Lipid raft integrity is required for human leukemia Jurkat T-cell migratory activity.

Lipid rafts are membrane microdomains featuring high cholesterol, sphingolipid, and protein content. These microdomains recruit various receptors, ion channels, and signaling molecules for coordination of various cellular functions, including synaptic transmission, immune response, cytoskeletal organization, adhesion, and migration. Many of these processes also depend on Ca2+ intake. We have previously shown in Jurkat cells that activity of transient receptor potential vanilloid, type 6 (TRPV6) calcium channel, and TRPV6-mediated Ca2+ influx, depend on lipid raft integrity. In this study, using the transwell cell migration assay and time-lapse video microscopy with Jurkat cells, we found that lipid raft destruction was associated with: inhibited cell adhesion and migration; and decreased mean speed, maximum speed, and trajectory length. Using String Server, we constructed a Protein Interaction Network (PIN). The network indicated that TRPV6 proteins interact with the highest probability (0.9) with Src family kinase members (SFKs) involved in processes related to cell migration. Analysis of detergent-resistant membrane fractions and immunoelectron microscopy data confirmed an association in lipid rafts between TRPV6 and Lck kinase, an SFKs member. Destruction of lipid rafts led to uncoupling of TRPV6 clusters with Lck and their departure from the plasma membrane into the cytosol of the cells. Src family kinases are generally associated with their roles in tumor invasion and progression, epithelial-mesenchymal transitions, angiogenesis, and metastatic development. We suggest that a functional interaction between TRPV6 calcium channels and SFKs members in lipid rafts is one of necessary elements of migration and oncogenic signaling in leukemia cells.

Sex differences and Tat expression affect dopaminergic receptor expression and response to antioxidant treatment in methamphetamine-sensitized HIV Tat transgenic mice.

Methamphetamine (Meth) abuse is a common HIV comorbidity. Males and females differ in their patterns of Meth use, associated behaviors, and responses, but the underlying mechanisms and impact of HIV infection are unclear. Transgenic mice with inducible HIV-1 Tat protein in the brain (iTat) replicate many neurological aspects of HIV infection in humans. We previously showed that Tat induction enhances the Meth sensitization response associated with perturbation of the dopaminergic system, in male iTat mice. Here, we used the iTat mouse model to investigate sex differences in individual and interactive effects of Tat and Meth challenge on locomotor sensitization, brain expression of dopamine receptors (DRDs) and regulatory adenosine receptors (ADORAs). Because Meth administration increases the production of reactive oxygen species (ROS), we also determined whether the effects of Meth could be rescued by concomitant treatment with the ROS scavenger N-acetyl cysteine (NAC). After Meth sensitization and a 7-day abstinence period, groups of Tat+ and Tat-male and female mice were challenged with Meth in combination with NAC. We confirmed that Tat expression and Meth challenge suppressed DRD mRNA and protein in males and females' brains, and showed that females were particularly susceptible to the effects of Meth on D1-like and D2-like DRD subtypes and ADORAs. The expression of these markers differed strikingly between males and females, and between females in different phases of the estrous cycle, in a Tat -dependent manner. NAC attenuated Meth-induced locomotor sensitization and preserved DRD expression in all groups except for Tat + females. These data identify complex interactions between sex, Meth use, and HIV infection on addiction responses, with potential implications for the treatment of male and female Meth users in the context of HIV, especially those with cognitive disorders.

Adenosine-5'-triphosphate suppresses proliferation and migration capacity of human endometrial stem cells.

Extracellular ATP through the activation of the P2X and P2Y purinergic receptors affects the migration, proliferation and differentiation of many types of cells, including stem cells. High plasticity, low immunogenicity and immunomodulation ability of mesenchymal stem cells derived from human endometrium (eMSCs) allow them to be considered a prominent tool for regenerative medicine. Here, we examined the role of ATP in the proliferation and migration of human eMSCs. Using a wound healing assay, we showed that ATP-induced activation of purinergic receptors suppressed the migration ability of eMSCs. We found the expression of one of the ATP receptors, the P2X7 receptor in eMSCs. In spite of this, cell activation with specific P2X7 receptor agonist, BzATP did not significantly affect the cell migration. The allosteric P2X7 receptor inhibitor, AZ10606120 also did not prevent ATP-induced inhibition of cell migration, confirming that inhibition occurs without P2X7 receptor involvement. Flow cytometry analysis showed that high concentrations of ATP did not have a cytotoxic effect on eMSCs. At the same time, ATP induced the cell cycle arrest, suppressed the proliferative and migration capacity of eMSCs and therefore could affect the regenerative potential of these cells.

Brain Reward Function after Chronic and Binge Methamphetamine Regimens in Mice Expressing the HIV-1 TAT Protein.

BACKGROUND: Methamphetamine abuse and human immunodeficiency virus (HIV) are common comorbidities. HIV-associated proteins, such as the regulatory protein TAT, may contribute to brain reward dysfunction, inducing an altered sensitivity to methamphetamine reward and/or withdrawal in this population. OBJECTIVE: These studies examined the combined effects of TAT protein expression and, chronic and binge methamphetamine regimens on brain reward function. METHODS: Transgenic mice with inducible brain expression of the TAT protein were exposed to either saline, a chronic, or a binge methamphetamine regimen. TAT expression was induced via doxycycline treatment during the last week of methamphetamine exposure. Brain reward function was assessed daily throughout the regimens, using the intracranial self-stimulation procedure, and after a subsequent acute methamphetamine challenge. RESULTS: Both methamphetamine regimens induced withdrawal-related decreases in reward function. TAT expression substantially, but not significantly increased the withdrawal associated with exposure to the binge regimen compared to the chronic regimen, but did not alter the response to acute methamphetamine challenge. TAT expression also led to persistent changes in adenosine 2B receptor expression in the caudate putamen, regardless of methamphetamine exposure. These results suggest that TAT expression may differentially affect brain reward function, dependent on the pattern of methamphetamine exposure. CONCLUSION: The subtle effects observed in these studies highlight that longer-term TAT expression, or its induction at earlier stages of methamphetamine exposure, may be more consequential at inducing behavioral and neurochemical effects.

Downregulation of TRPV6 channel activity by cholesterol depletion in Jurkat T cell line.

Transient receptor potential vanilloid 6 (TRPV6) channels are key players in calcium metabolism of healthy and cancerous cells. Nevertheless, the mechanisms controlling abundance of these channels in plasma membrane of the cells to regulate Ca2+ transport is still poorly understood. In this study, we provide the first evidence that TRPV6 calcium channels and Ca 2+ influx in Jurkat T cell line are modulated by cholesterol, a main lipid component of the plasma membrane. Using patch-clamp technique, we found that activity of TRPV6 channels decreased by cholesterol sequestration with methyl-ß-cyclodextrin (MßCD). Continuous measurement of intracellular Ca2+ revealed a reduction of Ca2+ influx into Jurkat cells following cholesterol depletion. Immunofluorescence and immunoelectron microscopy analyses of MßCD-treated cells detected the lower surface expression of the TRPV6 proteins in comparison with control cells. In general, our data showed that cholesterol regulates TRPV6 channel activity and TRPV6-mediated Ca2+ influx in cells, apparently affecting the localization and density of the calcium channels in the plasma membrane of Jurkat T cells.

Dopamine and its receptors play a role in the modulation of CCR5 expression in innate immune cells following exposure to Methamphetamine: Implications to HIV infection.

The Human Immunodeficiency Virus (HIV) infects cells in the Central Nervous System (CNS), where the access of antiretrovirals and antibodies that can kill the virus may be challenging. As a result of the early HIV entry in the brain, infected individuals develop inflammation and neurological deficits at various levels, which are aggravated by drugs of abuse. In the non-human primate model of HIV, we have previously shown that drugs of abuse such as Methamphetamine (Meth) increase brain viral load in correlation with a higher number of CCR5-expressing myeloid cells. CCR5 is a chemokine receptor that may be involved in increasing inflammation, but also, it is a co-receptor for viral entry into target cells. CCR5-expressing myeloid cells are the main targets of HIV in the CNS. Thus, the identification of factors and mechanisms that impact the expression of CCR5 in the brain is critical, as changes in CCR5 levels may affect the infection in the brain. Using a well-characterized in vitro system, with the THP1 human macrophage cell line, we have investigated the hypothesis that the expression of CCR5 is acutely affected by Meth, and examined pathways by which this effect could happen. We found that Meth plays a direct role by regulating the abundance and nuclear translocation of transcription factors with binding sites in the CCR5 promoter. However, we found that the main factor that modifies the CCR5 gene promoter at the epigenetic level towards transcription is Dopamine (DA), a neurotransmitter that is produced primarily in brain regions that are rich in dopaminergic neurons. In THP1 cells, the effect of DA on innate immune CCR5 transcription was mediated by DA receptors (DRDs), mainly DRD4. We also identified a role for DRD1 in suppressing CCR5 expression in this myeloid cell system, with potential implications for therapy. The effect of DA on innate immune CCR5 expression was also detectable on the cell surface during acute time-points, using low doses. In addition, HIV Tat acted by enhancing the surface expression of CCR5, in spite of its poor effect on transcription. Overall, our data suggests that the exposure of myeloid cells to Meth in the context of presence of HIV peptides such as Tat, may affect the number of HIV targets by modulating CCR5 expression, through a combination of DA-dependent and-independent mechanisms. Other drugs that increase DA may affect similar mechanisms. The implications of these epigenetic and translational mechanisms in enhancing HIV infection in the brain and elsewhere are demonstrated.

Differential effects of withdrawal from intermittent and continuous nicotine exposure on reward deficit and somatic aspects of nicotine withdrawal and expression of α4ß2* nAChRs in Wistar male rats.

BACKGROUND: Chronic nicotine exposure produces neuroadaptations in brain reward systems and α4ß2 nicotinic acetylcholine receptors (nAChRs) in the corticolimbic brain areas. We previously demonstrated opposite effects of nicotine exposure delivered by self-administration or pumps on brain reward thresholds that can be attributed to the different temporal pattern and contingency of nicotine exposure. We investigated the effects of these two factors on reward thresholds and somatic signs during nicotine withdrawal, and on nAChRs binding in corticolimbic brain areas. METHODS: The intracranial self-stimulation procedure was used to assess reward thresholds in rats prepared with pumps delivering various doses of nicotine continuously or intermittently. Separate group of rats were randomly exposed to nicotine via pumps (non-contingent) or nicotine self-administration (contingent) to determine [125I]-epibatidine binding at α4ß2* nAChRs. RESULTS: Withdrawal from continuous non-contingent nicotine exposure led to significant elevations in thresholds and increases in somatic signs in rats, while there was no significant effect of withdrawal from intermittent non-contingent nicotine exposure at the same doses. nAChRs were upregulated during withdrawal from continuous non-contingent nicotine exposure. α4ß2* nAChRs were upregulated in the ventral tegmental area and prelimbic cortex during withdrawal from non-contingent intermittent exposure and in the nucleus accumbens during withdrawal from contingent intermittent nicotine exposure to the same dose. CONCLUSIONS: During non-contingent nicotine exposure, the temporal pattern of nicotine delivery differentially affected thresholds and somatic signs of withdrawal. Upregulation of α4ß2* nAChRs was brain site-specific and depended on both temporal pattern and contingency of nicotine exposure.

Metabotropic Glutamate Receptor 5 as a Target for the Treatment of Depression and Smoking: Robust Preclinical Data but Inconclusive Clinical Efficacy.

The ability of novel pharmacological compounds to improve outcomes in preclinical models is often not translated into clinical efficacy. Psychiatric disorders do not have biological boundaries, and identifying mechanisms to improve the translational bottleneck between preclinical and clinical research domains is an important and challenging task. Glutamate transmission is disrupted in several neuropsychiatric disorders. Metabotropic glutamate (mGlu) receptors represent a diverse class of receptors that contribute to excitatory neurotransmission. Given the wide, yet region-specific manner of expression, developing pharmacological compounds to modulate mGlu receptor activity provides an opportunity to subtly and selectively modulate excitatory neurotransmission. This review focuses on the potential involvement of mGlu5 receptor disruption in major depressive disorder and substance and/or alcohol use disorders. We provide an overview of the justification of targeting mGlu5 receptors in the treatment of these disorders, summarize the preclinical evidence for negatively modulating mGlu5 receptors as a therapeutic target for major depressive disorders and nicotine dependence, and highlight the outcomes of recent clinical trials. While the evidence of mGlu5 receptor negative allosteric modulation has been promising in preclinical investigations, these beneficial effects have not translated into clinical efficacy. In this review, we identify key challenges that may contribute to poor clinical translation and provide suggested approaches moving forward to potentially improve the translation from preclinical to clinical domains. Such approaches may increase the success of clinical trials and may reduce the translational bottleneck that exists in drug discovery for psychiatric disorders.

Distribution, properties, and inhibitor sensitivity of zebrafish catechol-O-methyl transferases (COMT).

Catechol-O-methyltransferase (COMT; EC 2.1.1.6) is an enzyme with multiple functions in vertebrates. COMT methylates and thus inactivates catecholamine neurotransmitters and metabolizes xenobiotic catechols. Gene polymorphism rs4680 that influences the enzymatic activity of COMT affects cognition and behavior in humans. The zebrafish is widely used as an experimental animal in many areas of biomedical research, but most aspects of COMT function in this species have remained uncharacterized. We hypothesized that both comt genes play essential roles in zebrafish. Both comt-a and comt-b were widely expressed in zebrafish tissues, but their relative abundance varied considerably. Homogenates of zebrafish organs, including the brain, showed enzymatic COMT activity that was the highest in the liver and kidney. Treatment of larval zebrafish with the COMT inhibitor Ro41-0960 shifted the balance of catecholamine metabolic pathways towards increased oxidative metabolism. Whole-body concentrations of dioxyphenylacetic acid (DOPAC), a product of dopamine oxidation, were increased in the inhibitor-treated larvae, although the dopamine levels were unchanged. Thus, COMT is likely to participate in the processing of catecholamine neurotransmitters in the zebrafish, but the inhibition of COMT in larval fish is compensated efficiently and does not have pronounced effects on dopamine levels.

HIV-1 TAT protein enhances sensitization to methamphetamine by affecting dopaminergic function.

Methamphetamine abuse is common among humans with immunodeficiency virus (HIV). The HIV-1 regulatory protein TAT induces dysfunction of mesolimbic dopaminergic systems which may result in impaired reward processes and contribute to methamphetamine abuse. These studies investigated the impact of TAT expression on methamphetamine-induced locomotor sensitization, underlying changes in dopamine function and adenosine receptors in mesolimbic brain areas and neuroinflammation (microgliosis). Transgenic mice with doxycycline-induced TAT protein expression in the brain were tested for locomotor activity in response to repeated methamphetamine injections and methamphetamine challenge after a 7-day abstinence period. Dopamine function in the nucleus accumbens (Acb) was determined using high performance liquid chromatography. Expression of dopamine and/or adenosine A receptors (ADORA) in the Acb and caudate putamen (CPu) was assessed using RT-PCR and immunohistochemistry analyses. Microarrays with pathway analyses assessed dopamine and adenosine signaling in the CPu. Activity-dependent neurotransmitter switching of a reserve pool of non-dopaminergic neurons to a dopaminergic phenotype in the ventral tegmental area (VTA) was determined by immunohistochemistry and quantified with stereology. TAT expression enhanced methamphetamine-induced sensitization. TAT expression alone decreased striatal dopamine (D1, D2, D4, D5) and ADORA1A receptor expression, while increasing ADORA2A receptors expression. Moreover, TAT expression combined with methamphetamine exposure was associated with increased adenosine A receptors (ADORA1A) expression and increased recruitment of dopamine neurons in the VTA. TAT expression and methamphetamine exposure induced microglia activation with the largest effect after combined exposure. Our findings suggest that dopamine-adenosine receptor interactions and reserve pool neuronal recruitment may represent potential targets to develop new treatments for methamphetamine abuse in individuals with HIV.

Effects of HIV/TAT protein expression and chronic selegiline treatment on spatial memory, reversal learning and neurotransmitter levels in mice.

Neurotoxic viral protein TAT may contribute to deficits in dopaminergic and cognitive function in individuals infected with human immunodeficiency virus. Transgenic mice with brain-specific doxycycline-induced TAT expression (TAT+, TAT- control) show impaired cognition. However, previously reported TAT-induced deficits in reversal learning may be compromised by initial learning deficits. We investigated the effects of TAT expression on memory retention/recall and reversal learning, and neurotransmitter function. We also investigated if TAT-induced effects can be reversed by improving dopamine function with selegiline, a monoamine oxidase inhibitor. Mice were tested in the Barnes maze and TAT expression was induced after the task acquisition. Selegiline treatment continued throughout behavioral testing. Dopamine, serotonin and glutamate tissue levels in the prefrontal/orbitofrontal cortex, hippocampus and caudate putamen were measured using high performance liquid chromatography. Neither TAT expression nor selegiline altered memory retention. On day 2 of reversal learning testing, TAT+ mice made fewer errors and used more efficient search strategies than TAT- mice. TAT expression decreased dopamine turnover in the caudate putamen, increased serotonin turnover in the hippocampus and tended to increase the conversion of glutamate to glutamine in all regions. Selegiline decreased dopamine and serotonin metabolism in all regions and increased glutamate levels in the caudate putamen. In the absence of impaired learning, TAT expression does not impair spatial memory retention/recall, and actually facilitates reversal learning. Selegiline-induced increases in dopamine metabolism did not affect cognitive function. These findings suggest that TAT-induced alterations in glutamate signaling, but not alterations in monoamine metabolism, may underlie the facilitation of reversal learning.

TRPV5/V6 Channels Mediate Ca(2+) Influx in Jurkat T Cells Under the Control of Extracellular pH.

Regulation of cytoplasmic free calcium concentration [Ca(2+)]i is a key factor for the maintenance of cellular homeostasis in different cell types, including lymphocytes. During T lymphocyte activation as well as production of cytokines, sustained Ca(2+) influx is essential, however, it remains unclear how this influx is regulated. Previously, we reported the expression and functional activity of calcium channels TRPV5 and TRPV6 (transient receptor potential vanilloid type 5 and 6) in human leukemia Jurkat T cells. In this study, using single channel recordings, we found that activity of calcium channels TRPV5/V6 in Jurkat T cells is subject to strong control of external stimuli such as a low- or high-pH stressor. We showed that extracellular acidic pH reduces the activity of TRPV5/V6 channels, whereas alkaline pH increases the activity of TRPV5/V6 channels in Jurkat T cells. Using calcium imaging, we found that Ca(2+) influx in Jurkat T cells displayed sensitivity to extracellular pH, similar to that shown for the calcium channels TRPV5/V6. Double immunostaining of Jurkat T cells revealed that TRPV5 and TRPV6 channels colocalize with clathrin and the early endocytosis marker, EEA1. Moreover, we demonstrated that a specific inhibitor of clathrin-dependent endocytosis, dynasore, blocked TRPV5/V6 activity, and Ca(2+) influx into Jurkat T cells. Overall, our findings indicate that strong environmental cues may affect the intracellular calcium level in Jurkat T cells by influencing the traffic of TRPV5/V6 channels in lymphocytes.

Adolescent alcohol exposure decreased sensitivity to nicotine in adult Wistar rats.

Many adolescents engage in heavy alcohol use. Limited research in humans indicates that adolescent alcohol use predicts adult tobacco use. The present study investigated whether adolescent intermittent ethanol (AIE) exposure alters nicotine sensitivity in adulthood. Adolescent male Wistar rats (postnatal day 28-53) were exposed to AIE exposure that consisted of 5 g/kg of 25 percent ethanol three times per day in a 2 days on/2 days off regimen. Control rats received water with the same exposure regimen. In adulthood, separate groups of rats were tested for nicotine intravenous self-administration (IVSA), drug discrimination and conditioned taste aversion (CTA). The dose-response function for nicotine IVSA under a fixed-ratio schedule of reinforcement was similar in AIE-exposed and control rats. However, AIE-exposed rats self-administered less nicotine at the lowest dose, suggesting that low-dose nicotine was less reinforcing in AIE-exposed, compared with control rats. AIE-exposed rats self-administered less nicotine under a progressive-ratio schedule, suggesting decreased motivation for nicotine after AIE exposure. The discriminative stimulus effects of nicotine were diminished in AIE-exposed rats compared with control rats. No group differences in nicotine CTA were observed, suggesting that AIE exposure had no effect on the aversive properties of nicotine. Altogether, these results demonstrate that AIE exposure decreases sensitivity to the reinforcing, motivational and discriminative properties of nicotine while leaving the aversive properties of nicotine unaltered in adult rats. These findings suggest that drinking during adolescence may result in decreased sensitivity to nicotine in adult humans, which may in turn contribute to the higher rates of tobacco smoking.

Involvement of glutamatergic and GABAergic systems in nicotine dependence: Implications for novel pharmacotherapies for smoking cessation.

Tobacco smoking continues to be a major global health hazard despite significant public awareness of its harmful consequences. Although several treatment options are currently available for smoking cessation, these medications are effective in only a small subset of smokers, and relapse rates continue to be high. Therefore, a better understanding of the neurobiological mechanisms that mediate tobacco dependence is essential for the development of effective smoking cessation medications. Nicotine is the primary psychoactive component of tobacco that drives the harmful tobacco smoking habit. Nicotine binds to nicotinic acetylcholine receptors (nAChRs) in the brain, resulting in the release of a wide range of neurotransmitters, including glutamate and γ-aminobutyric acid (GABA). This review article focuses on the role of the excitatory glutamate system and inhibitory GABA system in nicotine dependence. Accumulating evidence suggests that blockade of glutamatergic transmission or facilitation of GABAergic transmission attenuates the positive reinforcing and incentive motivational aspects of nicotine, inhibits the reward-enhancing and conditioned rewarding effects of nicotine, and blocks nicotine-seeking behavior. Chronic nicotine exposure produced long-term neuroadaptations that contribute to nicotine withdrawal, but the role of GABA and glutamate transmission in nicotine withdrawal is less understood. Overall, the findings presented in this review provide strong converging evidence for the potential effectiveness of glutamatergic and GABAergic medications in nicotine dependence. This article is part of a Special Issue entitled 'NIDA 40th Anniversary Issue'.

Baseline impulsive choice predicts the effects of nicotine and nicotine withdrawal on impulsivity in rats.

Impulsive choice, a form of impulsivity, is associated with tobacco smoking in humans. Trait impulsivity may be a vulnerability factor for smoking, or smoking may lead to impulsive behaviors. We investigated the effects of 14-day nicotine exposure (6.32mg/kg/day base, subcutaneous minipumps) and spontaneous nicotine withdrawal on impulsive choice in low impulsive (LI) and high impulsive (HI) rats. Impulsive choice was measured in the delayed reward task in which rats choose between a small immediate reward and a large delayed reward. HI and LI rats were selected from the highest and lowest quartiles of the group before exposure to nicotine. In non-selected rats, nicotine or nicotine withdrawal had no effect on impulsive choice. In LI rats, chronic nicotine exposure decreased preference for the large reward with larger effects at longer delays, indicating increased impulsive choice. Impulsive choices for the smaller immediate rewards continued to increase during nicotine withdrawal in LI rats. In HI rats, nicotine exposure and nicotine withdrawal had no effect on impulsive choice, although there was a tendency for decreased preference for the large reward at short delays. These results indicate that nicotine- and nicotine withdrawal-induced increases in impulsive choice depend on trait impulsivity with more pronounced increases in impulsive choice in LI compared to HI subjects. Increased impulsivity during nicotine exposure may strengthen the addictive properties of nicotine and contribute to compulsive nicotine use.

Expression of transient receptor potential vanilloid channels TRPV5 and TRPV6 in human blood lymphocytes and Jurkat leukemia T cells.

Regulation of Ca(2+) entry is a key process for lymphocyte activation, cytokine synthesis and proliferation. Several members of the transient receptor potential (TRP) channel family can contribute to changes in [Ca(2+)](in); however, the properties and expression levels of these channels in human lymphocytes continue to be elusive. Here, we established and compared the expression of the most Ca(2+)-selective members of the TRPs, Ca(2+) channels transient receptor potential vanilloid 5 and 6 (TRPV5 and TRPV6), in human blood lymphocytes (HBLs) and leukemia Jurkat T cells. We found that TRPV6 and TRPV5 mRNAs are expressed in both Jurkat cells and quiescent HBLs; however, the levels of mRNAs were significantly higher in malignant cells than in quiescent lymphocytes. Western blot analysis showed TRPV5/V6 proteins in Jurkat T cells and TRPV5 protein in quiescent HBLs. However, the expression of TRPV6 protein was switched off in quiescent HBLs and turned on after mitogen stimulation of the cells with phytohemagglutinin. Inwardly directed monovalent currents that displayed characteristics of TRPV5/V6 currents were recorded in both Jurkat cells and normal HBLs. In outside-out patch-clamp studies, currents were reduced by ruthenium red, a nonspecific inhibitor of TRPV5/V6 channels. In addition, ruthenium red downregulated cell-cycle progression in both activated HBLs and Jurkat cells. Thus, we identified TRPV5 and TRPV6 calcium channels, which can be considered new candidates for Ca(2+) entry into human lymphocytes. The correlation between expression of TRPV6 channels and the proliferative status of lymphocytes suggests that TRPV6 may be involved in the physiological and/or pathological proliferation of lymphocytes.

Orally active metabotropic glutamate subtype 2 receptor positive allosteric modulators: structure-activity relationships and assessment in a rat model of nicotine dependence.

Compounds that modulate metabotropic glutamate subtype 2 (mGlu(2)) receptors have the potential to treat several disorders of the central nervous system (CNS) including drug dependence. Herein we describe the synthesis and structure-activity relationship (SAR) studies around a series of mGlu(2) receptor positive allosteric modulators (PAMs). The effects of N-substitution (R(1)) and substitutions on the aryl ring (R(2)) were identified as key areas for SAR exploration (Figure 3). Investigation of the effects of varying substituents in both the isoindolinone (2) and benzisothiazolone (3) series led to compounds with improved in vitro potency and/or efficacy. In addition, several analogues exhibited promising pharmacokinetic (PK) properties. Furthermore, compound 2 was shown to dose-dependently decrease nicotine self-administration in rats following oral administration. Our data, showing for the first time efficacy of an mGlu(2) receptor PAM in this in vivo model, suggest potential utility for the treatment of nicotine dependence in humans.

Mice lacking the ß4 subunit of the nicotinic acetylcholine receptor show memory deficits, altered anxiety- and depression-like behavior, and diminished nicotine-induced analgesia.

RATIONALE: The role of ß4-containing nicotinic acetylcholine receptors (nAChRs) in cognition, anxiety, depression, and analgesia in the absence of nicotine is unclear. METHODS: Wild-type (ß4(+/+)) and knockout (ß4(-/-)) mice for the nAChR ß4 subunit were tested in behavioral tests assessing cognitive function, affective behaviors, and nociception. RESULTS: There were no learning and memory deficits in ß4(-/-) mice compared with ß4(+/+) mice during the acquisition of the Barnes maze, contextual fear conditioning, and Y maze tasks. In the Barnes maze memory retention test, male ß4(-/-) mice showed reduced use of the spatial search strategy, indicating small spatial memory deficits compared with ß4(+/+) mice. In the cue-induced fear conditioning memory retention test, ß4(-/-) mice exhibited reduced freezing time compared with ß4(+/+) mice. Compared with ß4(+/+) mice, ß4(-/-) mice exhibited decreased anxiety-like behavior in the light-dark box. Depression-like behavior in ß4(-/-) mice was decreased in the tail suspension test and increased in the forced swim test compared with ß4(+/+) mice. ß4(-/-) mice did not differ from ß4(+/+) mice in basal nociception but were less sensitive to the antinociceptive effect of nicotine in 2 tests of acute thermal pain. CONCLUSIONS: Lack of ß4-containing nAChRs resulted in small deficits in hippocampus- and amygdala-dependent memory retention functions. ß4-containing nAChRs are involved in anxiety- and depression-like behaviors and contribute to the analgesic effects of nicotine.

The alpha2 adrenergic receptor antagonist idazoxan, but not the serotonin-2A receptor antagonist M100907, partially attenuated reward deficits associated with nicotine, but not amphetamine, withdrawal in rats.

Based on phenomenological similarities between anhedonia (reward deficits) associated with drug withdrawal and the negative symptoms of schizophrenia, we showed previously that the atypical antipsychotic clozapine attenuated reward deficits associated with psychostimulant withdrawal. Antagonism of alpha(2) adrenergic and 5-HT(2A) receptors may contribute to these effects of clozapine. We investigated here whether blockade of alpha(2) or 5-HT(2A) receptors by idazoxan and M100907, respectively, would reverse anhedonic aspects of psychostimulant withdrawal. Idazoxan treatment facilitated recovery from spontaneous nicotine, but not amphetamine, withdrawal by attenuating reward deficits and increase the number of somatic signs. Thus, alpha(2) adrenoceptor blockade may have beneficial effects against nicotine withdrawal and may be involved in the effects of clozapine previously observed. M100907 worsened the anhedonia associated with nicotine and amphetamine withdrawal, suggesting that monotherapy with M100907 may exacerbate the expression of the negative symptoms of schizophrenia or nicotine withdrawal symptoms in people, including schizophrenia patients, attempting to quit smoking.

Endogenous expression of TRPV5 and TRPV6 calcium channels in human leukemia K562 cells.

In blood cells, changes in intracellular Ca(2+) concentration ([Ca(2+)](i)) are associated with multiple cellular events, including activation of cellular kinases and phosphatases, degranulation, regulation of cytoskeleton binding proteins, transcriptional control, and modulation of surface receptors. Although there is no doubt as to the significance of Ca(2+) signaling in blood cells, there is sparse knowledge about the molecular identities of the plasmalemmal Ca(2+) permeable channels that control Ca(2+) fluxes across the plasma membrane and mediate changes in [Ca(2+)](i) in blood cells. Using RNA expression analysis, we have shown that human leukemia K562 cells endogenously coexpress transient receptor potential vanilloid channels type 5 (TRPV5) and type 6 (TRPV6) mRNAs. Moreover, we demonstrated that TRPV5 and TRPV6 channel proteins are present in both the total lysates and the crude membrane preparations from leukemia cells. Immunoprecipitation revealed that a physical interaction between TRPV5 and TRPV6 may take place. Single-channel patch-clamp experiments demonstrated the presence of inwardly rectifying monovalent currents that displayed kinetic characteristics of unitary TRPV5 and/or TRPV6 currents and were blocked by extracellular Ca(2+) and ruthenium red. Taken together, our data strongly indicate that human myeloid leukemia cells coexpress functional TRPV5 and TRPV6 calcium channels that may interact with each other and contribute into intracellular Ca(2+) signaling.