein levels than BD patients, even though no differences were found in FKBP5 basal mRNA levels.

Conclusions:

Our data suggest that the epigenetic modulation of the FKBP5 gene, along with increased FKBP51 levels, is associated with the GR hyporesponsiveness seen in BD patients. Our findings are consistent with the notion that unaffected first-degree relatives of BD patients share biological factors that influence the disorder, and that such changes are more pronounced in the late stages of the illness.     

Chronic Lithium Treatment Enhances the Number of Quiescent Neural Progenitors but Not the Number of DCX-Positive Immature Neurons

Background:

The term adult neurogenesis constitutes a series of developmental steps including the birth, survival, differentiation, maturation, and even death of newborn progenitor cells within neurogenic niches. Within the hippocampus progenitors reside in the neurogenic niche of the subgranular zone in the dentate gyrus subfield. At the different stages, designated type-I, type-IIa, type-IIb, type-III, and granule cell neurons, the cells express a series of markers enabling their identification and visualization. Lithium has been shown to increase hippocampal cell proliferation in the subgranular zone of the hippocampal dentate gyrus subfield of adult rodents and to stimulate the proliferation of hippocampal progenitor cells in vitro, but data regarding lithium’s ability to increase neuronal differentiation and survival is equivocal.

Methods:

To clarify the effect of lithium on adult hippocampal neurogenesis, we identified the effect of chronic lithium treatment on distinct stages of hippocampal progenitor development using adult Nestin-green fluorescent protein transgenic mice and immunofluorescent techniques.

Results:

The present observations confirm that lithium targets the initial stages of progenitor development enhancing the turnover of quiescent neural progenitors/putative stem-cells, corroborating previous reports. However, the enhanced quiescent neural progenitor-turnover does not translate into an increased number of immature neurons. We also observed a steep decline in the number of type-III immature neurons with complex tertiary-dendrites, suggesting that lithium alters the morphological maturation of newborn neurons.

Conclusions:

Our results do not corroborate previous reports of lithium-induced enhanced numbers of newly generated neurons.     

The N-Methyl-D-Aspartate Receptor as a Neurobiological Intersection Between Bipolar Disorder and Alcohol Use: A Longitudinal Mismatch Negativity Study

Background:

Comorbid risky alcohol use in bipolar disorder (BD) is recognized for its high prevalence and clinical relevance, though understanding of its neurobiological underpinning is limited. The N-methyl-D-aspartate (NMDA) receptor has recognized alterations in BD and is a major site of ethanol’s effects in the brain. The present study aimed to examine the NMDA receptor system in adolescents and young adults with BD by evaluating the longitudinal changes in a robust marker of NMDA function, mismatch negativity (MMN), in relation to changes in alcohol use patterns.

Methods:

Forty-six BD patients (aged 16–30) were recruited at baseline and 59% (n = 27) returned for follow-up 17.9 +/- 7.3 months later. At both time-points a two-tone, passive, duration-deviant MMN paradigm was conducted and alcohol measures were collected. Pearson’s correlations were performed between changes in MMN amplitudes and changes in alcohol use. Multiple regression was used to assess whether MMN amplitudes at baseline could predict alcohol use at follow-up.

Results:

Reduction in risky drinking patterns was associated with increased temporal MMN and decreased fronto-central MMN. Larger temporal MMN at baseline was a significant predictor of greater alcohol use at follow-up.

Conclusions:

Results suggest risky alcohol use in BD may further compound pre-existing NMDA receptor abnormalities and, importantly, reducing alcohol use early in stages of illness is associated with changes in MMN. This highlights the importance of monitoring alcohol use from first presentation. In addition, preliminary results present an exciting potential for utility of MMN as a neurobiological marker used to determine risk for alcohol misuse in BD.     

Treatment Implications of Predominant Polarity and the Polarity Index: A Comprehensive Review

Background:

Bipolar disorder (BD) is a serious and recurring condition that affects approximately 2.4% of the global population. About half of BD sufferers have an illness course characterized by either a manic or a depressive predominance. This predominant polarity in BD may be differentially associated with several clinical correlates. The concept of a polarity index (PI) has been recently proposed as an index of the antimanic versus antidepressive efficacy of various maintenance treatments for BD. Notwithstanding its potential clinical utility, predominant polarity was not included in the DSM-5 as a BD course specifier.

Methods:

Here we searched computerized databases for original clinical studies on the role of predominant polarity for selection of and response to pharmacological treatments for BD. Furthermore, we systematically searched the Pubmed database for maintenance randomized controlled trials (RCTs) for BD to determine the PI of the various pharmacological agents for BD.

Results:

We found support from naturalistic studies that bipolar patients with a predominantly depressive polarity are more likely to be treated with an antidepressive stabilization package, while BD patients with a manic-predominant polarity are more frequently treated with an antimanic stabilization package. Furthermore, predominantly manic BD patients received therapeutic regimens with a higher mean PI. The calculated PI varied from 0.4 (for lamotrigine) to 12.1 (for aripiprazole).

Conclusions:

This review supports the clinical relevance of predominant polarity as a course specifier for BD. Future studies should investigate the role of baseline, predominant polarity as an outcome predictor of BD maintenance RCTs.     

K(+) depolarization evokes ATP, adenosine and glutamate release from glia in rat hippocampus: a microelectrode biosensor study

BACKGROUND AND PURPOSE

This study was undertaken to characterize the ATP, adenosine and glutamate outflow evoked by depolarization with high K⁺ concentrations, in slices of rat hippocampus.

EXPERIMENTAL APPROACH

We utilized the microelectrode biosensor technique and extracellular electrophysiological recording for the real-time monitoring of the efflux of ATP, adenosine and glutamate.

KEY RESULTS

ATP, adenosine and glutamate sensors exhibited transient and reversible current during depolarization with 25 mM K⁺, with distinct kinetics. The ecto-ATPase inhibitor {"type":"entrez-protein","attrs":{"text":"ARL67156","term_id":"1186396857","term_text":"ARL67156"}}ARL67156 enhanced the extracellular level of ATP and inhibited the prolonged adenosine efflux, suggesting that generation of adenosine may derive from the extracellular breakdown of ATP. Stimulation-evoked ATP, adenosine and glutamate efflux was inhibited by tetrodotoxin, while exposure to Ca²⁺-free medium abolished ATP and adenosine efflux from hippocampal slices. Extracellular elevation of ATP and adenosine were decreased in the presence of NMDA receptor antagonists, D-AP-5 and ifenprodil, whereas non-NMDA receptor blockade by CNQX inhibited glutamate but not ATP and adenosine efflux. The gliotoxin fluoroacetate and P2X7 receptor antagonists inhibited the K⁺-evoked ATP, adenosine and glutamate efflux, while carbenoxolone in low concentration and probenecid decreased only the adenosine efflux.

CONCLUSIONS AND IMPLICATIONS

Our results demonstrated activity-dependent gliotransmitter release in the hippocampus in response to ongoing neuronal activity. ATP and glutamate were released by P2X7 receptor activation into extracellular space. Although the increased extracellular levels of adenosine did derive from released ATP, adenosine might also be released directly via pannexin hemichannels.

LINKED ARTICLE

This article is commented on by Sershen, pp. 1000–1002 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2012.02072.x     

Etomidate reduces glutamate uptake in rat cultured glial cells: involvement of PKA

Background and purpose:

Glutamate is the main excitatory neurotransmitter in the vertebrate CNS. Removal of the transmitter from the synaptic cleft by glial and neuronal glutamate transporters (GLTs) has an important function in terminating glutamatergic neurotransmission and neurological disorders. Five distinct excitatory amino-acid transporters have been characterized, among which the glial transporters excitatory amino-acid transporter 1 (EAAT1) (glutamate aspartate transporter) and EAAT2 (GLT1) are most important for the removal of extracellular glutamate. The purpose of this study was to describe the effect of the commonly used anaesthetic etomidate on glutamate uptake in cultures of glial cells.

Experimental approach:

The activity of the transporters was determined electrophysiologically using the whole cell configuration of the patch-clamp recording technique.

Key results:

Glutamate uptake was suppressed by etomidate (3–100 μM) in a time- and concentration-dependent manner with a half-maximum effect occurring at 2.4±0.6 μM. Maximum inhibition was approximately 50% with respect to the control. Etomidate led to a significant decrease of V_max whereas the K_m of the transporter was unaffected. In all cases, suppression of glutamate uptake was reversible within a few minutes upon washout. Furthermore, both GF 109203X, a nonselective inhibitor of PKs, and H89, a selective blocker of PKA, completely abolished the inhibitory effect of etomidate.

Conclusion and implications:

Inhibition of glutamate uptake by etomidate at clinically relevant concentrations may affect glutamatergic neurotransmission by increasing the glutamate concentration in the synaptic cleft and may compromise patients suffering from acute or chronic neurological disorders such as CNS trauma or epilepsy.     

Modulatory effects of neuropsychopharmaca on intracellular pH of hippocampal neurones in vitro

Background and purpose:

The intracellular pH (pHi) of neurones is tightly regulated by, for example, membrane-bound acid-exchangers and loaders. Nevertheless, excessive bioelectric activity lowers steady-state pHi. In turn, even a moderate acidification can inhibit neuronal activity, a process believed to be part of a negative feedback loop controlling neuronal excitation. As moclobemide, an antidepressant, and also some antiepileptic drugs can reduce neuronal pHi in hippocampus slices in vitro, we screened a panel of currently used neuropsychopharmaca for comparable effects.

Experimental approach:

BCECF-AM loaded hippocampal slices were superfused with 16 different neuroleptics, antidepressants and antiepileptics under bicarbonate-buffered conditions. Changes in steady-state pHi of CA3 neurones were measured fluorometrically.

Key results:

The antipsychotics haloperidol, clozapine, ziprasidone, and the antidepressants amitriptyline, doxepin, trimipramine, citalopram, mirtazapine, as well as the anticonvulsive drug tiagabine reversibly reduced the steady-state pHi by up to 0.35 pH-units in concentrations of 5–50 µM. In contrast, venlafaxine, the anticonvulsants carbamazepine, clonazepam, gabapentin, lamotrigine, zonisamide, and the mood stabilizer lithium had no effect on neuronal pHi.

Conclusion and implications:

These data substantiate the view that clinically relevant concentrations of neuroleptics and antidepressants can mediate changes in neuronal pHi, which may contribute to their pharmacological mode of action. Effects on pHi should be taken into account when therapeutic or even harmful effects of these drugs are evaluated.     

The Mood Stabilizer Lithium Potentiates the Antidepressant-Like Effects and Ameliorates Oxidative Stress Induced by Acute Ketamine in a Mouse Model of Stress

Background:

Evidence suggests that mammalian target of rapamycin activation mediates ketamine’s rapid but transient antidepressant effects and that glycogen synthase kinase-3β inhibits this pathway. However, ketamine has associated psychotomimetic effects and a high risk of abuse. The mood stabilizer lithium is a glycogen synthase kinase-3 inhibitor with strong antisuicidal properties. Here, we used a mouse stress model to investigate whether adjunct lithium treatment would potentiate ketamine’s antidepressant-like effects.

Methods:

Mice received chronic restraint stress and long-term pre- or postketamine lithium treatment in drinking water. The effects of lithium on ketamine-induced antidepressant-like effects, activation of the mammalian target of rapamycin/brain-derived neurotrophic factor signaling pathways, oxidative stress, and dendritic spine density in the brain of mice were investigated.

Results:

Subtherapeutic (600mg/L) lithium-pretreated mice exhibited an antidepressant-like response to an ineffective ketamine (2.5mg/kg, intraperitoneally) challenge in the forced swim test. Both the antidepressant-like effects and restoration of dendritic spine density in the medial prefrontal cortex of stressed mice induced by a single ketamine (50mg/kg) injection were sustained by postketamine treatment with 1200mg/L of lithium for at least 2 weeks. These benefits of lithium treatments were associated with activation of the mammalian target of rapamycin/brain-derived neurotrophic factor signaling pathways in the prefrontal cortex. Acute ketamine (50mg/kg) injection also significantly increased lipid peroxidation, catalase activity, and oxidized glutathione levels in stressed mice. Notably, these oxidative stress markers were completely abolished by pretreatment with 1200mg/L of lithium.

Conclusions:

Our results suggest a novel therapeutic strategy and justify the use of lithium in patients who benefit from ketamine.     

Hippocampal-Dependent Antidepressant Action of the H3 Receptor Antagonist Clobenpropit in a Rat Model of Depression

Background:

Histamine is a modulatory neurotransmitter regulating neuronal activity. Antidepressant drugs target modulatory neurotransmitters, thus ultimately regulating glutamatergic transmission and plasticity. Histamine H₃ receptor (H₃R) antagonists have both pro-cognitive and antidepressant effects; however, the mechanism by which they modulate glutamate transmission is not clear. We measured the effects of the H₃R antagonist clobenpropit in the Flinders Sensitive Line (FSL), a rat model of depression with impaired memory and altered glutamatergic transmission.

Methods:

Behavioral tests included the forced swim test, memory tasks (passive avoidance, novel object recognition tests), and anxiety-related paradigms (novelty suppressed feeding, social interaction, light/dark box tests). Hippocampal protein levels were detected by Western blot. Hippocampal plasticity was studied by in slice field recording of CA3-CA1 long-term synaptic potentiation (LTP), and glutamatergic transmission by whole-cell patch clamp recording of excitatory postsynaptic currents (EPSCs) in CA1 pyramidal neurons.

Results:

Clobenpropit, administered systemically or directly into the hippocampus, decreased immobility during the forced swim test; systemic injections reversed memory deficits and increased hippocampal GluN2A protein levels. FSL rats displayed anxiety-related behaviors not affected by clobenpropit treatment. Clobenpropit enhanced hippocampal plasticity, but did not affect EPSCs. H₁R and H₂R antagonists prevented the clobenpropit-induced increase in LTP and, injected locally into the hippocampus, blocked clobenpropit’s effect in the forced swim test.

Conclusions:

Clobenpropit’s antidepressant effects and the enhanced synaptic plasticity require hippocampal H₁R and H₂R activation, suggesting that clobenpropit acts through disinhibition of histamine release. Clobenpropit reverses memory deficits and increases hippocampal GluN2A expression without modifying anxiety-related phenotypes or EPSCs in CA1 pyramidal neurons.     

Influence of the hippocampus on amino acid utilizing and cholinergic neurons within the nucleus accumbens is promoted by histamine via H1 receptors

Background and Purpose

The influence of the neurotransmitter histamine on spontaneous and stimulation-evoked release of glutamate, aspartate, GABA and ACh in the nucleus accumbens (NAc) was investigated in vivo.

Experimental Approach

Using the push–pull superfusion technique, histaminergic compounds were applied to the NAc and neurotransmitter release was assessed. In some experiments, the fornix/fimbria of the hippocampus was electrically stimulated by a microelectrode and evoked potentials were monitored in the NAc.

Key Results

Superfusion of the NAc with the H₁ receptor antagonist triprolidine (50 μM) decreased spontaneous outflow of glutamate, aspartate and ACh, while release of GABA remained unaffected. Superfusion with histamine elevated release of ACh, without influencing that of the amino acids. Electrical stimulation of the fornix/fimbria enhanced the output of amino acids and ACh within the NAc. The evoked outflow of glutamate and ACh was diminished on superfusion with triprolidine, while release of aspartate and GABA was not affected. Superfusion of the NAc with histamine intensified the stimulation-evoked release of glutamate and Ach. Histamine also elevated the stimulation-induced release of aspartate, without influencing that of GABA. Presuperfusion with triprolidine abolished the reinforced effect of histamine on stimulation-evoked transmitter release within the NAc.

Conclusion and Implications

Neuronal histamine activates H₁ receptors and increases spontaneous release of glutamate, aspartate and ACh within the NAc. Stimulation of the hippocampal fornix/fimbria tract also enhances release of glutamate and ACh within the NAc and this effect is intensified by H₁ receptor stimulation within the NAc. The latter effects, which are mediated by hippocampal afferences, might play an important role in mnemonic performance and in emotional processes such as anxiety and stress disorders.

Linked Articles

This article is part of a themed issue on Histamine Pharmacology Update. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2013.170.issue-1     

Acute desipramine restores presynaptic cortical defects in murine experimental autoimmune encephalomyelitis by suppressing central CCL5 overproduction

Background and Purpose

Altered glutamate exocytosis and cAMP production in cortical terminals of experimental autoimmune encephalomyelitis (EAE) mice occur at the early stage of disease (13 days post-immunization, d.p.i.). Neuronal defects were paralleled by overexpression of the central chemokine CCL5 (also known as RANTES), suggesting it has a role in presynaptic impairments. We propose that drugs able to restore CCL5 content to physiological levels could also restore presynaptic defects. Because of its efficacy in controlling CCL5 overexpression, desipramine (DMI) appeared to be a suitable candidate to test our hypothesis.

Experimental Approach

Control and EAE mice at 13 d.p.i. were acutely or chronically administered DMI and monitored for behaviour and clinical scores. Noradrenaline and glutamate release, cAMP, CCL5 and TNF-α production were quantified in cortical synaptosomes and homogenates. Peripheral cytokine production was also determined.

Key Results

Noradrenaline exocytosis and α₂-adrenoeceptor-mediated activity were unmodified in EAE mice at 13 d.p.i. when compared with control. Acute, but not chronic, DMI reduced CCL5 levels in cortical homogenates of EAE mice at 13 d.p.i., but did not affect peripheral IL-17 and TNF-α contents or CCL5 plasma levels. Acute DMI caused a long-lasting restoration of glutamate exocytosis, restored endogenous cAMP production and impeded the shift from inhibition to facilitation of the CCL5-mediated control of glutamate exocytosis. Finally, DMI ameliorated anxiety-related behaviour but not motor activity or severity of clinical signs.

Conclusions

We propose DMI as an add-on therapy to normalize neuropsychiatric symptoms in multiple sclerosis patients at the early stage of the disease.     

Inhibition of endothelial cell Ca2+ entry and transient receptor potential channels by Sigma-1 receptor ligands

Background and Purpose

The Sigma-1 receptor (Sig1R) impacts on calcium ion signalling and has a plethora of ligands. This study investigated Sig1R and its ligands in relation to endogenous calcium events of endothelial cells and transient receptor potential (TRP) channels.

Experimental Approach

Intracellular calcium and patch clamp measurements were made from human saphenous vein endothelial cells and HEK 293 cells expressing exogenous human TRPC5, TRPM2 or TRPM3. Sig1R ligands were applied and short interfering RNA was used to deplete Sig1R. TRP channels tagged with fluorescent proteins were used for subcellular localization studies.

Key Results

In endothelial cells, 10–100 μM of the Sig1R antagonist BD1063 inhibited sustained but not transient calcium responses evoked by histamine. The Sig1R agonist 4-IBP and related antagonist BD1047 were also inhibitory. The Sig1R agonist {"type":"entrez-protein","attrs":{"text":"SKF10047","term_id":"1156210965","term_text":"SKF10047"}}SKF10047 had no effect. Sustained calcium entry evoked by VEGF or hydrogen peroxide was also inhibited by BD1063, BD1047 or 4-IBP, but not {"type":"entrez-protein","attrs":{"text":"SKF10047","term_id":"1156210965","term_text":"SKF10047"}}SKF10047. 4-IBP, BD1047 and BD1063 inhibited TRPC5 or TRPM3, but not TRPM2. Inhibitory effects of BD1047 were rapid in onset and readily reversed on washout. {"type":"entrez-protein","attrs":{"text":"SKF10047","term_id":"1156210965","term_text":"SKF10047"}}SKF10047 inhibited TRPC5 but not TRPM3 or TRPM2. Depletion of Sig1R did not prevent the inhibitory actions of BD1063 or BD1047 and Sig1R did not co-localize with TRPC5 or TRPM3.

Conclusions and Implications

The data suggest that two types of Sig1R ligand (BD1047/BD1063 and 4-IBP) are inhibitors of receptor- or chemically activated calcium entry channels, acting relatively directly and independently of the Sig1R. Chemical foundations for TRP channel inhibitors are suggested.     

A Placebo-Controlled Trial of Dextromethorphan as an Adjunct in Opioid-Dependent Patients Undergoing Methadone Maintenance Treatment

Background:

Low-dose dextromethorphan (DM) might have anti-inflammatory and neurotrophic effects mechanistically remote from an NMDA receptor. In a randomized, double-blind, controlled 12 week study, we investigated whether add-on dextromethorphan reduced cytokine levels and benefitted opioid-dependent patients undergoing methadone maintenance therapy (MMT).

Methods:

Patients were randomly assigned to a group: DM60 (60mg/day dextromethorphan; n = 65), DM120 (120mg/day dextromethorphan; n = 65), or placebo (n = 66). Primary outcomes were the methadone dose required, plasma morphine level, and retention in treatment. Plasma tumor necrosis factor (TNF)-α, C-reactive protein, interleukin (IL)-6, IL-8, transforming growth factor–β1, and brain-derived neurotrophic factor (BDNF) levels were examined during weeks 0, 1, 4, 8, and 12. Multiple linear regressions with generalized estimating equation methods were used to examine the therapeutic effect.

Results:

After 12 weeks, the DM60 group had significantly longer treatment retention and lower plasma morphine levels than did the placebo group. Plasma TNF-α was significantly decreased in the DM60 group compared to the placebo group. However, changes in plasma cytokine levels, BDNF levels, and the methadone dose required in the three groups were not significantly different.

Conclusions:

We provide evidence—decreased concomitant heroin use—of low-dose add-on DM’s efficacy for treating opioid-dependent patients undergoing MMT.     

Galectin-3 gene silencing inhibits migration and invasion of human tongue cancer cells in vitro via downregulating β-catenin

Aim:

Galectin-3 (Gal-3) is a member of the carbohydrate-binding protein family that contributes to neoplastic transformation, tumor survival, angiogenesis, and metastasis. The aim of this study is to investigate the role of Gal-3 in human tongue cancer progression.

Methods:

Human tongue cancer cell lines (SCC-4 and CAL27) were transfected with a small-interfering RNA against Gal-3 (Gal-3-siRNA). The migration and invasion of the cells were examined using a scratch assay and BD BioCoat Matrigel Invasion Chamber, respectively. The mRNA and protein levels of β-catenin, Akt/pAkt, GSK-3β/pGSK-3β, MMP-9 in the cells were measured using RT-PCR and Western blotting, respectively.

Results:

Transient silencing of Gal-3 gene for 48 h significantly suppressed the migration and invasion of both SCC-4 and CAL27 cells. Silencing of Gal-3 gene significantly decreased the protein level of β-catenin, leaving the mRNA level of β-catenin unaffected. Furthermore, silencing Gal-3 gene significantly decreased the levels of phosphorylated Akt and GSK-3β, and suppressed the mRNA and protein levels of MMP-9 in the cells.

Conclusion:

Our data suggest that Gal-3 mediates the migration and invasion of tongue cancer cells in vitro via regulating the Wnt/β-catenin signaling pathway and Akt phosphorylation.     

Stereoselective binding of doxazosin enantiomers to plasma proteins from rats,dogs and humans in vitro

Aim:

(±)Doxazosin is a long-lasting inhibitor of α1-adrenoceptors that is widely used to treat benign prostatic hyperplasia and lower urinary tract symptoms. In this study we investigated the stereoselective binding of doxazosin enantiomers to the plasma proteins of rats, dogs and humans in vitro.

Methods:

Human, dog and rat plasma were prepared. Equilibrium dialysis was used to determine the plasma protein binding of each enantiomer in vitro. Chiral HPLC with fluorescence detection was used to measure the drug concentrations on each side of the dialysis membrane bag.

Results:

Both the enantiomers were highly bound to the plasma proteins of rats, dogs and humans [(−)doxazosin: 89.4%–94.3%; (+)doxazosin: 90.9%–95.4%]. (+)Doxazosin exhibited significantly higher protein binding capacities than (−)doxazosin in all the three species, and the difference in the bound concentration (C_b) between the two enantiomers was enhanced as their concentrations were increased. Although the percentage of the plasma protein binding in the dog plasma was significantly lower than that in the human plasma at 400 and 800 ng/mL, the corrected percentage of plasma protein binding was dog>human>rat.

Conclusion:

(−)Doxazosin and (+)doxazosin show stereoselective plasma protein binding with a significant species difference among rats, dogs and humans.