RESUMO
The pancreatic islets are essential microorgans controlling the glucose level in the blood. The islets consist of different cell types which communicate with each other by means of auto- and paracrine interactions. One of the communication molecules produced by and released within the islets is γ-aminobutyric acid (GABA), a well-known inhibitor of neuronal excitability in the mammalian nervous system. Interestingly, GABA is also present in the blood in the nanomolar concentration range. Thus, GABA can affect not only islet function per se (e.g. hormone secretion) but also interactions between immune cells and the pancreatic islet cells in physiological conditions and in pathological states (particularly in type 1 diabetes). In the last decade the interest in GABA signalling in islets has increased. The broad research scope ranges from fundamental physiological studies at the molecular and cellular level to pathological implications and clinical trials. The aim of this mini-review is to outline the current status of the islet GABA field mostly in relation to human islets, to identify the gaps in the current knowledge and what clinical implications GABA signalling may have in islets.
Assuntos
Diabetes Mellitus Tipo 1 , Ilhotas Pancreáticas , Animais , Humanos , Ilhotas Pancreáticas/metabolismo , Ácido gama-Aminobutírico/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Transdução de Sinais , Mamíferos/metabolismoRESUMO
A new series of dual low nanomolar benzothiazole inhibitors of bacterial DNA gyrase and topoisomerase IV were developed. The resulting compounds show excellent broad-spectrum antibacterial activities against Gram-positive Enterococcus faecalis, Enterococcus faecium and multidrug resistant (MDR) Staphylococcus aureus strains [best compound minimal inhibitory concentrations (MICs): range, <0.03125-0.25 µg/mL] and against the Gram-negatives Acinetobacter baumannii and Klebsiella pneumoniae (best compound MICs: range, 1-4 µg/mL). Lead compound 7a was identified with favorable solubility and plasma protein binding, good metabolic stability, selectivity for bacterial topoisomerases, and no toxicity issues. The crystal structure of 7a in complex with Pseudomonas aeruginosa GyrB24 revealed its binding mode at the ATP-binding site. Expanded profiling of 7a and 7h showed potent antibacterial activity against over 100 MDR and non-MDR strains of A. baumannii and several other Gram-positive and Gram-negative strains. Ultimately, in vivo efficacy of 7a in a mouse model of vancomycin-intermediate S. aureus thigh infection was also demonstrated.
Assuntos
Staphylococcus aureus , Staphylococcus aureus Resistente à Vancomicina , Animais , Camundongos , Staphylococcus aureus/metabolismo , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Antibacterianos/química , DNA Girase/metabolismo , DNA Topoisomerase IV , Testes de Sensibilidade MicrobianaRESUMO
We have developed compounds with a promising activity against Acinetobacter baumannii and Pseudomonas aeruginosa, which are both on the WHO priority list of antibiotic-resistant bacteria. Starting from DNA gyrase inhibitor 1, we identified compound 27, featuring a 10-fold improved aqueous solubility, a 10-fold improved inhibition of topoisomerase IV from A. baumannii and P. aeruginosa, a 10-fold decreased inhibition of human topoisomerase IIα, and no cross-resistance to novobiocin. Cocrystal structures of 1 in complex with Escherichia coli GyrB24 and (S)-27 in complex with A. baumannii GyrB23 and P. aeruginosa GyrB24 revealed their binding to the ATP-binding pocket of the GyrB subunit. In further optimization steps, solubility, plasma free fraction, and other ADME properties of 27 were improved by fine-tuning of lipophilicity. In particular, analogs of 27 with retained anti-Gram-negative activity and improved plasma free fraction were identified. The series was found to be nongenotoxic, nonmutagenic, devoid of mitochondrial toxicity, and possessed no ion channel liabilities.
Assuntos
Acinetobacter baumannii , Inibidores da Topoisomerase II , Humanos , Inibidores da Topoisomerase II/farmacologia , Inibidores da Topoisomerase II/química , Pseudomonas aeruginosa/metabolismo , Acinetobacter baumannii/metabolismo , Antibacterianos/farmacologia , Antibacterianos/química , Escherichia coli/metabolismo , Benzotiazóis , Testes de Sensibilidade Microbiana , DNA Girase/metabolismoRESUMO
Mowat-Wilson syndrome (MWS) is a severe neurodevelopmental disorder caused by heterozygous variants in the gene encoding transcription factor ZEB2. Affected individuals present with structural brain abnormalities, speech delay and epilepsy. In mice, conditional loss of Zeb2 causes hippocampal degeneration, altered migration and differentiation of GABAergic interneurons, a heterogeneous population of mainly inhibitory neurons of importance for maintaining normal excitability. To get insights into GABAergic development and function in MWS we investigated ZEB2 haploinsufficient induced pluripotent stem cells (iPSC) of MWS subjects together with iPSC of healthy donors. Analysis of RNA-sequencing data at two time points of GABAergic development revealed an attenuated interneuronal identity in MWS subject derived iPSC with enrichment of differentially expressed genes required for transcriptional regulation, cell fate transition and forebrain patterning. The ZEB2 haploinsufficient neural stem cells (NSCs) showed downregulation of genes required for ventral telencephalon specification, such as FOXG1, accompanied by an impaired migratory capacity. Further differentiation into GABAergic interneuronal cells uncovered upregulation of transcription factors promoting pallial and excitatory neurons whereas cortical markers were downregulated. The differentially expressed genes formed a neural protein-protein network with extensive connections to well-established epilepsy genes. Analysis of electrophysiological properties in ZEB2 haploinsufficient GABAergic cells revealed overt perturbations manifested as impaired firing of repeated action potentials. Our iPSC model of ZEB2 haploinsufficient GABAergic development thus uncovers a dysregulated gene network leading to immature interneurons with mixed identity and altered electrophysiological properties, suggesting mechanisms contributing to the neuropathogenesis and seizures in MWS.
RESUMO
AIM: We examined if tonic γ-aminobutyric acid (GABA)-activated currents in primary hippocampal neurons were modulated by insulin in wild-type and tg-APPSwe mice, an Alzheimer's disease (AD) model. METHODS: GABA-activated currents were recorded in dentate gyrus (DG) granule cells and CA3 pyramidal neurons in hippocampal brain slices, from 8 to 10 weeks old (young) wild-type mice and in dorsal DG granule cells in adult, 5-6 and 10-12 (aged) months old wild-type and tg-APPSwe mice, in the absence or presence of insulin, by whole-cell patch-clamp electrophysiology. RESULTS: In young mice, insulin (1 nmol/L) enhanced the total spontaneous inhibitory postsynaptic current (sIPSCT ) density in both dorsal and ventral DG granule cells. The extrasynaptic current density was only increased by insulin in dorsal CA3 pyramidal neurons. In absence of action potentials, insulin enhanced DG granule cells and dorsal CA3 pyramidal neurons miniature IPSC (mIPSC) frequency, consistent with insulin regulation of presynaptic GABA release. sIPSCT densities in DG granule cells were similar in wild-type and tg-APPSwe mice at 5-6 months but significantly decreased in aged tg-APPSwe mice where insulin normalized currents to wild-type levels. The extrasynaptic current density was increased in tg-APPSwe mice relative to wild-type littermates but, only in aged tg-APPSwe mice did insulin decrease and normalize the current. CONCLUSION: Insulin effects on GABA signalling in hippocampal neurons are selective while multifaceted and context-based. Not only is the response to insulin related to cell-type, hippocampal axis-location, age of animals and disease but also to the subtype of neuronal inhibition involved, synaptic or extrasynaptic GABAA receptors-activated currents.
Assuntos
Doença de Alzheimer , Animais , Hipocampo , Insulina/farmacologia , Camundongos , Neurônios/metabolismo , Receptores de GABA-A/metabolismo , Ácido gama-AminobutíricoRESUMO
The hippocampus is a medial temporal lobe structure in the brain and is widely studied for its role in memory and learning, in particular, spacial memory and emotional responses. It was thought to be a homogenous structure but emerging evidence shows differentiation along the dorsoventral axis and even microdomains for functional and cellular markers. We have examined in two cell-types of the hippocampal projection neurons, the dentate gyrus (DG) granule cells and CA3 pyramidal neurons, if the GABA-activated tonic current density varied between the dorsal (septal) and the ventral (temporal) poles of the male mouse hippocampus. Tonic synaptic currents, arising from spontaneous and miniature inhibitory postsynaptic currents (sIPSC, mIPSC), and extrasynaptic tonic currents were evaluated. The results revealed different levels of sIPSC but not mIPSC density between the dorsal and ventral hippocampal neurons for both the DG granule cells and the CA3 pyramidal neurons. The extrasynaptic tonic current density was larger in the DG granule cells as compared to the CA3 pyramidal neurons but did not vary between the dorsal and ventral regions. IPSC bursting was observed in both cell-types in the ventral hippocampus but was more common in the CA3 pyramidal neurons. Only in the dorsal DG granule cells was the level of the sIPSC and mIPSC density similar. The results indicate that the tonic GABAergic inhibition is particularly strong in the ventral hippocampal DG granule cells and enhanced in the dorsal as compared to the ventral hippocampal CA3 pyramidal neurons.
Assuntos
Região CA3 Hipocampal/fisiologia , Giro Denteado/fisiologia , Potenciais Pós-Sinápticos Inibidores/fisiologia , Células Piramidais/fisiologia , Receptores de GABA-A/fisiologia , Sinapses/fisiologia , Animais , Fórnice/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Técnicas de Cultura de Órgãos , Potenciais Sinápticos/fisiologia , Ácido gama-Aminobutírico/fisiologiaRESUMO
In pancreatic islets, the major cell-types are α, ß and δ cells. The γ-aminobutyric acid (GABA) signalling system is expressed in human pancreatic islets. In single hormone transcript-expressing cells, we have previously characterized the functional properties of islet GABAA receptors (iGABAARs). Here, we extended these studies to islet cells expressing mRNAs for more than one hormone and sought for correlation between iGABAAR activity level and relative mRNA expression ratio. The single-cell RT-PCR in combination with the patch-clamp current recordings was used to examine functional properties of iGABAARs in the multiple hormone mRNA-expressing cells. We detected cells expressing double (α/ß, α/δ, ß/δ cell-types) and triple (α/ß/δ cell-type) hormone transcripts. The most common mixed-identity cell-type was the α/ß group where the cells could be grouped into ß- and α-like subgroups. The ß-like cells had low GCG/INS expression ratio (<0.6) and significantly higher frequency of iGABAAR single-channel openings than the α-like cells where the GCG/INS expression ratio was high (>1.2). The hormone expression levels and iGABAAR single-channel characteristics varied in the α/ß/δ cell-type. Clearly, multiple hormone transcripts can be expressed in islet cells whereas iGABAAR single-channel functional properties appear to be α or ß cell specific.
Assuntos
Diabetes Mellitus Tipo 2/fisiopatologia , Ativação do Canal Iônico , Ilhotas Pancreáticas/fisiologia , Hormônios Pancreáticos/metabolismo , RNA Mensageiro/metabolismo , Receptores de GABA-A/metabolismo , Estudos de Casos e Controles , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Humanos , Ilhotas Pancreáticas/citologia , Hormônios Pancreáticos/genética , RNA Mensageiro/genética , Receptores de GABA-A/genética , Transdução de Sinais , Ácido gama-Aminobutírico/metabolismoRESUMO
Immunomodulation is increasingly being recognised as a part of mental diseases. Here, we examined whether levels of immunological protein markers changed with depression, age, or the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). An analysis of plasma samples from patients with a major depressive episode and control blood donors (CBD) revealed the expression of 67 inflammatory markers. Thirteen of these markers displayed augmented levels in patients compared to CBD. Twenty-one markers correlated with the age of the patients, whereas 10 markers correlated with the age of CBD. Interestingly, CST5 and CDCP1 showed the strongest correlation with age in the patients and CBD, respectively. IL-18 was the only marker that correlated with the MADRS-S scores of the patients. Neuronal growth factors (NGFs) were significantly enhanced in plasma from the patients, as was the average plasma GABA concentration. GABA modulated the release of seven cytokines in anti-CD3-stimulated peripheral blood mononuclear cells (PBMCs) from the patients. The study reveals significant changes in the plasma composition of small molecules during depression and identifies potential peripheral biomarkers of the disease.
Assuntos
Biomarcadores/sangue , Biomarcadores/metabolismo , Transtorno Depressivo Maior/sangue , Transtorno Depressivo Maior/metabolismo , Inflamação/sangue , Inflamação/metabolismo , Leucócitos Mononucleares/metabolismo , Ácido gama-Aminobutírico/metabolismo , Adulto , Fatores Etários , Idoso , Depressão , Transtorno Depressivo Maior/imunologia , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
The neural transmission and plasticity can be differentially modulated by various elements of the immune system. Interferon-γ (IFN-γ) is a "pro-inflammatory" cytokine mainly produced by T lymphocytes, activates its corresponding receptor and plays important roles under both homeostatic and inflammatory conditions. However, the impact of IFN-γ on the γ-aminobutyric acid (GABA)-mediated currents in the hippocampus, a major brain region involved in the cognitive function, has not been investigated. Here we detected abundant expression of both IFN-γ receptor subunit gene transcripts (Ifngr1 and Ifngr2) in the rat hippocampus by quantitative PCR. In addition, we pre-incubated rat hippocampal slices with IFN-γ (100â¯ng/ml) and recorded GABA-activated spontaneous and miniature postsynaptic inhibitory currents (sIPSCs and mIPSCs) and tonic currents in hippocampal CA1 pyramidal neurons by the whole-cell patch-clamp method. The pre-incubation with IFN-γ increased the frequency but not the mean amplitude, rise time or decay time of both sIPSCs and mIPSCs in hippocampal CA1 pyramidal neurons, suggesting a presynaptic effect of IFN-γ. Moreover, the GABA-activated tonic currents were enhanced by IFN-γ. In conclusion, the potentiation of GABAergic currents in hippocampal neurons by IFN-γ may contribute to the disturbed neuronal excitability and cognitive dysfunction during neuroinflammation.
Assuntos
Região CA1 Hipocampal/fisiologia , Hipocampo/fisiologia , Interferon gama/farmacologia , Inibição Neural/fisiologia , Células Piramidais/fisiologia , Receptores de GABA-A/fisiologia , Animais , Região CA1 Hipocampal/efeitos dos fármacos , Antagonistas de Receptores de GABA-A/farmacologia , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Potenciais Pós-Sinápticos Inibidores/efeitos dos fármacos , Potenciais Pós-Sinápticos Inibidores/fisiologia , Masculino , Inibição Neural/efeitos dos fármacos , Técnicas de Cultura de Órgãos , Células Piramidais/efeitos dos fármacos , Ratos , Ratos WistarRESUMO
We generated human iPS derived neural stem cells and differentiated cells from healthy control individuals and an individual with autism spectrum disorder carrying bi-allelic NRXN1-alpha deletion. We investigated the expression of NRXN1-alpha during neural induction and neural differentiation and observed a pivotal role for NRXN1-alpha during early neural induction and neuronal differentiation. Single cell RNA-seq pinpointed neural stem cells carrying NRXN1-alpha deletion shifting towards radial glia-like cell identity and revealed higher proportion of differentiated astroglia. Furthermore, neuronal cells carrying NRXN1-alpha deletion were identified as immature by single cell RNA-seq analysis, displayed significant depression in calcium signaling activity and presented impaired maturation action potential profile in neurons investigated with electrophysiology. Our observations propose NRXN1-alpha plays an important role for the efficient establishment of neural stem cells, in neuronal differentiation and in maturation of functional excitatory neuronal cells.
Assuntos
Transtorno Autístico/patologia , Proteínas de Ligação ao Cálcio/genética , Deleção de Genes , Células-Tronco Pluripotentes Induzidas/patologia , Proteínas do Tecido Nervoso/genética , Moléculas de Adesão de Célula Nervosa/genética , Células-Tronco Neurais/patologia , Análise de Célula Única/métodos , Potenciais de Ação , Alelos , Transtorno Autístico/genética , Diferenciação Celular , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Neurais/metabolismo , Neurogênese/genéticaRESUMO
Calcium (Ca2+) is an important ion in physiology and is found both outside and inside cells. The intracellular concentration of Ca2+ is tightly regulated as it is an intracellular signal molecule and can affect a variety of cellular processes. In immune cells Ca2+ has been shown to regulate e.g. gene transcription, cytokine secretion, proliferation and migration. Ca2+ can enter the cytoplasm either from intracellular stores or from outside the cells when Ca2+ permeable ion channels in the plasma membrane open. The Ca2+ release-activated (CRAC) channel is the most prominent Ca2+ ion channel in the plasma membrane. It is formed by ORAI1-3 and the channel is opened by the endoplasmic reticulum Ca2+ sensor proteins stromal interaction molecules (STIM) 1 and 2. Another group of Ca2+ channels in the plasma membrane are the voltage-gated Ca2+ (CaV) channels. We examined if a change in immunological tolerance is accompanied by altered ORAI, STIM and CaV gene expression in peripheral blood mononuclear cells (PBMCs) in pregnant women and in type 1 diabetic individuals. Our results show that in pregnancy and type 1 diabetes ORAI1-3 are up-regulated whereas STIM1 and 2 are down-regulated in pregnancy but only STIM2 in type 1 diabetes. Expression of L-, P/Q-, R- and T-type voltage-gated Ca2+ channels was detected in the PBMCs where the CaV2.3 gene was up-regulated in pregnancy and type 1 diabetes whereas the CaV 2.1 and CaV3.2 genes were up-regulated only in pregnancy and the CaV1.3 gene in type 1 diabetes. The results are consistent with that expression of ORAI, STIM and CaV genes correlate with a shift in immunological status of the individual in health, as during pregnancy, and in the autoimmune disease type 1 diabetes. Whether the changes are in general protective or in type 1 diabetes include some pathogenic components remains to be clarified.
Assuntos
Canais de Cálcio Ativados pela Liberação de Cálcio/genética , Diabetes Mellitus Tipo 1/genética , Regulação da Expressão Gênica , Leucócitos Mononucleares/metabolismo , Adolescente , Adulto , Cálcio/metabolismo , Estudos de Casos e Controles , Criança , Pré-Escolar , Diabetes Mellitus Tipo 1/sangue , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Gravidez , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Adulto JovemRESUMO
The neurotransmitter γ-aminobutyric acid (GABA) is an extracellular signaling molecule in the brain and in pancreatic islets. Here, we demonstrate that GABA regulates cytokine secretion from human peripheral blood mononuclear cells (PBMCs) and CD4+ T cells. In anti-CD3 stimulated PBMCs, GABA (100nM) inhibited release of 47 cytokines in cells from patients with type 1 diabetes (T1D), but only 16 cytokines in cells from nondiabetic (ND) individuals. CD4+ T cells from ND individuals were grouped into responder or non-responder T cells according to effects of GABA (100nM, 500nM) on the cell proliferation. In the responder T cells, GABA decreased proliferation, and inhibited secretion of 37 cytokines in a concentration-dependent manner. In the non-responder T cells, GABA modulated release of 8 cytokines. GABA concentrations in plasma from T1D patients and ND individuals were correlated with 10 cytokines where 7 were increased in plasma of T1D patients. GABA inhibited secretion of 5 of these cytokines from both T1D PBMCs and ND responder T cells. The results identify GABA as a potent regulator of both Th1- and Th2-type cytokine secretion from human PBMCs and CD4+ T cells where GABA generally decreases the secretion.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Citocinas/sangue , Citocinas/metabolismo , Diabetes Mellitus Tipo 1/sangue , Terapia de Imunossupressão , Mediadores da Inflamação/metabolismo , Leucócitos Mononucleares/metabolismo , Ácido gama-Aminobutírico/farmacologia , Anti-Inflamatórios/metabolismo , Vias Biossintéticas/efeitos dos fármacos , Vias Biossintéticas/genética , Linfócitos T CD4-Positivos/efeitos dos fármacos , Estudos de Casos e Controles , Proliferação de Células/efeitos dos fármacos , Colesterol/biossíntese , Diabetes Mellitus Tipo 1/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Ácido gama-Aminobutírico/sangueRESUMO
In human pancreatic islets, the neurotransmitter γ-aminobutyric acid (GABA) is an extracellular signaling molecule synthesized by and released from the insulin-secreting ß cells. The effective, physiological GABA concentration range within human islets is unknown. Here we use native GABAA receptors in human islet ß cells as biological sensors and reveal that 100-1000nM GABA elicit the maximal opening frequency of the single-channels. In saturating GABA, the channels desensitized and stopped working. GABA modulated insulin exocytosis and glucose-stimulated insulin secretion. GABAA receptor currents were enhanced by the benzodiazepine diazepam, the anesthetic propofol and the incretin glucagon-like peptide-1 (GLP-1) but not affected by the hypnotic zolpidem. In type 2 diabetes (T2D) islets, single-channel analysis revealed higher GABA affinity of the receptors. The findings reveal unique GABAA receptors signaling in human islets ß cells that is GABA concentration-dependent, differentially regulated by drugs, modulates insulin secretion and is altered in T2D.
Assuntos
Células Secretoras de Insulina/metabolismo , Receptores de GABA-A/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Exocitose/efeitos dos fármacos , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Ativação do Canal Iônico/efeitos dos fármacos , Cinética , Modelos Biológicos , Subunidades Proteicas/metabolismo , Ácido gama-Aminobutírico/farmacologiaRESUMO
Insulin, a pancreatic hormone, can access the central nervous system, activate insulin receptors distributed in selective brain regions and affect various cellular functions such as neurotransmission. We have previously shown that physiologically relevant concentration of insulin potentiates the GABAA receptor-mediated tonic inhibition and reduces excitability of rat hippocampal CA1 neurons. The central nucleus of the amygdala (CeA) comprises heterogeneous neuronal populations that can respond to hormonal stimulus. Using quantitative PCR and immunofluorescent labeling, we report that the mRNA and protein of the insulin receptor are abundantly expressed in the rat CeA. The insulin receptor mRNA is also detected in the CeA from post-mortem human brain samples. Furthermore, our whole-cell patch-clamp recordings show that the application of insulin (5 and 50â¯nM) selectively enhances the frequency and amplitude of spontaneous inhibitory postsynaptic currents (sIPSCs) in rat CeA neurons. Our findings reveal that GABAergic synaptic transmission is a target in the CeA for insulin receptor signaling that may underlie insulin modulation of emotion- and feeding-related behaviors.
Assuntos
Núcleo Central da Amígdala/metabolismo , Hipoglicemiantes/farmacologia , Potenciais Pós-Sinápticos Inibidores/efeitos dos fármacos , Insulina/farmacologia , Neurônios/metabolismo , Receptor de Insulina/metabolismo , Receptores de GABA-A/metabolismo , Animais , Núcleo Central da Amígdala/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp , Ratos , Ratos Wistar , Receptor de Insulina/genética , Transmissão Sináptica/efeitos dos fármacosRESUMO
BACKGROUND: γ-Aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the brain where it regulates activity of neuronal networks. The receptor for glucagon-like peptide-1 (GLP-1) is expressed in the hippocampus, which is the center for memory and learning. In this study we examined effects of liraglutide, a GLP-1 analog, on GABA signaling in CA3 hippocampal pyramidal neurons. METHODS: We used patch-clamp electrophysiology to record synaptic and tonic GABA-activated currents in CA3 pyramidal neurons in rat hippocampal brain slices. RESULTS: We examined the effects of liraglutide on the neurons at concentrations ranging from one nM to one µM. Significant changes of the spontaneous inhibitory postsynaptic currents (sIPSCs) were only recorded with 100 nM liraglutide and then in just ≈50% of the neurons tested at this concentration. In neurons affected by liraglutide both the sIPSC frequency and the most probable amplitudes increased. When the action potential firing was inhibited by tetrodotoxin (TTX) the frequency and amplitude of IPSCs in TTX and in TTX plus 100 nM liraglutide were similar. CONCLUSIONS: The results demonstrate that liraglutide regulation of GABA signaling of CA3 pyramidal neurons is predominantly presynaptic and more limited than has been observed for GLP-1 and exendin-4 in hippocampal neurons.
Assuntos
Hipoglicemiantes/farmacologia , Liraglutida/farmacologia , Terminações Pré-Sinápticas/efeitos dos fármacos , Células Piramidais/efeitos dos fármacos , Animais , Região CA3 Hipocampal/citologia , Terminações Pré-Sinápticas/fisiologia , Células Piramidais/fisiologia , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Ácido gama-Aminobutírico/fisiologiaRESUMO
The amino acid glutamate opens cation permeable ion channels, the iGlu receptors. These ion channels are abundantly expressed in the mammalian brain where glutamate is the main excitatory neurotransmitter. The neurotransmitters and their receptors are being increasingly detected in the cells of immune system. Here we examined the expression of the 18 known subunits of the iGlu receptors families; α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), kainate, N-methyl-d-aspartate (NMDA) and delta in human peripheral blood mononuclear cells (PBMCs). We compared the expression of the subunits between four groups: men, non-pregnant women, healthy pregnant women and depressed pregnant women. Out of 18 subunits of the iGlu receptors, mRNAs for 11 subunits were detected in PBMCs from men and non-pregnant women; AMPA: GluA3, GluA4, kainate: GluK2, GluK4, GluK5, NMDA: GluN1, GluN2C, GluN2D, GluN3A, GluN3B, and delta: GluD1. In the healthy and the depressed pregnant women, in addition, the delta GluD2 subunit was identified. The mRNAs for GluK4, GluK5, GluN2C and GluN2D were expressed at a higher level than other subunits. Gender, pregnancy or depression during pregnancy altered the expression of GluA3, GluK4, GluN2D, GluN3B and GluD1 iGlu subunit mRNAs. The greatest changes recorded were the lower GluA3 and GluK4 mRNA levels in pregnant women and the higher GluN2D mRNA level in healthy but not in depressed pregnant women as compared to non-pregnant individuals. Using subunit specific antibodies, the GluK4, GluK5, GluN1, GluN2C and GluN2D subunit proteins were identified in the PBMCs. The results show expression of specific iGlu receptor subunit in the PBMCs and support the idea of physiology-driven changes of iGlu receptors subtypes in the immune cells.
Assuntos
Depressão/sangue , Regulação da Expressão Gênica/fisiologia , Leucócitos Mononucleares/metabolismo , Gravidez/fisiologia , Subunidades Proteicas/metabolismo , Receptores Ionotrópicos de Glutamato/metabolismo , Adulto , Feminino , Humanos , Masculino , Microscopia Confocal , Gravidez/sangue , Subunidades Proteicas/genética , RNA Mensageiro/metabolismo , Receptores de AMPA , Receptores Ionotrópicos de Glutamato/genética , Receptores de Ácido Caínico , Receptores de N-Metil-D-Aspartato , Fatores Sexuais , Estatísticas não Paramétricas , Adulto JovemRESUMO
TNFα is a very potent and pleiotropic pro-inflammatory cytokine, essential to the immune system for eradicating cancer and microorganisms, and to the nervous system, for brain development and ongoing function. Yet, excess and/or chronic TNFα secretion causes massive tissue damage in autoimmune, inflammatory and neurological diseases and injuries. Therefore, many patients with autoimmune/inflammatory diseases receive anti-TNFα medications. TNFα is secreted primarily by CD4(+) T cells, macrophages, monocytes, neutrophils and NK cells, mainly after immune stimulation. Yet, the cause for the pathologically high and chronic TNFα secretion is unknown. Can blocking of a particular ion channel in T cells induce by itself TNFα secretion? Such phenomenon was never revealed or even hypothesized. In this interdisciplinary study we discovered that: (1) normal human T cells express Kv1.1 voltage-gated potassium channel mRNA, and the Kv1.1 membrane-anchored protein channel; (2) Kv1.1 is expressed in most CD4(+)CD3(+) helper T cells (mean CD4(+)CD3(+)Kv1.1(+) T cells of 7 healthy subjects: 53.09 ± 22.17 %), but not in CD8(+)CD3(+) cytotoxic T cells (mean CD8(+)CD3(+)Kv1.1(+) T cells: 4.12 ± 3.04 %); (3) electrophysiological whole-cell recordings in normal human T cells revealed Kv currents; (4) Dendrotoxin-K (DTX-K), a highly selective Kv1.1 blocker derived from snake toxin, increases the rate of rise and decay of Kv currents in both resting and activated T cells, without affecting the peak current; (5) DTX-K by itself induces robust TNFα production and secretion by normal human T cells, without elevating IFNγ, IL-4 and IL-10; (6) intact Ca(2+) channels are required for DTX-induced TNFα secretion; (7) selective anti-Kv1.1 antibodies also induce by themselves TNFα secretion; (8) DTX-K activates NFκB in normal human T cells via the unique non-canonical-pathway; (9) injection of Kv1.1-blocked human T cells to SCID mice, causes recruitment of resident mouse cells into the liver, alike reported after TNFα injection into the brain. Based on our discoveries we speculate that abnormally blocked Kv1.1 in T cells (and other immune cells?), due to either anti-Kv1.1 autoimmune antibodies, or Kv1.1-blocking toxins alike DTX-K, or Kv1.1-blocking genetic mutations, may be responsible for the chronic/excessive TNFα in autoimmune/inflammatory diseases. Independently, we also hypothesize that selective block of Kv1.1 in CD4(+) T cells of patients with cancer or chronic infectious diseases could be therapeutic, since it may: a. augment beneficial secretion and delivery of TNFα to the disease-affected sites; b. induce recruitment and extravasation of curative immune cells and factors; c. improve accessibility of drugs to the brain and few peripheral organs thanks to TNFα-induced increased permeability of organ's barriers.
Assuntos
Doenças Autoimunes , Linfócitos T CD4-Positivos/metabolismo , Inflamação , Canal de Potássio Kv1.1/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Doenças Autoimunes/imunologia , Doenças Autoimunes/metabolismo , Western Blotting , Linfócitos T CD4-Positivos/imunologia , Ensaio de Desvio de Mobilidade Eletroforética , Feminino , Humanos , Imuno-Histoquímica , Inflamação/imunologia , Inflamação/metabolismo , Masculino , Camundongos , Camundongos SCID , NF-kappa B/imunologia , NF-kappa B/metabolismo , Técnicas de Patch-Clamp , Reação em Cadeia da Polimerase , Transdução de Sinais/imunologiaRESUMO
Glucagon-like peptide-1 (GLP-1) is a metabolic hormone that is secreted in a glucose-dependent manner and enhances insulin secretion. GLP-1 receptors are also found in the brain where their signalling affects neuronal activity. We have previously shown that the GLP-1 receptor agonists, GLP-1 and exendin-4 enhanced GABA-activated synaptic and tonic currents in rat hippocampal CA3 pyramidal neurons. The hippocampus is the centre for memory and learning and is important for cognition. Here we examined if exendin-4 similarly enhanced the GABA-activated currents in the presence of the benzodiazepine diazepam. In whole-cell recordings in rat brain slices, diazepam (1 µM), an allosteric positive modulator of GABAA receptors, alone enhanced the spontaneous inhibitory postsynaptic current (sIPSC) amplitude and frequency by a factor of 1.3 and 1.6, respectively, and doubled the tonic GABAA current normally recorded in the CA3 pyramidal cells. Importantly, in the presence of exendin-4 (10 nM) plus diazepam (1 µM), only the tonic but not the sIPSC currents transiently increased as compared to currents recorded in the presence of diazepam alone. The results suggest that exendin-4 potentiates a subpopulation of extrasynaptic GABAA receptors in the CA3 pyramidal neurons.
Assuntos
Região CA3 Hipocampal/citologia , Diazepam/farmacologia , Agonistas de Receptores de GABA-A/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Peptídeos/farmacologia , Receptores de GABA-A/metabolismo , Peçonhas/farmacologia , Animais , Região CA3 Hipocampal/efeitos dos fármacos , Eletrofisiologia , Exenatida , Técnicas de Patch-Clamp , Ratos , Ratos Wistar , Potenciais Sinápticos/efeitos dos fármacosRESUMO
Glucagon-like peptide-1 (GLP-1) is a hormone that stimulates insulin secretion. Receptors for GLP-1 are also found in the brain, including the hippocampus, the center for memory and learning. Diabetes is a risk factor for decreased memory functions. We studied effects of GLP-1 and exendin-4, a GLP-1 receptor agonist, on γ-aminobutyric acid (GABA) signaling in hippocampal CA3 pyramidal neurons. GABA is the main inhibitory neurotransmitter and decreases neuronal excitability. GLP-1 (0.01-1 nmol/L) transiently enhanced synaptic and tonic currents, and the effects were blocked by exendin (9-39). Ten pmol/L GLP-1 increased both the spontaneous inhibitory postsynaptic current (sIPSC) amplitudes and frequency by a factor of 1.8. In 0.1, 1 nmol/L GLP-1 or 10, 50, or 100 nmol/L exendin-4, only the sIPSC frequency increased. The tonic current was enhanced by 0.01-1 nmol/L GLP-1 and by 0.5-100 nmol/L exendin-4. When action potentials were inhibited by tetrodotoxin (TTX), inhibitory postsynaptic currents decreased and currents were no longer potentiated by GLP-1 or exendin-4. In contrast, although the tonic current decreased in TTX, it was still enhanced by GLP-1 or exendin-4. The results demonstrate GLP-1 receptor regulation of hippocampal function and are consistent with GLP-1 receptor agonists enhancing GABAA signaling by pre- and postsynaptic mechanisms.
Assuntos
Região CA3 Hipocampal/fisiologia , Peptídeo 1 Semelhante ao Glucagon/fisiologia , Potenciais Pós-Sinápticos Inibidores/fisiologia , Peptídeos/farmacologia , Receptores de GABA-A/fisiologia , Receptores de Glucagon/fisiologia , Peçonhas/farmacologia , Animais , Região CA3 Hipocampal/citologia , Região CA3 Hipocampal/efeitos dos fármacos , Exenatida , Peptídeo 1 Semelhante ao Glucagon/farmacologia , Receptor do Peptídeo Semelhante ao Glucagon 1 , Potenciais Pós-Sinápticos Inibidores/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Memória/fisiologia , Técnicas de Cultura de Órgãos , Técnicas de Patch-Clamp , Peptídeos/fisiologia , Células Piramidais/efeitos dos fármacos , Células Piramidais/fisiologia , Ratos Wistar , Receptores de Glucagon/agonistas , Receptores de Glucagon/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Bloqueadores dos Canais de Sódio/farmacologia , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologia , Tetrodotoxina/farmacologia , Ácido gama-Aminobutírico/metabolismoRESUMO
GABAA receptors are pentameric chloride ion channels that are opened by GABA. We have screened a cell line derived from human glioblastoma, U3047MG, for expression of GABAA receptor subunit isoforms and formation of functional ion channels. We identified GABAA receptors subunit α2, α3, α5, ß1, ß2, ß3, δ, γ3, π, and θ mRNAs in the U3047MG cell line. Whole-cell GABA-activated currents were recorded and the half-maximal concentration (EC50) for the GABA-activated current was 36 µM. The currents were activated by THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol) and enhanced by the benzodiazepine diazepam (1 µM) and the general anesthetics etomidate and propofol (50 µM). In line with the expressed GABAA receptors containing at least the α3ß3θ subunits, the receptors were highly sensitive to etomidate (EC50=55 nM). Immunocytochemistry identified expression of the α3 and ß3 subunit proteins. Our results show that the GABAA receptors in the glial cell line are functional and are modulated by classical GABAA receptor drugs. We propose that the U3047MG cell line may be used as a model system to study GABAA receptors function and pharmacology in glial cells.