Resveratrol ameliorates ortho- polychlorinated biphenyls' induced toxicity in ovary cells.

Polychlorinated biphenyls (PCBs) can induce chronic oxidative stress, inflammation, and cell death, leading to coronary heart disease, endothelial dysfunction, neurotoxicity, cancer, obesity, type 2 diabetes, reproductive dysfunction, etc. The aim of this study was to investigate possible protective effect of resveratrol (2.5-20 µM) in ovarian cells exposed to PCBs. An emphasis was on identifying mechanisms of resveratrol action upon distinct structure of the individual PCB congener-planar dioxin-like PCB 77 and non-planar di-ortho-substituted PCB 153. Multiple toxicity endpoint analysis was performed. Cell viability/proliferation was assessed by Trypan Blue exclusion method, Neutral Red, Kenacid Blue, and MTT bioassays. The level of oxidative stress was measured by fluorescent probes, and flow cytometry was applied to evaluate the mode of cell death. Resveratrol applied alone did not affect cell proliferation and viability in doses up to 20 µM, although significant antioxidative activity was observed. Toxic effects of ortho-PCB 153 (cytotoxicity, oxidative stress, and cell death) were mitigated by resveratrol. On the contrary pre-incubation with resveratrol did not result in cell viability protection when planar PCB 77 was applied. This indicates that resveratrol efficacy may be linked to specific structure of the individual congener, suggesting nutritional modulation of environmental insults caused by ortho-PCBs. We point out the importance of resveratrol dosage considering that synergistic cytotoxic effect with both PCB congeners is observed at concentrations ≥ 10 µM.

Dehydroepiandrosterone (DHEA): Pharmacological Effects and Potential Therapeutic Application.

Dehydroepiandrosterone (DHEA) is the most abundant steroid hormone in primates, which is predominantly synthesized in the adrenal cortex. A characteristic curve of growth and decline of its synthesis during life was observed, together with the corresponding formation of its sulphate ester (DHEAS). High levels of plasma circulating DHEA are suggested as a marker of human longevity, and various pathophysiological conditions lead to a decreased DHEA level, including adrenal insufficiency, severe systemic diseases, acute stress, and anorexia. More recent studies have established the importance of DHEA in the central nervous system (CNS). A specific intranuclear receptor for DHEA has not yet been identified; however, highly specific membrane receptors have been detected in endothelial cells, the heart, kidney, liver, and the brain. Research shows that DHEA and DHEAS, as well as their metabolites, have a wide range of effects on numerous organs and organ systems, which places them in the group of potential pharmacological agents useful in various clinical entities. Their action as neurosteroids is especially interesting due to potential neuroprotective, pro-cognitive, anxiolytic, and antidepressant effects. Evidence from clinical studies supports the use of DHEA in hypoadrenal individuals and in treating depression and associated cognitive disorders. However, there is also an increasing trend of recreational DHEA misuse in healthy people, as it is classified as a dietary supplement in some countries. This article aims to provide a critical review regarding the biological and pharmacological effects of DHEA, its mechanism of action, and potential therapeutic use, especially in CNS disorders.

Plasma lipidomics in subjects with combat posttraumatic stress disorder.

Posttraumatic stress disorder (PTSD) is complex neuropsychiatric disorder triggered by a traumatic event and characterized by the symptoms that represent large burden to patients, as well as to society. Lipidomic approach can be applied as a useful tool for discovery of novel diagnostic, prognostic and therapeutic lipid biomarkers of various disorders, whose etiology is complex and still unknown, including PTSD. Since changes in the levels of lipid metabolites might indicate impairments in various metabolic pathways and cellular processes, the aim of this lipidomic study was to determine altered levels of lipid compounds in PTSD. The study enrolled 235 male patients with combat PTSD and 241 healthy male control subjects. Targeted lipidomic analysis of plasma samples was conducted using reverse-phase liquid chromatography coupled with mass spectrometry. Lipids that have been analyzed belong to the group of ceramides, cholesterol esters, diacylglycerols, lysophosphatidylcholines, lysophosphatidylethanolamines, phosphatidylcholines, phosphatidylethanolamines, sphingomyelins and triglycerides. The levels of fifteen lipid compounds were found to be significantly different between PTSD patients and healthy control subjects, including four phosphatidylcholines, two phosphatidylethanolamines, five sphingomyelins, two cholesterol esters and two ceramides. The lipid metabolites whose levels significantly differed between patients with PTSD and control subjects are associated with various biological processes, including impairments of membrane integrity and function, mitochondrial dysfunction, inflammation and oxidative stress. As these processes might be associated with development and progression of PTSD, altered lipid compounds represent potential biomarkers that could facilitate the diagnosis of PTSD, prediction of the disease, as well as identification of novel treatment approaches in PTSD.

IL-1ß, IL-6, IL-10, and TNFα Single Nucleotide Polymorphisms in Human Influence the Susceptibility to Alzheimer's Disease Pathology.

BACKGROUND: Neuroinflammation plays an important role in Alzheimer's disease (AD). During this process, activated microglia release pro-inflammatory cytokines such as interleukin (IL)-1α, IL-1ß, IL-6, and tumor necrosis factor α (TNFα) that participate in neuron damage, but also anti-inflammatory cytokines (such as IL-10), which maintain homeostasis of immune response. Previous studies showed the association of IL-1α -889C/T (rs1800587), IL-1ß-1473G/C (rs1143623), IL-6 -174C/G (rs1800795), IL-10 -1082G/A (rs1800896), and TNFα -308A/G (rs1800629) polymorphisms with AD. OBJECTIVE: We aimed to investigate whether people with certain IL-1α, IL-1ß, IL-6, IL-10, and TNFα genotypes in these polymorphisms are more prone to develop AD-related pathology, reflected by pathological levels of cerebrospinal fluid (CSF) AD biomarkers including amyloid-ß1-42, total tau (t-tau), tau phosphorylated at Thr 181 (p-tau181), Ser 199 (p-tau199), and Thr 231 (p-tau231), and visinin-like protein 1 (VILIP-1). METHODS: The study included 115 AD patients, 53 patients with mild cognitive impairment, and 11 healthy controls. The polymorphisms were determined using real-time polymerase chain reaction. Levels of CSF biomarkers were determined by enzyme-linked immunosorbent assay. RESULTS: A significant increase in p-tau CSF levels was found in patients with the AA IL-10 -1082G/A and GG TNFα -308A/G genotypes, and in carriers of a G allele in IL-1ß -1473C/G and IL-6 -174C/G polymorphisms. t-tau levels were increased in carriers of a G allele in IL-1ß -1473C/G polymorphism. An increase in VILIP-1 levels was observed in patients with CG and GG IL-1ß -1473C/G, GC IL-6 -174C/G, and GG TNFα -308A/G genotype. CONCLUSION: These results suggest that persons carrying certain genotypes in IL10 (-1082G/A), IL1ß (1473C/G), IL6 (-174C/G), and TNFIα (-308A/G) could be more vulnerable to development of neuroinflammation, and consequently of AD.

Association between reduced brain-derived neurotrophic factor concentration & coronary heart disease.

Background & objectives: Brain-derived neurotrophic factor (BDNF) facilitates neuronal survival, differentiation and synaptic connectivity and affects neurotransmission throughout the brain. However, it has also a modulatory role in energy homeostasis, obesity and cardiovascular function. Obesity, high body mass index (BMI) and dyslipidaemia, among other factors, contribute to coronary heart disease (CHD) development. The exact role of BDNF in development of CHD is not well defined. This study was aimed to evaluate if plasma BDNF concentration was associated with CHD in ethnically homogeneous groups of patients and to correlate plasma BDNF levels with known risk factors for CHD. Methods: Plasma BDNF concentration, BDNF Val66Met polymorphism and other biological and anthropological risk factors for CHD were determined in 208 patients with CHD and 156 healthy controls. Results: Plasma BDNF concentration was significantly (P <0.01) reduced in patients with CHD compared to controls, and it was not influenced by gender, age, smoking or BDNF Val66Met polymorphism. It was considerably correlated with cholesterol (P=0.004), low-density lipoprotein (P=0.006), and diastolic blood pressure (P=0.018) in patients with CHD and with platelet number (P=0.003) in healthy controls. Interpretation & conclusions: The results revealed lower plasma BDNF concentration in patients with CHD, suggesting that decreased plasma BDNF concentration might be associated with CHD pathogenesis. Longitudinal studies with a large sample need to be conducted to confirm these findings.

Catechol-O-methyltransferase, Cognition and Alzheimer's Disease.

OBJECTIVE: Cognition is a complex trait representing a set of all mental abilities and processes related to knowledge. Although diverse brain regions are involved, most cognitive processes appear to engage cortical regions. The activity of dopaminergic neurons in prefrontal cortex represents a biological substrate underlying cognitive functions. Alzheimer's Disease (AD) is the most frequent dementia associated with cognitive impairments. Cognitive impairment in AD starts slowly with discrete deterioration in memory, language, thinking and reasoning, but it progresses into more severe and debilitating cognitive dysfunction. Cognitive function is affected by the complex interactions between various genetic, epigenetic, developmental and environmental factors. One of the most studied genes, associated with cognitive disturbances, is the gene coding for Catechol-O-methyltransferase (COMT), the enzyme with major role in dopamine metabolism and modulation of different brain functions. Therefore, COMT is studied as a target for many neuropsychiatric disorders, including dementias and AD. The COMT Val158/108Met functional polymorphism affects significantly the enzyme activity and consequently cognitive performance associated with altered dopamine function. The association of COMT Val158/108Met polymorphism with some cognitive domains and psychosis in AD was reported in some but not in all studies. Besides COMT Val158/108Met polymorphism, other risk genotypes or haplotypes should be evaluated to determine the association of COMT with cognitive decline in AD. CONCLUSION: Better understanding of the role of COMT in cognitive processes in AD, as well as integration of neurobiological, genetic, genomic and epigenetic data, might help in developing new potential therapies of cognitive impairments and psychotic symptoms, characteristic features of AD.

Theranostic Biomarkers for Schizophrenia.

Schizophrenia is a highly heritable, chronic, severe, disabling neurodevelopmental brain disorder with a heterogeneous genetic and neurobiological background, which is still poorly understood. To allow better diagnostic procedures and therapeutic strategies in schizophrenia patients, use of easy accessible biomarkers is suggested. The most frequently used biomarkers in schizophrenia are those associated with the neuroimmune and neuroendocrine system, metabolism, different neurotransmitter systems and neurotrophic factors. However, there are still no validated and reliable biomarkers in clinical use for schizophrenia. This review will address potential biomarkers in schizophrenia. It will discuss biomarkers in schizophrenia and propose the use of specific blood-based panels that will include a set of markers associated with immune processes, metabolic disorders, and neuroendocrine/neurotrophin/neurotransmitter alterations. The combination of different markers, or complex multi-marker panels, might help in the discrimination of patients with different underlying pathologies and in the better classification of the more homogenous groups. Therefore, the development of the diagnostic, prognostic and theranostic biomarkers is an urgent and an unmet need in psychiatry, with the aim of improving diagnosis, therapy monitoring, prediction of treatment outcome and focus on the personal medicine approach in order to improve the quality of life in patients with schizophrenia and decrease health costs worldwide.

Association of GABAA receptor α2 subunit gene (GABRA2) with alcohol dependence-related aggressive behavior.

Alcohol dependence is a common chronic disorder precipitated by the complex interaction between biological, genetic and environmental risk factors. Recent studies have demonstrated that polymorphisms of the gene encoding the GABAA receptor α2 subunit (GABRA2) are associated with alcohol dependence in different populations of European ancestry. As aggression often occurs in the context of alcohol dependence, the aim of this study was to examine the allelic and haplotypic association of GABRA2 gene with alcohol dependence and related aggressive behavior in subjects of Eastern European (Croatian) origin. Genotyping of the 3 single nucleotide polymorphisms (SNPs) across the GABRA2 gene (rs567926, rs279858 and rs9291283) was performed in patients with alcohol dependence (N=654) and healthy control subjects (N=574). Alcohol-dependent participants were additionally subdivided according to the presence/absence of aggressive behavior and type of alcohol dependence according to the Cloninger's classification. The association of rs279858 with alcohol dependence yielded nominal significance level. Haplotype analysis revealed a high degree of linkage disequilibrium (LD) for rs567926 and rs279858, but not for rs9291283 polymorphism in the GABRA2 gene. In patients with alcohol dependence, the A-C (rs567926 and rs279858) haplotype carriers were more likely to demonstrate aggressive behavior. The same haplotype (present only in 1.6% of all subjects) was significantly more often present in patients with a combination of early onset alcohol abuse and aggression, corresponding to the Cloninger's type II alcoholism subgroup. These findings support the involvement of GABRA2 gene in alcohol dependence-related aggressive behavior.

Neurotransmitter measures in the cerebrospinal fluid of patients with Alzheimer's disease: a review.

BACKGROUND: Alzheimer's disease (AD) is a severe neurodegenerative disorder characterized by progressive cognitive and functional decline, as well as by a variety of neuropsychiatric and psychological symptoms and behavioral dysfunctions. Various studies proposed the role of different neurotransmitter systems not only in AD-related cognitive, but also psychotic symptoms and behavioral and emotional deficits. Due to the close proximity, pathological neurochemical changes in brain occurring in AD are likely to be reflected in the cerebrospinal fluid (CSF). The purpose of this review is to provide a summary of the CSF neurotransmitter correlates of AD in order to get further insights into the potential role of altered neurotransmitters in the pathophysiology of AD and to offer novel AD biomarkers. METHODS: PubMed and MEDLINE data bases were searched for English-language articles by using "Alzheimer's disease", "CSF" and "neurotransmitter" as primary terms. No time or article type constraints were applied. Moreover, the lists of references were searched manually for additional articles. RESULTS: Changes in various correlates of cholinergic, monoaminergic and amino acid neurotransmitter systems, as well as neuropeptides, have been observed in CSF of AD patients. However, as the results of these studies have been controversial, the importance of CSF neurotransmitter parameters as potential biomarkers in AD remains quite unclear. The observed discrepancies could be bypassed by implementation of new sensitive methods, such as novel proteomics approaches that include protein separation techniques, mass spectroscopy and targeted multiplex panels of specific analytes. CONCLUSION: Although no individual CSF neurotransmitter correlate was demonstrated as suitable biomarker of AD, a combined profile of several CSF neurochemical parameters might show enhanced sensitivity and specificity and thus contribute to earlier and more accurate diagnosis of AD, crucial for application of effective treatments.

DMCM, a benzodiazepine site inverse agonist, improves active avoidance and motivation in the rat.

There are several modulatory sites at GABA(A) receptors, which mediate the actions of many drugs, among them benzodiazepine. Three kinds of allosteric modulators act through the benzodiazepine binding site: positive (agonist), neutral (antagonist), and negative (inverse agonist). The goal of the present study was to examine the influence of the inverse agonist methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) acting on α GABA(A) receptor and compare its dose-response effects on memory and depression-like behavior. We independently studied the effects of DMCM (0.05-1.0 mg/kg) on retention versus acquisition of active avoidance and depression-like behavior in the forced swim test. Throughout the study, drugs were given intraperitoneally, 30 min before testing. ANOVA has showed that treatment with DMCM significantly affected retrieval of avoidance response (p<0.05), exerted promnesic effects in inverted U-shape manner. Dunnett's test indicated that the DMCM avoidance-facilitatory dose was 0.1mg/kg. At the dose facilitating retrieval of avoidance memory, DMCM significantly (p<0.05, comparison of regression coefficients by Student's t-test) and progressively increased acquisition rate during 5 days training, compared to the saline group. In forced swim test, ANOVA indicated statistically significant effects of DMCM (p<0.05). Dunnett's analysis showed that DMCM significantly decreased immobility time at the dose of 0.1mg/kg, exerted acute antidepressant-like effects. Our results experimentally support the findings that under certain circumstances, nonselective benzodiazepine site inverse agonists, produce memory-enhancing and antidepressant-like effects. The molecular and neuronal substrates linking the actions of specific GABA-benzodiazepine receptor complex subunits remains to be further elucidated.

Differential effects of short- and long-term zolpidem treatment on recombinant α1ß2γ2s subtype of GABA(A) receptors in vitro.

AIM: Zolpidem is a non-benzodiazepine agonist at benzodiazepine binding site in GABA(A) receptors, which is increasingly prescribed. Recent studies suggest that prolonged zolpidem treatment induces tolerance. The aim of this study was to explore the adaptive changes in GABA(A) receptors following short and long-term exposure to zolpidem in vitro. METHODS: Human embryonic kidney (HEK) 293 cells stably expressing recombinant α1ß2γ2s GABA(A) receptors were exposed to zolpidem (1 and 10 µmol/L) for short-term (2 h daily for 1, 2, or 3 consecutive days) or long-term (continuously for 48 h). Radioligand binding studies were used to determine the parameters of [(3)H]flunitrazepam binding sites. RESULTS: A single (2 h) or repeated (2 h daily for 2 or 3 d) short-term exposure to zolpidem affected neither the maximum number of [(3)H]flunitrazepam binding sites nor the affinity. In both control and short-term zolpidem treated groups, addition of GABA (1 nmol/L-1 mmol/L) enhanced [(3)H]flunitrazepam binding in a concentration-dependent manner. The maximum enhancement of [(3)H]flunitrazepam binding in short-term zolpidem treated group was not significantly different from that in the control group. In contrast, long-term exposure to zolpidem resulted in significantly increase in the maximum number of [(3)H]flunitrazepam binding sites without changing the affinity. Furthermore, long-term exposure to zolpidem significantly decreased the ability of GABA to stimulate [(3)H]flunitrazepam binding. CONCLUSION: The results suggest that continuous, but not intermittent and short-term, zolpidem-exposure is able to induce adaptive changes in GABA(A) receptors that could be related to the development of tolerance and dependence.

The effects of zolpidem treatment and withdrawal on the in vitro expression of recombinant alpha1beta2gamma2s GABA(A) receptors expressed in HEK 293 cells.

Zolpidem, a widely used hypnotic drug which acts through benzodiazepine binding sites, is a positive allosteric modulator of gamma-aminobutyric acid (GABA) action with preferential affinity for GABA(A) receptors containing alpha1 subunit. The pharmacological profile of zolpidem is different from that of classical benzodiazepines. The aim of this study was to find out whether zolpidem treatment triggers adaptive changes in the recombinant alpha1 subunit-containing GABA(A) receptors other than those observed following treatment with classical benzodiazepine-diazepam. Radioligand binding studies showed that 2-day exposure of human embryonic kidney (HEK) 293 cells stably expressing recombinant alpha1beta2gamma2s GABA(A) receptors to zolpidem (10 muM) up-regulated the maximum number (B (max)) of [(3)H]flunitrazepam, [(3)H]muscimol, and [(3)H]t-butylbicycloorthobenzoate ([(3)H]TBOB) binding sites without changing their affinity (K (d)), suggesting an increase in total GABA(A) receptor number. Semi-quantitative RT-PCR analysis demonstrated increased levels of alpha1 subunit mRNA, while Western blot demonstrated up-regulated gamma2 subunit proteins, suggesting that zolpidem induced de novo synthesis of receptors proteins, at both the transcriptional and translational levels. GABA-induced potentiation of [(3)H]flunitrazepam binding to membranes obtained from zolpidem-treated cells was markedly reduced, indicating allosteric uncoupling between GABA and benzodiazepine binding sites. The number of benzodiazepine and convulsant binding sites as well as the functional coupling between GABA and benzodiazepine binding sites normalized in 24 h following discontinuation of zolpidem treatment. The results of our in vitro studies suggest that a 2-day exposure of recombinant alpha1 subunit-containing GABA(A) receptors stably transfected in HEK 293 cells to zolpidem induces adaptive changes in this selective GABA(A) receptor subtype, which are not substantially different from those obtained after prolonged exposure of cells to high concentrations of diazepam.

Sedative and anticonvulsant effects of zolpidem in adult and aged mice.

To evaluate the possible age-related differences in the behavioral effects of zolpidem, a widely used hypnotic, we compared the effects of zolpidem on the locomotor activity and on the seizure threshold for pentylenetetrazole (PTZ) and picrotoxin (given by i.v. infusion) between adult (3 months) and aged (13 months) mice. Zolpidem (10 mg/kg) produced similar enhancements of the seizure threshold in adult and aged mice. The drug was more potent against PTZ- than against picrotoxin-induced seizures. Diazepam (1 mg/kg), which was taken for comparison, had a weaker effect on picrotoxin-induced tonic seizures in aged than in adult mice. Sedative effect of zolpidem (10 mg/kg), as assessed by its effect on the locomotor activity, was very strong in both groups of mice. The results suggest that sedative and antiseizure effects of zolpidem are not changed in aged mice.

Interaction of diazepam and swim stress.

Many people take diazepam to prevent or counteract the consequences of exposure to different stressors. However, the interaction of diazepam and stress is not clear. Several studies have suggested that swim stress alters the properties of benzodiazepines. Both diazepam and swim stress have anticonvulsant properties. To determine whether swim stress alters the activity of diazepam, we tested the anticonvulsant effect of diazepam given in combination with swim stress. The mice were, prior to exposure to swim stress (10 or 5 min swimming at ambient temperature) and the intravenous infusion of picrotoxin, pre-treated with diazepam or allopregnanolone, or 24 h after exposure to stress treated with diazepam. The latency to the onset of clonic and tonic seizures was registered and the threshold for picrotoxin-induced clonic and tonic seizures calculated. In swim stressed (10 min) mice diazepam (0.5 and 1.0 mg/kg) enhanced the threshold for picrotoxin-induced tonic seizures more than in unstressed mice. The effect of allopregnanolone (20 mg/kg) in combination with 10-min swim stress on the threshold for picrotoxin-induced tonic seizures was also supraadditive. When diazepam (1 mg/kg) was given 20 min prior to 5-min swim stress, or 24 h after 10-min swim stress, there was no significant change in its anticonvulsant activity between unstressed and swim stressed mice. The results demonstrate that the anticonvulsant and possibly other behavioral properties of diazepam given in combination with swim stress can be either potentiated or unchanged, depending on the duration of stress and the time of diazepam administration in relation to stress.

Allosteric uncoupling and up-regulation of benzodiazepine and GABA recognition sites following chronic diazepam treatment of HEK 293 cells stably transfected with alpha1beta2gamma2S subunits of GABA (A) receptors.

Benzodiazepines are drugs known to produce tolerance and dependence and also to be abused and co-abused. The aim of this study was to further explore the mechanisms that underlie adaptive changes in GABA(A) receptors following prolonged exposure to these drugs. Human embryonic kidney (HEK 293) cells stably expressing recombinant alpha1beta2gamma2s GABA(A) receptors were exposed for 72 h to a high concentration of diazepam (50 microM) in the absence or presence of other drugs. Radioligand binding studies were used to determine the parameters of [(3)H]flunitrazepam and [(3)H]muscimol binding sites and allosteric interactions between these sites. Prolonged treatment with diazepam increased the maximum number (B (max)) of [(3)H]flunitrazepam and [(3)H]muscimol binding sites in the membranes, and of [(3)H]muscimol binding sites on the surface of HEK 293 cells. There was no change in the affinity (K (d)) of binding sites. The diazepam-induced increase in the B (max) value of [(3)H]flunitrazepam binding sites was reduced by two GABA(A) receptor antagonists, gabazine (1 and 10 microM) and picrotoxin (100 microM). In addition, it was reduced by cycloheximide (5 microg/ml), a protein synthesis inhibitor, and actinomycin D (7.5 microg/ml), an RNA synthesis inhibitor. Flumazenil (5 microM), the antagonist of benzodiazepine binding sites, also up-regulated [(3)H]flunitrazepam recognition sites. Simultaneous treatment with diazepam and flumazenil failed to produce an additive up-regulation. GABA (1 nM - 1 mM)-induced potentiation of [(3)H]flunitrazepam binding to membranes obtained from diazepam (50 microM)-pretreated cells was markedly reduced, suggesting functional uncoupling between GABA and benzodiazepine binding sites. The results suggest that diazepam up-regulated benzodiazepine binding sites on stably expressed GABA(A) receptors by stimulating their synthesis at both the transcriptional and translational levels. A comparable increase of [(3)H]muscimol binding sites expressed on the surface of intact HEK 293 cells suggests that internalisation of surface receptors presumably can not explain the uncoupling.

Chronic treatment with flumazenil enhances binding sites for convulsants at recombinant alpha(1)beta(2)gamma(2S) GABA(A) receptors.

GABA(A) receptors mediate most of the fast inhibitory neurotransmission in the brain. Prolonged occupancy of these receptors by ligands leads to regulatory changes often resulting in reduction of receptor function. The mechanism of these changes is still unknown. In this study, stably transfected human embryonic kidney (HEK) 293 cells were used as a model to study the effects of prolonged flumazenil (antagonist of benzodiazepine binding sites at GABA(A) receptors) exposure on the recombinant alpha(1)beta(2)gamma(2S) GABA(A) receptors, the most common type of GABA(A) receptors found in the brain. Exposure (48 h) of HEK 293 cells stably expressing recombinant alpha(1)beta(2)gamma(2S) GABA(A) receptors to flumazenil (1 or 5 microM) in the presence of GABA (1 microM), enhanced the maximum number (B(max)) without affecting the affinity (K(d)) of [(3)H]TBOB labeled binding sites for convulsants. Diazepam (1 nM-1 mM) in the presence of GABA (1 microM) modulated [(3)H]TBOB binding to control and flumazenil pretreated cells according to a two-site model. No significant differences between the groups were observed in either the potency or efficacy of diazepam to modulate [(3)H]TBOB binding, as evidenced by a lack of significant changes between their IC(50) and I(max) values. The results suggest that chronic exposure of HEK 293 cells stably expressing recombinant alpha(1)beta(2)gamma(2S) GABA(A) receptors to flumazenil up-regulates the binding sites for convulsants, but it does not appear to affect the functional coupling between these sites and benzodiazepine binding sites. Along with our recent data, these results suggest that chronic treatment with flumazenil enhances the number of GABA(A) receptors.

Chronic exposure of cells expressing recombinant GABAA receptors to benzodiazepine antagonist flumazenil enhances the maximum number of benzodiazepine binding sites.

The aim of this study was to better understand the mechanisms that underlie adaptive changes in GABAA receptors following their prolonged exposure to drugs. Exposure (48 h) of human embryonic kidney (HEK) 293 cells stably expressing recombinant alpha1beta2gamma2S GABAA receptors to flumazenil (1 or 5 microM) in the presence of GABA (1 microM) enhanced the maximum number (Bmax) of [3H]flunitrazepam binding sites without affecting their affinity (Kd). The flumazenil-induced enhancement in Bmax was not counteracted by diazepam (1 microM). GABA (1 nM-1 mM) enhanced [3H]flunitrazepam binding to membranes obtained from control and flumazenil-pretreated cells in a concentration-dependent manner. No significant differences were observed in either the potency (EC50) or efficacy (Emax) of GABA to potentiate [3H]flunitrazepam binding. However, in flumazenil pretreated cells the basal [3H]flunitrazepam and [3H]TBOB binding were markedly enhanced. GABA produced almost complete inhibition of [3H]TBOB binding to membranes obtained from control and flumazenil treated cells. The potencies of GABA to inhibit this binding, as shown by a lack of significant changes in the IC50 values, were not different between vehicle and drug treated cells. The results suggest that chronic exposure of HEK 293 cells stably expressing recombinant alpha1beta2gamma2S GABAA receptors to flumazenil (in the presence of GABA) up-regulates benzodiazepine and convulsant binding sites, but it does not affect the allosteric interactions between these sites and the GABA binding site. Further studies are needed to elucidate these phenomena.

Prolonged exposure to gamma-aminobutyric acid up-regulates stably expressed recombinant alpha 1 beta 2 gamma 2s GABAA receptors.

The aim of this study was to better understand the mechanisms that underlie adaptive changes in GABA(A) receptors following their prolonged exposure to drugs. Exposure (48 and/or 96 h) of human embryonic kidney (HEK 293) cells stably expressing recombinant alpha1beta2gamma2s GABA(A) receptors for gamma-aminobutyric (GABA, 1 mM) and muscimol (100 microM), but not for diazepam (1 microM), enhanced the maximum number (B(max)) of [3H]flunitrazepam binding sites without affecting their affinity (K(d)). The GABA-induced enhancement in B(max) was reduced by the GABA receptor antagonist, bicuculline (100 microM), and by cycloheximide (10 microl/ml), a protein synthesis inhibitor. GABA (100 microM) enhanced the affinity of [3H]flunitrazepam binding to vehicle- and GABA-pretreated, but not to diazepam-pretreated, HEK 293 cells. The results suggest that chronic GABA treatment up-regulates stably expressed GABA(A) receptors, presumably by stimulating their synthesis. Unlike chronic diazepam, which produced functional uncoupling of GABA and benzodiazepine binding sites, chronic GABA failed to produce this effect.