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1.
Proc Natl Acad Sci U S A ; 119(20): e2120870119, 2022 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-35544691

RESUMO

Transient receptor potential canonical 4 (TRPC4) is a receptor-operated cation channel codependent on both the Gq/11­phospholipase C signaling pathway and Gi/o proteins for activation. This makes TRPC4 an excellent coincidence sensor of neurotransmission through Gq/11- and Gi/o-coupled receptors. In whole-cell slice recordings of lateral septal neurons, TRPC4 mediates a strong depolarizing plateau that shuts down action potential firing, which may or may not be followed by a hyperpolarization that extends the firing pause to varying durations depending on the strength of Gi/o stimulation. We show that the depolarizing plateau is codependent on Gq/11-coupled group I metabotropic glutamate receptors and on Gi/o-coupled γ-aminobutyric acid type B receptors. The hyperpolarization is mediated by Gi/o activation of G protein­activated inwardly rectifying K+ (GIRK) channels. Moreover, the firing patterns, elicited by either electrical stimulation or receptor agonists, encode information about the relative strengths of Gq/11 and Gi/o inputs in the following fashion. Pure Gq/11 input produces weak depolarization accompanied by firing acceleration, whereas pure Gi/o input causes hyperpolarization that pauses firing. Although coincident Gq/11­Gi/o inputs also pause firing, the pause is preceded by a burst, and both the pause duration and firing recovery patterns reflect the relative strengths of Gq/11 versus Gi/o inputs. Computer simulations demonstrate that different combinations of TRPC4 and GIRK conductances are sufficient to produce the range of firing patterns observed experimentally. Thus, concurrent neurotransmission through the Gq/11 and Gi/o pathways is converted to discernible electrical responses by the joint actions of TRPC4 and GIRK for communication to downstream neurons.


Assuntos
Potenciais de Ação , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP , Subunidades alfa de Proteínas de Ligação ao GTP , Neurônios , Transmissão Sináptica , Canais de Cátion TRPC , Animais , Comunicação Celular , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/fisiologia , Subunidades alfa de Proteínas de Ligação ao GTP/fisiologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/fisiologia , Camundongos , Neurônios/fisiologia , Canais de Cátion TRPC/fisiologia
2.
Mol Brain ; 14(1): 144, 2021 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-34544455

RESUMO

Astrocytes express a plethora of G protein-coupled receptors (GPCRs) that are crucial for shaping synaptic activity. Upon GPCR activation, astrocytes can respond with transient variations in intracellular Ca2+. In addition, Ca2+-dependent and/or Ca2+-independent release of gliotransmitters can occur, allowing them to engage in bidirectional neuron-astrocyte communication. The development of designer receptors exclusively activated by designer drugs (DREADDs) has facilitated many new discoveries on the roles of astrocytes in both physiological and pathological conditions. They are an excellent tool, as they can target endogenous GPCR-mediated intracellular signal transduction pathways specifically in astrocytes. With increasing interest and accumulating research on this topic, several discrepancies on astrocytic Ca2+ signalling and astrocyte-mediated effects on synaptic plasticity have emerged, preventing a clear-cut consensus about the downstream effects of DREADDs in astrocytes. In the present study, we performed a side-by-side evaluation of the effects of bath application of the DREADD agonist, clozapine-N-oxide (10 µM), on Gq- and Gi-DREADD activation in mouse CA1 hippocampal astrocytes. In doing so, we aimed to avoid confounding factors, such as differences in experimental procedures, and to directly compare the actions of both DREADDs on astrocytic intracellular Ca2+ dynamics and synaptic plasticity in acute hippocampal slices. We used an adeno-associated viral vector approach to transduce dorsal hippocampi of male, 8-week-old C57BL6/J mice, to drive expression of either the Gq-DREADD or Gi-DREADD in CA1 astrocytes. A viral vector lacking the DREADD construct was used to generate controls. Here, we show that agonism of Gq-DREADDs, but not Gi-DREADDs, induced consistent increases in spontaneous astrocytic Ca2+ events. Moreover, we demonstrate that both Gq-DREADD as well as Gi-DREADD-mediated activation of CA1 astrocytes induces long-lasting synaptic potentiation in the hippocampal CA1 Schaffer collateral pathway in the absence of a high frequency stimulus. Moreover, we report for the first time that astrocytic Gi-DREADD activation is sufficient to elicit de novo potentiation. Our data demonstrate that activation of either Gq or Gi pathways drives synaptic potentiation through Ca2+-dependent and Ca2+-independent mechanisms, respectively.


Assuntos
Astrócitos/fisiologia , Região CA1 Hipocampal/fisiologia , Sinalização do Cálcio/fisiologia , Clozapina/análogos & derivados , Drogas Desenhadas/farmacologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/fisiologia , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/fisiologia , Receptores Acoplados a Proteínas G/fisiologia , Animais , Astrócitos/efeitos dos fármacos , Região CA1 Hipocampal/citologia , Clozapina/farmacologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/efeitos dos fármacos , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/efeitos dos fármacos , Vetores Genéticos/farmacologia , Potenciação de Longa Duração/efeitos dos fármacos , Potenciação de Longa Duração/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Plasticidade Neuronal/fisiologia , Receptores Acoplados a Proteínas G/efeitos dos fármacos
3.
Theranostics ; 11(17): 8535-8549, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34373757

RESUMO

Neuroligin-3 (NLGN3) is necessary and sufficient to promote glioma cell growth. The recruitment of Gαi1/3 to the ligand-activated receptor tyrosine kinases (RTKs) is essential for mediating oncogenic signaling. Methods: Various genetic strategies were utilized to examine the requirement of Gαi1/3 in NLGN3-driven glioma cell growth. Results: NLGN3-induced Akt-mTORC1 and Erk activation was inhibited by decreasing Gαi1/3 expression. In contrast ectopic Gαi1/3 overexpression enhanced NLGN3-induced signaling. In glioma cells, NLGN3-induced cell growth, proliferation and migration were attenuated by Gαi1/3 depletion with shRNA, but facilitated with Gαi1/3 overexpression. Significantly, Gαi1/3 silencing inhibited orthotopic growth of patient-derived glioma xenografts in mouse brain, whereas forced Gαi1/3-overexpression in primary glioma xenografts significantly enhanced growth. The growth of brain-metastatic human lung cancer cells in mouse brain was largely inhibited with Gαi1/3 silencing. It was however expedited with ectopic Gαi1/3 overexpression. In human glioma Gαi3 upregulation was detected, correlating with poor prognosis. Conclusion: Gαi1/3 mediation of NLGN3-induced signaling is essential for neuronal-driven glioma growth.


Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Glioma/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Idoso , Animais , Neoplasias Encefálicas/patologia , Moléculas de Adesão Celular Neuronais/fisiologia , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/fisiologia , Glioma/genética , Glioma/patologia , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas de Membrana/fisiologia , Camundongos , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/fisiologia , Neurônios/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Extratos Vegetais , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais
4.
Neural Plast ; 2021: 7806370, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34306063

RESUMO

Chronic stress is one of the main risk factors of bone loss. While the neurons and neural circuits of the ventromedial hypothalamus (VMH) mediate bone loss induced by chronic stress, the detailed intrinsic mechanisms within the VMH nucleus still need to be explored. Astrocytes in brain regions play important roles in the regulation of metabolism and anxiety-like behavior through interactions with surrounding neurons. However, whether astrocytes in the VMH affect neuronal activity and therefore regulate chronic stress-induced anxiety and bone loss remain elusive. In this study, we found that VMH astrocytes were activated during chronic stress-induced anxiety and bone loss. Pharmacogenetic activation of the Gi and Gq pathways in VMH astrocytes reduced and increased the levels of anxiety and bone loss, respectively. Furthermore, activation of VMH astrocytes by optogenetics induced depolarization in neighboring steroidogenic factor-1 (SF-1) neurons, which was diminished by administration of N-methyl-D-aspartic acid (NMDA) receptor blocker but not by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor blocker. These results suggest that there may be a functional "glial-neuron microcircuit" in VMH nuclei that mediates anxiety and bone loss induced by chronic stress. This study not only advances our understanding of glial cell function but also provides a potential intervention target for chronic stress-induced anxiety and bone loss therapy.


Assuntos
Transtornos de Ansiedade/fisiopatologia , Astrócitos/fisiologia , Reabsorção Óssea/fisiopatologia , Estresse Psicológico/complicações , Núcleo Hipotalâmico Ventromedial/patologia , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Densidade Óssea , Reabsorção Óssea/etiologia , Reabsorção Óssea/prevenção & controle , Doença Crônica , Clozapina/farmacologia , Clozapina/uso terapêutico , Teste de Labirinto em Cruz Elevado , Emoções , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/agonistas , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/fisiologia , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/fisiologia , Genes Reporter , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/fisiologia , Teste de Campo Aberto , Optogenética , Proteínas Proto-Oncogênicas c-fos/biossíntese , Proteínas Proto-Oncogênicas c-fos/genética , Distribuição Aleatória , Receptores de N-Metil-D-Aspartato/fisiologia , Núcleo Hipotalâmico Ventromedial/fisiopatologia
5.
Mol Pharmacol ; 100(3): 217-223, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34135098

RESUMO

Regulators of G protein signaling (RGS) proteins modulate signaling by G protein-coupled receptors. Using a knock-in transgenic mouse model with a mutation in Gαo that does not bind RGS proteins (RGS-insensitive), we determined the effect of RGS proteins on presynaptic µ opioid receptor (MOR)-mediated inhibition of GABA release in the ventrolateral periaqueductal gray (vlPAG). The MOR agonists [d-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO) and met-enkephalin (ME) inhibited evoked inhibitory postsynaptic currents (eIPSCs) in the RGS-insensitive mice compared with wild-type (WT) littermates, respectively. Fentanyl inhibited eIPSCs similarly in both WT and RGS-insensitive mice. There were no differences in opioid agonist inhibition of spontaneous GABA release between the genotypes. To further probe the mechanism underlying these differences between opioid inhibition of evoked and spontaneous GABA release, specific myristoylated Gα peptide inhibitors for Gαo1 and Gαi1-3 that block receptor-G protein interactions were used to test the preference of agonists for MOR-Gα complexes. The Gαo1 inhibitor reduced DAMGO inhibition of eIPSCs, but Gαi1-3 inhibitors had no effect. Both Gαo1 and Gαi1-3 inhibitors separately reduced fentanyl inhibition of eIPSCs but had no effects on ME inhibition. Gαi1-3 inhibitors blocked the inhibitory effects of ME and fentanyl on miniature postsynaptic current (mIPSC) frequency, but both Gαo1 and Gαi1-3 inhibitors were needed to block the effects of DAMGO. Finally, baclofen-mediated inhibition of GABA release is unaffected in the RGS-insensitive mice and in the presence of Gαo1 and Gαi1-3 inhibitor peptides, suggesting that GABAB receptor coupling to G proteins in vlPAG presynaptic terminals is different than MOR coupling. SIGNIFICANCE STATEMENT: Presynaptic µ opioid receptors (MORs) in the ventrolateral periaqueductal gray are critical for opioid analgesia and are negatively regulated by RGS proteins. These data in RGS-insensitive mice provide evidence that MOR agonists differ in preference for Gαo versus Gαi and regulation by RGS proteins in presynaptic terminals, providing a mechanism for functional selectivity between agonists. The results further define important differences in MOR and GABAB receptor coupling to G proteins that could be exploited for new pain therapies.


Assuntos
Subunidade alfa Gi2 de Proteína de Ligação ao GTP/fisiologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/fisiologia , Terminações Pré-Sinápticas/fisiologia , Receptores Opioides mu/fisiologia , Ácido gama-Aminobutírico/metabolismo , Analgésicos Opioides/farmacologia , Animais , Baclofeno/farmacologia , Feminino , Subunidade alfa Gi2 de Proteína de Ligação ao GTP/antagonistas & inibidores , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/antagonistas & inibidores , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Potenciais Pós-Sinápticos Inibidores/efeitos dos fármacos , Potenciais Pós-Sinápticos Inibidores/fisiologia , Masculino , Camundongos , Camundongos Transgênicos , Modelos Animais , Proteínas RGS/metabolismo , Receptores de GABA-B/metabolismo , Receptores Opioides mu/agonistas
6.
Commun Biol ; 4(1): 662, 2021 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-34079054

RESUMO

Pathological impulsivity is a debilitating symptom of multiple psychiatric diseases with few effective treatment options. To identify druggable receptors with anti-impulsive action we developed a systematic target discovery approach combining behavioural chemogenetics and gene expression analysis. Spatially restricted inhibition of three subdivisions of the prefrontal cortex of mice revealed that the anterior cingulate cortex (ACC) regulates premature responding, a form of motor impulsivity. Probing three G-protein cascades with designer receptors, we found that the activation of Gi-signalling in layer-5 pyramidal cells (L5-PCs) of the ACC strongly, reproducibly, and selectively decreased challenge-induced impulsivity. Differential gene expression analysis across murine ACC cell-types and 402 GPCRs revealed that - among Gi-coupled receptor-encoding genes - Grm2 is the most selectively expressed in L5-PCs while alternative targets were scarce. Validating our approach, we confirmed that mGluR2 activation reduced premature responding. These results suggest Gi-coupled receptors in ACC L5-PCs as therapeutic targets for impulse control disorders.


Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/fisiologia , Giro do Cíngulo/citologia , Giro do Cíngulo/fisiologia , Células Piramidais/fisiologia , Animais , Clozapina/análogos & derivados , Clozapina/farmacologia , Feminino , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/efeitos dos fármacos , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Expressão Gênica/efeitos dos fármacos , Giro do Cíngulo/efeitos dos fármacos , Humanos , Comportamento Impulsivo/efeitos dos fármacos , Comportamento Impulsivo/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Células Piramidais/citologia , Células Piramidais/efeitos dos fármacos , Receptores de Glutamato Metabotrópico/efeitos dos fármacos , Receptores de Glutamato Metabotrópico/genética , Receptores de Glutamato Metabotrópico/fisiologia , Transdução de Sinais
7.
Thromb Haemost ; 120(11): 1536-1547, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32854120

RESUMO

Platelet activation plays a pivotal role in physiological hemostasis and pathological thrombosis causing heart attack and stroke. Previous studies conclude that simultaneous activation of Gi and G12/13 signaling pathways is sufficient to cause platelet aggregation. However, using Gq knockout mice and Gq-specific inhibitors, we here demonstrated that platelet aggregation downstream of coactivation of Gi and G12/13 depends on agonist concentrations; coactivation of Gi and G12/13 pathways only induces platelet aggregation under higher agonist concentrations. We confirmed Gi and G12/13 pathway activation by showing cAMP (cyclic adenosine monophosphate) decrease and RhoA activation in platelets stimulated at both low and high agonist concentrations. Interestingly, we found that though Akt and PAK (p21-activated kinase) translocate to the platelet membrane upon both low and high agonist stimulation, membrane-translocated Akt and PAK only phosphorylate at high agonist concentrations, correlating well with platelet aggregation downstream of concomitant Gi and G12/13 pathway activation. PAK inhibitor abolishes Akt phosphorylation, inhibits platelet aggregation in vitro and arterial thrombus formation in vivo. We propose that the PAK-PI3K/Akt pathway mediates platelet aggregation downstream of Gi and G12/13, and PAK may represent a potential antiplatelet and antithrombotic target.


Assuntos
Agregação Plaquetária , Transdução de Sinais/fisiologia , Quinases Ativadas por p21/fisiologia , Difosfato de Adenosina/farmacologia , Animais , Forma Celular , Relação Dose-Resposta a Droga , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/fisiologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/fisiologia , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/deficiência , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/fisiologia , Humanos , Camundongos , Camundongos Knockout , Oligopeptídeos/farmacologia , Fragmentos de Peptídeos/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Transporte Proteico , Proteínas Proto-Oncogênicas c-akt/fisiologia , Ratos , Tromboxano A2/farmacologia , Proteína rhoA de Ligação ao GTP/metabolismo
8.
Mol Metab ; 40: 101029, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32480042

RESUMO

OBJECTIVES: Typically, obesity results from an inappropriate balance between energy uptake from nutrient consumption and burning of calories, which leads to a pathological increase in fat mass. Obesity is a major cause of insulin resistance and diabetes. Inhibitory G proteins (Gαi) form a subfamily that is involved in the regulation of adipose tissue function. Among the three Gαi members, i.e. Gαi1, Gαi2, Gαi3, the Gαi2, protein is predominantly expressed in adipose tissue. However, the functions of the Gαi2 isoform in adipose tissue and its impact on the development of obesity are poorly understood. METHODS: By using AdipoqCreERT2 mice, we generated adipocyte-specific Gnai2-deficient mice to study Gαi2 function, specifically in white and brown adipocytes. These mice were fed either a control diet (CD) or a high fat diet (HFD). Mice were examined for obesity development, insulin resistance and glucose intolerance. We examined adipocyte morphology and the development of inflammation in the white adipose tissue. Finally, intracellular cAMP levels as an indicator of Gαi signaling and glycerol release as an indicator of lipolysis rates were measured to verify the impact of Gαi2 on the signaling pathway in brown and white adipocytes. RESULTS: An adipocyte-specific deficiency of Gαi2 significantly reduced diet-induced obesity, leading to decreased fat masses, smaller adipocytes and decreased inflammation in the white adipose tissue relative to littermate controls. Concurrently, oxygen consumption of brown adipocytes and in vivo measured energy expenditure were significantly enhanced. In addition, glucose tolerance and insulin sensitivity of HFD-fed adipocyte-specific Gnai2-deficient mice were improved compared to the respective controls. In the absence of Gαi2, adrenergic stimulation of intracellular adipocyte cAMP levels was increased, which correlated with increased lipolysis and energy expenditure. CONCLUSION: We conclude that adipocyte Gαi2 is a major regulator of adipocyte lipid content in diet-induced obesity by inhibiting adipocyte lipolysis in a cAMP-dependent manner resulting in increased energy expenditure.


Assuntos
Tecido Adiposo/metabolismo , Subunidade alfa Gi2 de Proteína de Ligação ao GTP/metabolismo , Obesidade/metabolismo , Adipócitos Marrons/metabolismo , Adipócitos Brancos/metabolismo , Tecido Adiposo/fisiologia , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/metabolismo , Animais , Dieta Hiperlipídica , Metabolismo Energético , Subunidade alfa Gi2 de Proteína de Ligação ao GTP/fisiologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/fisiologia , Glucose/metabolismo , Intolerância à Glucose/metabolismo , Insulina/metabolismo , Resistência à Insulina/fisiologia , Lipólise , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/genética , Consumo de Oxigênio
10.
Front Immunol ; 9: 687, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29696016

RESUMO

The follicular (FO) versus marginal zone (MZ) B cell fate decision in the spleen depends upon BCR, BAFF, and Notch2 signaling. Whether or how Gi signaling affects this fate decision is unknown. Here, we show that direct contact with Notch ligand expressing stromal cells (OP9-Delta-like 1) cannot promote normal MZ B cell development when progenitor B cells lack Gαi proteins, or if Gi signaling is disabled. Consistent with faulty ADAM10-dependent Notch2 processing, Gαi-deficient transitional B cells had low ADAM10 membrane expression levels and reduced Notch2 target gene expression. Immunoblotting Gαi-deficient B cell lysates revealed a reduction in mature, processed ADAM10. Suggesting that Gαi signaling promotes ADAM10 membrane expression, stimulating normal transitional B cells with CXCL12 raised it, while inhibiting Gαi nucleotide exchange blocked its upregulation. Surprisingly, inhibiting Gαi nucleotide exchange in transitional B cells also impaired the upregulation of ADAM10 that occurs following antigen receptor crosslinking. These results indicate that Gαi signaling supports ADAM10 maturation and activity in transitional B cells, and ultimately Notch2 signaling to promote MZ B cell development.


Assuntos
Proteína ADAM10/fisiologia , Secretases da Proteína Precursora do Amiloide/fisiologia , Linfócitos B/fisiologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/fisiologia , Proteínas de Membrana/fisiologia , Animais , Células Cultivadas , Feminino , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Toxina Pertussis/farmacologia , Receptor Notch2/fisiologia , Transdução de Sinais , Baço/citologia
11.
Naunyn Schmiedebergs Arch Pharmacol ; 391(5): 513-522, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29470593

RESUMO

Hypertension is a risk factor for cardiovascular diseases, which have been associated with dysfunction of sympathetic and purinergic neurotransmission. Therefore, herein, we evaluated whether modifications of adenosine receptor signaling may contribute to the cardiac dysfunction observed in hypertension. Isolated right atria from spontaneously hypertensive (SHR) or normotensive Wistar rats (NWR) were used to investigate the influence of adenosine receptor signaling cascade in the cardiac chronotropism. Our results showed that adenosine, the endogenous agonist of adenosine receptors, and CPA, a selective agonist of A1 receptor, decreased the atrial chronotropism of NWR and SHR in a concentration- and time-dependent manner, culminating in cardiac arrest (0 bpm). Interestingly, a 3-fold lower concentration of adenosine was required to induce the negative chronotropic effect in SHR atria. Pre-incubation of tissues from both strains with DPCPX, a selective A1 receptor antagonist, inhibited the negative chronotropic effect of CPA, while simultaneous inhibition of A2 and A3 receptors, with ZM241385 and MRS1523, did not change the adenosine chronotropic effects. Moreover, 1 µg/ml pertussis toxin, which inactivates the Gαi protein subunit, reduced by 80% the negative chronotropic effects of adenosine in the NWR atrium, with minor effects in SHR tissue. These data indicate that the negative chronotropic effect of adenosine in right atrium depends exclusively on the activation of A1 receptors. Moreover, the distinct responsiveness of NWR and SHR atria to pertussis toxin reveals that the enhanced negative chronotropic response of SHR right atrium is probably due to an increased activity of Gαi protein-mediated.


Assuntos
Adenosina/fisiologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/fisiologia , Átrios do Coração/fisiopatologia , Hipertensão/fisiopatologia , Receptor A1 de Adenosina/fisiologia , Animais , Masculino , Ratos Endogâmicos SHR , Ratos Wistar , Transdução de Sinais
12.
Neurobiol Aging ; 64: 44-57, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29331876

RESUMO

Deposition of amyloid-ß (Aß), the proteolytic product of the amyloid precursor protein (APP), might cause neurodegeneration and cognitive decline in Alzheimer's disease (AD). However, the direct involvement of APP in the mechanism of Aß-induced degeneration in AD remains on debate. Here, we analyzed the interaction of APP with heterotrimeric Go protein in primary hippocampal cultures and found that Aß deposition dramatically enhanced APP-Go protein interaction in dystrophic neurites. APP overexpression rendered neurons vulnerable to Aß toxicity by a mechanism that required Go-Gßγ complex signaling and p38-mitogen-activated protein kinase activation. Gallein, a selective pharmacological inhibitor of Gßγ complex, inhibited Aß-induced dendritic and axonal dystrophy, abnormal tau phosphorylation, synaptic loss, and neuronal cell death in hippocampal neurons expressing endogenous protein levels. In the 3xTg-AD mice, intrahippocampal application of gallein reversed memory impairment associated with early Aß pathology. Our data provide further evidence for the involvement of APP/Go protein in Aß-induced degeneration and reveal that Gßγ complex is a signaling target potentially relevant for developing therapies for halting Aß degeneration in AD.


Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/fisiologia , Encéfalo/metabolismo , Disfunção Cognitiva/genética , Disfunção Cognitiva/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Doença de Alzheimer/patologia , Doença de Alzheimer/terapia , Animais , Células Cultivadas , Disfunção Cognitiva/patologia , Disfunção Cognitiva/terapia , Modelos Animais de Doenças , Hipocampo , Camundongos Transgênicos , Terapia de Alvo Molecular , Complexos Multiproteicos , Ratos
13.
J Neurosci ; 37(42): 10063-10073, 2017 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-28912160

RESUMO

Dopaminergic modulation of prefrontal cortex (PFC) is thought to play key roles in many cognitive functions and to be disrupted in pathological conditions, such as schizophrenia. We have previously described a phenomenon whereby dopamine D2 receptor (D2R) activation elicits afterdepolarizations (ADPs) in subcortically projecting (SC) pyramidal neurons within L5 of the PFC. These D2R-induced ADPs only occur following synaptic input, which activates NMDARs, even when the delay between the synaptic input and ADPs is relatively long (e.g., several hundred milliseconds). Here, we use a combination of electrophysiological, optogenetic, pharmacological, transgenic, and chemogenetic approaches to elucidate cellular mechanisms underlying this phenomenon in male and female mice. We find that knocking out D2Rs eliminates the ADP in a cell-autonomous fashion, confirming that this ADP depends on D2Rs. Hyperpolarizing current injection, but not AMPA receptor blockade, prevents synaptic stimulation from facilitating D2R-induced ADPs, suggesting that this phenomenon depends on the recruitment of voltage-dependent currents (e.g., NMDAR-mediated Ca2+ influx) by synaptic input. Finally, the D2R-induced ADP is blocked by inhibitors of cAMP/PKA signaling, insensitive to pertussis toxin or ß-arrestin knock-out, and mimicked by Gs-DREADD stimulation, suggesting that D2R activation elicits the ADP by stimulating cAMP/PKA signaling. These results show that this unusual physiological phenomenon, in which D2Rs enhance cellular excitability in a manner that depends on synaptic input, is mediated at the cellular level through the recruitment of signaling pathways associated with Gs, rather than the Gi/o-associated mechanisms that have classically been ascribed to D2Rs.SIGNIFICANCE STATEMENT Dopamine D2 receptors (D2Rs) in the prefrontal cortex (PFC) are thought to play important roles in behaviors, including working memory and cognitive flexibility. Variation in D2Rs has also been implicated in schizophrenia, Tourette syndrome, and bipolar disorder. Recently, we described a new mechanism through which D2R activation can enhance the excitability of pyramidal neurons in the PFC. Here, we explore the underlying cellular mechanisms. Surprisingly, although D2Rs are classically assumed to signal through Gi/o-coupled G-proteins and/or scaffolding proteins, such as ß-arrestin, we find that the effects of D2Rs on prefrontal pyramidal neurons are actually mediated by pathways associated with Gs-mediated signaling. Furthermore, we show how, via this D2R-dependent phenomenon, synaptic input can enhance the excitability of prefrontal neurons over timescales on the order of seconds. These results elucidate cellular mechanisms underlying a novel signaling pathway downstream of D2Rs that may contribute to prefrontal function under normal and pathological conditions.


Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/fisiologia , Córtex Pré-Frontal/citologia , Córtex Pré-Frontal/fisiologia , Células Piramidais/fisiologia , Receptores de Dopamina D2/fisiologia , Transdução de Sinais/fisiologia , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , N-Metilaspartato/farmacologia , Técnicas de Cultura de Órgãos , Córtex Pré-Frontal/efeitos dos fármacos , Células Piramidais/efeitos dos fármacos , Quimpirol/farmacologia , Receptores de Dopamina D2/agonistas , Transdução de Sinais/efeitos dos fármacos
14.
Endocrinology ; 158(6): 1715-1726, 2017 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-28407060

RESUMO

Age-related bone loss is an important risk factor for fractures in the elderly; it results from an imbalance in bone remodeling mainly due to decreased bone formation. We have previously demonstrated that endogenous G protein-coupled receptor (GPCR)-driven Gi signaling in osteoblasts (Obs) restrains bone formation in mice during growth. Here, we launched a longitudinal study to test the hypothesis that Gi signaling in Obs restrains bone formation in aging mice, thereby promoting bone loss. Our approach was to block Gi signaling in maturing Obs by the induced expression of the catalytic subunit of pertussis toxin (PTX) after the achievement of peak bone mass. In contrast to the progressive cancellous bone loss seen in aging sex-matched littermate control mice, aging female Col1(2.3)+/PTX+ mice showed an age-related increase in bone volume. Increased bone volume was associated with increased bone formation at both trabecular and endocortical surfaces as well as increased bending strength of the femoral middiaphyses. In contrast, male Col1(2.3)+/PTX+ mice were not protected from age-related bone loss. Our results indicate that Gi signaling markedly restrains bone formation at cancellous and endosteal bone surfaces in female mice during aging. Blockade of the relevant Gi-coupled GPCRs represents an approach for the development of osteoporosis therapies-at least in the long bones of aging women.


Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/fisiologia , Osteoblastos/metabolismo , Osteoporose/genética , Animais , Densidade Óssea/efeitos dos fármacos , Densidade Óssea/genética , Remodelação Óssea/efeitos dos fármacos , Remodelação Óssea/genética , Colágeno Tipo I/genética , Cadeia alfa 1 do Colágeno Tipo I , Feminino , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Osteoporose/metabolismo , Osteoporose/patologia , Toxina Pertussis/genética , Transdução de Sinais/fisiologia
15.
Mol Pharmacol ; 92(3): 265-277, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28424220

RESUMO

G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs) act in concert to regulate cell growth, proliferation, survival, and migration. Metabotropic GABAB receptor (GABABR) is the GPCR for the main inhibitory neurotransmitter GABA in the central nervous system. Increased expression of GABABR has been detected in human cancer tissues and cancer cell lines, but the role of GABABR in these cells is controversial and the underlying mechanism remains poorly understood. Here, we investigated whether GABABR hijacks RTK signaling to modulate the fates of human prostate cancer cells. RTK array analysis revealed that the GABABR-specific agonist baclofen selectively induced the transactivation of EGFR in PC-3 cells. EGFR transactivation resulted in the activation of ERK1/2 by a mechanism that is dependent on Gi/o protein and that requires matrix metalloproteinase-mediated proligand shedding. Positive allosteric modulators (PAMs) of GABABR, such as CGP7930, rac-BHFF, and GS39783, can function as PAM agonists to induce EGFR transactivation and subsequent ERK1/2 activation. Moreover, both baclofen and CGP7930 promoted cell migration and invasion through EGFR signaling. In summary, our observations demonstrated that GABABR transactivated EGFR in a ligand-dependent mechanism to promote prostate cancer cell migration and invasion, thus providing new insights into developing a novel strategy for prostate cancer treatment by targeting neurotransmitter signaling.


Assuntos
Receptores ErbB/genética , Neoplasias da Próstata/patologia , Ativação Transcricional , Regulação Alostérica , Linhagem Celular Tumoral , Movimento Celular , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/fisiologia , Humanos , Masculino , Invasividade Neoplásica , Receptores de GABA-B/fisiologia
16.
J Am Heart Assoc ; 5(11)2016 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-27912212

RESUMO

BACKGROUND: We previously showed that the levels of both Giα-2 and Giα-3 proteins were augmented in spontaneously hypertensive rats (SHRs) before the onset of hypertension. In addition, intraperitoneal injection of pertussis toxin, which inactivates both Giα proteins, prevented the development of hypertension in SHRs. The aim of the present study was to determine the specific contributions of Giα-2 and Giα-3 proteins to the development of hypertension. METHODS AND RESULTS: Antisense oligodeoxynucleotide of Giα-2 and Giα-3 encapsulated in PEG/DOTAP/DOPE cationic liposomes were administrated intravenously into 3-week-old prehypertensive SHRs and Wistar Kyoto rats, whereas the control Wistar Kyoto rats and SHRs received PBS, empty liposomes, or sense. The knockdown of Giα-2 but not Giα-3 protein attenuated tachycardia and prevented the development of hypertension up to age 6 weeks; thereafter, blood pressure started increasing and reached the same level as that of untreated SHRs at 9 weeks. Furthermore, Giα-2 and Giα-3 antisense oligodeoxynucleotide treatments significantly decreased the enhanced levels of Giα-2 and Giα-3 proteins, respectively, and enhanced levels of superoxide anion and NADPH oxidase activity in heart, aorta, and kidney and hyperproliferation of vascular smooth muscle cells from SHRs aged 6 weeks. In addition, antisense oligodeoxynucleotide treatment with Giα-2 but not Giα-3 restored enhanced inhibition of adenylyl cyclase by oxotremorine to WKY levels. CONCLUSIONS: These results suggested that the enhanced expression of Giα-2 but not Giα-3 protein plays an important role in the pathogenesis of hypertension and tachycardia in SHRs.


Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/fisiologia , Hipertensão/prevenção & controle , Oligodesoxirribonucleotídeos Antissenso/fisiologia , Taquicardia/prevenção & controle , Inibidores de Adenilil Ciclases/farmacologia , Animais , Aorta/metabolismo , Pressão Sanguínea/fisiologia , Células Cultivadas , Modelos Animais de Doenças , Subunidade alfa Gi2 de Proteína de Ligação ao GTP/deficiência , Subunidade alfa Gi2 de Proteína de Ligação ao GTP/fisiologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/deficiência , Técnicas de Silenciamento de Genes , Frequência Cardíaca/fisiologia , Rim/metabolismo , Lipossomos/administração & dosagem , Masculino , Músculo Liso Vascular/metabolismo , Miocárdio/metabolismo , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Transdução de Sinais/fisiologia , Transfecção/métodos
17.
Development ; 143(21): 3926-3932, 2016 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-27660326

RESUMO

Sensory perception in the inner ear relies on the hair bundle, the highly polarized brush of movement detectors that crowns hair cells. We previously showed that, in the mouse cochlea, the edge of the forming bundle is defined by the 'bare zone', a microvilli-free sub-region of apical membrane specified by the Insc-LGN-Gαi protein complex. We now report that LGN and Gαi also occupy the very tip of stereocilia that directly abut the bare zone. We demonstrate that LGN and Gαi are both essential for promoting the elongation and differential identity of stereocilia across rows. Interestingly, we also reveal that total LGN-Gαi protein amounts are actively balanced between the bare zone and stereocilia tips, suggesting that early planar asymmetry of protein enrichment at the bare zone confers adjacent stereocilia their tallest identity. We propose that LGN and Gαi participate in a long-inferred signal that originates outside the bundle to model its staircase-like architecture, a property that is essential for direction sensitivity to mechanical deflection and hearing.


Assuntos
Padronização Corporal , Proteínas de Transporte/fisiologia , Polaridade Celular , Cóclea/embriologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/fisiologia , Células Ciliadas Auditivas/fisiologia , Animais , Padronização Corporal/genética , Proteínas de Transporte/genética , Proteínas de Ciclo Celular , Polaridade Celular/genética , Cóclea/citologia , Surdez/embriologia , Surdez/genética , Embrião de Mamíferos , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos
18.
Physiol Rep ; 4(15)2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27528004

RESUMO

The heart is controlled by the sympathetic and parasympathetic limbs of the autonomic nervous system with inhibitory signaling mechanisms recruited in both limbs. The aim of this study was to determine the role of inhibitory heterotrimeric G proteins in the central nervous mechanisms underlying autonomic control of the heart and its potential role in arrhythmogenesis. Mice with conditional deletion of the inhibitory heterotrimeric G protein GαO in the presympathetic area of the rostral ventral lateral medulla (RVLM) were generated to determine the role of GαO-mediated signalling in autonomic control and electrophysiological properties of the heart. GαO deletion within the RVLM was not associated with changes in heart rate (HR) or the arterial blood pressure at rest (home cage, normal behavior). However, exposure to stressful conditions (novel environment, hypoxia, or hypercapnia) in these mice was associated with abnormal HR responses and an increased baroreflex gain when assessed under urethane anesthesia. This was associated with shortening of the ventricular effective refractory period. This phenotype was reversed by systemic beta-adrenoceptor blockade, suggesting that GαO depletion in the RVLM increases central sympathetic drive. The data obtained support the hypothesis that GαO-mediated signaling within the presympathetic circuits of the RVLM contributes to the autonomic control of the heart. GαO deficiency in the RVLM has a significant impact on cardiovascular responses to stress, cardiovascular reflexes and electrical properties of the heart.


Assuntos
Sistema Nervoso Autônomo/fisiologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/fisiologia , Coração/fisiologia , Bulbo/fisiologia , Animais , Pressão Sanguínea , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Frequência Cardíaca , Hemodinâmica , Hipercapnia/fisiopatologia , Hipóxia/fisiopatologia , Camundongos , Camundongos Transgênicos , Respiração , Transdução de Sinais , Função Ventricular
20.
Proc Natl Acad Sci U S A ; 113(4): 1092-7, 2016 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-26755577

RESUMO

Transient Receptor Potential Canonical (TRPC) proteins form nonselective cation channels commonly known to be activated downstream from receptors that signal through phospholipase C (PLC). Although TRPC3/C6/C7 can be directly activated by diacylglycerols produced by PLC breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2), the mechanism by which the PLC pathway activates TRPC4/C5 remains unclear. We show here that TRPC4 activation requires coincident stimulation of Gi/o subgroup of G proteins and PLCδ, with a preference for PLCδ1 over PLCδ3, but not necessarily the PLCß pathway commonly thought to be involved in receptor-operated TRPC activation. In HEK293 cells coexpressing TRPC4 and Gi/o-coupled µ opioid receptor, µ agonist elicited currents biphasically, with an initial slow phase preceding a rapidly developing phase. The currents were dependent on intracellular Ca(2+) and PIP2. Reducing PIP2 through phosphatases abolished the biphasic kinetics and increased the probability of channel activation by weak Gi/o stimulation. In both HEK293 cells heterologously expressing TRPC4 and renal carcinoma-derived A-498 cells endogenously expressing TRPC4, channel activation was inhibited by knocking down PLCδ1 levels and almost completely eliminated by a dominant-negative PLCδ1 mutant and a constitutively active RhoA mutant. Conversely, the slow phase of Gi/o-mediated TRPC4 activation was diminished by inhibiting RhoA or enhancing PLCδ function. Our data reveal an integrative mechanism of TRPC4 on detection of coincident Gi/o, Ca(2+), and PLC signaling, which is further modulated by the small GTPase RhoA. This mechanism is not shared with the closely related TRPC5, implicating unique roles of TRPC4 in signal integration in brain and other systems.


Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/fisiologia , Fosfolipase C delta/fisiologia , Canais de Cátion TRPC/fisiologia , Cálcio/metabolismo , Carbacol/farmacologia , Ala(2)-MePhe(4)-Gly(5)-Encefalina/farmacologia , Células HEK293 , Humanos , Transdução de Sinais , Proteína rhoA de Ligação ao GTP/fisiologia
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