RESUMO
G-protein-coupled receptors (GPCRs), also known as seven transmembrane receptors (7TMRs), typically interact with two distinct signal-transducers, i.e., G proteins and ß-arrestins (ßarrs). Interestingly, there are some non-canonical 7TMRs that lack G protein coupling but interact with ßarrs, although an understanding of their transducer coupling preference, downstream signaling, and structural mechanism remains elusive. Here, we characterize two such non-canonical 7TMRs, namely, the decoy D6 receptor (D6R) and the complement C5a receptor subtype 2 (C5aR2), in parallel with their canonical GPCR counterparts. We discover that D6R and C5aR2 efficiently couple to ßarrs, exhibit distinct engagement of GPCR kinases (GRKs), and activate non-canonical downstream signaling pathways. We also observe that ßarrs adopt distinct conformations for D6R and C5aR2, compared to their canonical GPCR counterparts, in response to common natural agonists. Our study establishes D6R and C5aR2 as ßarr-coupled 7TMRs and provides key insights into their regulation and signaling with direct implication for biased agonism.
Assuntos
Membrana Celular/metabolismo , Conformação Proteica , Transdução de Sinais , beta-Arrestinas/química , Animais , Proteínas de Ligação ao GTP/química , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Ligação Proteica , Domínios Proteicos , Estrutura Secundária de Proteína , Transporte Proteico , Receptor da Anafilatoxina C5a/metabolismoRESUMO
G protein-coupled receptors (GPCRs) are intricately involved in a diverse array of physiological processes and pathophysiological conditions. They constitute the largest class of drug target in the human genome, which highlights the importance of understanding the molecular basis of their activation, downstream signalling and regulation. In the past few years, considerable progress has been made in our ability to visualize GPCRs and their signalling complexes at the structural level. This is due to a series of methodological developments, improvements in technology and the use of highly innovative approaches, such as protein engineering, new detergents, lipidic cubic phase-based crystallization and microfocus synchrotron beamlines. These advances suggest that an unprecedented amount of structural information will become available in the field of GPCR biology in the coming years.
Assuntos
Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Animais , Humanos , Conformação Proteica , Engenharia de Proteínas/métodos , Transdução de Sinais/genéticaRESUMO
The D2 dopamine receptor (DRD2) is a therapeutic target for Parkinson's disease1 and antipsychotic drugs2. DRD2 is activated by the endogenous neurotransmitter dopamine and synthetic agonist drugs such as bromocriptine3, leading to stimulation of Gi and inhibition of adenylyl cyclase. Here we used cryo-electron microscopy to elucidate the structure of an agonist-bound activated DRD2-Gi complex reconstituted into a phospholipid membrane. The extracellular ligand-binding site of DRD2 is remodelled in response to agonist binding, with conformational changes in extracellular loop 2, transmembrane domain 5 (TM5), TM6 and TM7, propagating to opening of the intracellular Gi-binding site. The DRD2-Gi structure represents, to our knowledge, the first experimental model of a G-protein-coupled receptor-G-protein complex embedded in a phospholipid bilayer, which serves as a benchmark to validate the interactions seen in previous detergent-bound structures. The structure also reveals interactions that are unique to the membrane-embedded complex, including helix 8 burial in the inner leaflet, ordered lysine and arginine side chains in the membrane interfacial regions, and lipid anchoring of the G protein in the membrane. Our model of the activated DRD2 will help to inform the design of subtype-selective DRD2 ligands for multiple human central nervous system disorders.
Assuntos
Microscopia Crioeletrônica , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/ultraestrutura , Lipídeos de Membrana/metabolismo , Membranas Artificiais , Receptores de Dopamina D2/química , Receptores de Dopamina D2/ultraestrutura , Bromocriptina/química , Bromocriptina/metabolismo , Dopamina/química , Dopamina/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Humanos , Lipídeos de Membrana/química , Modelos Moleculares , Conformação Proteica , Receptores de Dopamina D2/agonistas , Receptores de Dopamina D2/metabolismo , Transdução de SinaisRESUMO
In this issue of Molecular Cell, crystal structures of a prostaglandin D2 receptor determined by Wang et al. (2018) reveal novel insights into differential ligand recognition among the members of lipid-binding GPCRs, and provide a structural framework for the identification of novel therapeutics in inflammatory disorders.
Assuntos
Receptores Imunológicos , Receptores de Prostaglandina , Humanos , LigantesRESUMO
FFAR1 is a G-protein-coupled receptor (GPCR) that responds to circulating free fatty acids to enhance glucose-stimulated insulin secretion and release of incretin hormones. Due to the glucose-lowering effect of FFAR1 activation, potent agonists for this receptor have been developed for the treatment of diabetes. Previous structural and biochemical studies of FFAR1 showed multiple sites of ligand binding to the inactive state but left the mechanism of fatty acid interaction and receptor activation unknown. We used cryo-electron microscopy to elucidate structures of activated FFAR1 bound to a Gq mimetic, which were induced either by the endogenous FFA ligand docosahexaenoic acid or γ-linolenic acid and the agonist drug TAK-875. Our data identify the orthosteric pocket for fatty acids and show how both endogenous hormones and synthetic agonists induce changes in helical packing along the outside of the receptor that propagate to exposure of the G-protein-coupling site. These structures show how FFAR1 functions without the highly conserved "DRY" and "NPXXY" motifs of class A GPCRs and also illustrate how the orthosteric site of a receptor can be bypassed by membrane-embedded drugs to confer full activation of G protein signaling.
Assuntos
Ácidos Graxos , Insulina , Insulina/metabolismo , Ligantes , Microscopia Crioeletrônica , Receptores Acoplados a Proteínas G/metabolismo , Ácidos Graxos não Esterificados , GlucoseRESUMO
ß-arrestins (ßarrs) are key regulators of G protein-coupled receptor (GPCR) signaling and trafficking, and their knockdown typically leads to a decrease in agonist-induced ERK1/2 MAP kinase activation. Interestingly, for some GPCRs, knockdown of ßarr1 augments agonist-induced ERK1/2 phosphorylation although a mechanistic basis for this intriguing phenomenon is unclear. Here, we use selected GPCRs to explore a possible correlation between the spatial positioning of receptor phosphorylation sites and the contribution of ßarr1 in ERK1/2 activation. We discover that engineering a spatially positioned double-phosphorylation-site cluster in the bradykinin receptor (B2 R), analogous to that present in the vasopressin receptor (V2 R), reverses the contribution of ßarr1 in ERK1/2 activation from inhibitory to promotive. An intrabody sensor suggests a conformational mechanism for this role reversal of ßarr1, and molecular dynamics simulation reveals a bifurcated salt bridge between this double-phosphorylation site cluster and Lys294 in the lariat loop of ßarr1, which directs the orientation of the lariat loop. Our findings provide novel insights into the opposite roles of ßarr1 in ERK1/2 activation for different GPCRs with a direct relevance to biased agonism and novel therapeutics.
Assuntos
Sistema de Sinalização das MAP Quinases , Receptores Acoplados a Proteínas G , Células HEK293 , Humanos , Fosforilação , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , beta-Arrestina 1/metabolismo , beta-Arrestinas/metabolismoRESUMO
Agonist stimulation of G-protein-coupled receptors (GPCRs) typically leads to phosphorylation of GPCRs and binding to multifunctional proteins called ß-arrestins (ßarrs). The GPCR-ßarr interaction critically contributes to GPCR desensitization, endocytosis, and downstream signaling, and GPCR-ßarr complex formation can be used as a generic readout of GPCR and ßarr activation. Although several methods are currently available to monitor GPCR-ßarr interactions, additional sensors to visualize them may expand the toolbox and complement existing methods. We have previously described antibody fragments (FABs) that recognize activated ßarr1 upon its interaction with the vasopressin V2 receptor C-terminal phosphopeptide (V2Rpp). Here, we demonstrate that these FABs efficiently report the formation of a GPCR-ßarr1 complex for a broad set of chimeric GPCRs harboring the V2R C terminus. We adapted these FABs to an intrabody format by converting them to single-chain variable fragments and used them to monitor the localization and trafficking of ßarr1 in live cells. We observed that upon agonist simulation of cells expressing chimeric GPCRs, these intrabodies first translocate to the cell surface, followed by trafficking into intracellular vesicles. The translocation pattern of intrabodies mirrored that of ßarr1, and the intrabodies co-localized with ßarr1 at the cell surface and in intracellular vesicles. Interestingly, we discovered that intrabody sensors can also report ßarr1 recruitment and trafficking for several unmodified GPCRs. Our characterization of intrabody sensors for ßarr1 recruitment and trafficking expands currently available approaches to visualize GPCR-ßarr1 binding, which may help decipher additional aspects of GPCR signaling and regulation.
Assuntos
Receptores Acoplados a Proteínas G/metabolismo , beta-Arrestina 1/metabolismo , Células HEK293 , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/metabolismo , Transporte Proteico , Receptores Acoplados a Proteínas G/genética , beta-Arrestina 1/genéticaRESUMO
The human complement component, C5a, binds two different seven-transmembrane receptors termed C5aR1 and C5aR2. C5aR1 is a prototypical G-protein-coupled receptor that couples to the Gαi subfamily of heterotrimeric G-proteins and ß-arrestins (ßarrs) following C5a stimulation. Peptide fragments derived from the C terminus of C5a can still interact with the receptor, albeit with lower affinity, and can act as agonists or antagonists. However, whether such fragments might display ligand bias at C5aR1 remains unexplored. Here, we compare C5a and a modified C-terminal fragment of C5a, C5apep, in terms of G-protein coupling, ßarr recruitment, endocytosis, and extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase activation at the human C5aR1. We discover that C5apep acts as a full agonist for Gαi coupling as measured by cAMP response and extracellular signal-regulated kinase 1/2 phosphorylation, but it displays partial agonism for ßarr recruitment and receptor endocytosis. Interestingly, C5apep exhibits full-agonist efficacy with respect to inhibiting lipopolysaccharide-induced interleukin-6 secretion in human macrophages, but its ability to induce human neutrophil migration is substantially lower compared with C5a, although both these responses are sensitive to pertussis toxin treatment. Taken together, our data reveal that compared with C5a, C5apep exerts partial efficacy for ßarr recruitment, receptor trafficking, and neutrophil migration. Our findings therefore uncover functional bias at C5aR1 and also provide a framework that can potentially be extended to chemokine receptors, which also typically interact with chemokines through a biphasic mechanism.
Assuntos
Complemento C5a/metabolismo , Endocitose , Receptor da Anafilatoxina C5a/metabolismo , beta-Arrestinas/metabolismo , Sequência de Aminoácidos , Movimento Celular , Complemento C5a/genética , Células HEK293 , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Neutrófilos/metabolismo , Fosforilação , Ligação Proteica , Receptor da Anafilatoxina C5a/genética , Homologia de Sequência , Transdução de Sinais , beta-Arrestinas/genéticaRESUMO
The past 20 years have resulted in unprecedented progress in understanding brain energy metabolism and its role in health and disease. In this review, which was initiated at the 14th International Society for Neurochemistry Advanced School, we address the basic concepts of brain energy metabolism and approach the question of why the brain has high energy expenditure. Our review illustrates that the vertebrate brain has a high need for energy because of the high number of neurons and the need to maintain a delicate interplay between energy metabolism, neurotransmission, and plasticity. Disturbances to the energetic balance, to mitochondria quality control or to glia-neuron metabolic interaction may lead to brain circuit malfunction or even severe disorders of the CNS. We cover neuronal energy consumption in neural transmission and basic ('housekeeping') cellular processes. Additionally, we describe the most common (glucose) and alternative sources of energy namely glutamate, lactate, ketone bodies, and medium chain fatty acids. We discuss the multifaceted role of non-neuronal cells in the transport of energy substrates from circulation (pericytes and astrocytes) and in the supply (astrocytes and microglia) and usage of different energy fuels. Finally, we address pathological consequences of disrupted energy homeostasis in the CNS.
Assuntos
Encéfalo/metabolismo , Metabolismo Energético/fisiologia , Neuroquímica/educação , Estudantes , Animais , Astrócitos/metabolismo , Congressos como Assunto/tendências , Humanos , Neuroglia/metabolismo , Neurônios/metabolismoRESUMO
BACKGROUND: Sleep deprivation (SD) leads to cognitive impairment. Neuroinflammation could be a significant contributing factor in the same. An increase in regional brain pro-inflammatory cytokines induces cognitive deficits, however, the magnitude of the effect under SD is not apparent. It is plausible that microglia activation could be involved in the SD-induced cognitive impairment by modulation of neuronal cell proliferation, differentiation, and brain-derived neuronal factor (BDNF) level. The present study aimed to evaluate the possible beneficial effect of minocycline in amelioration of spatial memory decline during SD by its anti-inflammatory and neuroprotective actions. We scrutinized the effect of minocycline on the inflammatory cytokine levels associated with glial cells (microglia and astrocytes) activity and neurogenesis markers crucial for behavioral functions during SD. METHODS: Male Sprague-Dawley rats weighing 230-250 g were sleep deprived for 48 h using automated cage shaking apparatus. The spatial memory was tested using MWM apparatus immediately after completion of SD with and without minocycline. The animals were euthanized, blood was collected, and brain was extracted for neuroinflammation and neurogenesis studies. The set of experiments were also conducted with use of temozolomide, a neurogenesis blocker. RESULTS: Minocycline treatment increased the body weight, food intake, and spatial memory performance which declined during SD. It reduced the pro-inflammatory and increased the anti-inflammatory cytokine levels in hippocampus and plasma and inhibited the reactive gliosis in the hippocampus evidenced by improved cell count, morphology, and immunoreactivity. Additionally, minocycline administration promoted neurogenesis at different stages: proliferation (BrdU, Ki-67), differentiation (DCX) cells and growth factor (BDNF). However, no significant change was observed in maturation (NeuN) during SD. In addition, molecules related to behavior, inflammation, and neurogenesis were shown to be more affected after temozolomide administration during SD, and changes were restored with minocycline treatment. We observed a significant correlation of neurogenesis with microglial activation, cytokine levels, and spatial memory during SD. CONCLUSION: The present study demonstrated that the SD-induced decline in spatial memory, neuronal cells proliferation, differentiation, and BDNF level could be attributed to upregulation of neuroinflammatory molecules, and minocycline may be an effective intervention to counteract these changes. Microglial activation is involved in SD-induced changes in inflammatory molecules, neurogenesis, and spatial memory.
Assuntos
Hipocampo/imunologia , Microglia/patologia , Neurogênese/imunologia , Privação do Sono/complicações , Memória Espacial/fisiologia , Animais , Transtornos Cognitivos/imunologia , Proteína Duplacortina , Hipocampo/patologia , Masculino , Aprendizagem em Labirinto , Microglia/imunologia , Ratos , Ratos Sprague-Dawley , Privação do Sono/imunologiaRESUMO
Low oxygen environments, like hypobaric hypoxia (HH), are common nodes in a number of diseases characterized by neuroinflammation, which is detrimental to the structural and functional aspects of hippocampal circuitry. Hypoxic conditions lead to elevation of inflammasome-mediated inflammation that may contribute to cognitive deficits. However, a systematic investigation of the impact of inflammasome-mediated neuroinflammation on the components of neurogenic niche during HH remains to be elusive. Cerebral hypoxia was induced in adult male Sprague Dawley rats via decreasing partial pressure of oxygen. The effect of HH (1, 3, and 7 days at 25,000 ft) on social memory, anxiety, adult neurogenesis, and NLRP3- (NLR family pyrin domain containing 3) mediated neuroinflammation in the dentate gyrus (DG) was explored in detail. Furthermore, we explored the therapeutic efficacy of cyclooxygenase-1 inhibitor (valeryl salicylate, 5 mg/kg/day, i.p.) and EP1 receptor (EP1R) antagonist (SC19220, 1 mg/kg/day, i.p.) on HH-induced deficits. Seven days of HH exposure induced alteration in social and anxiety-like behavior along with perturbation in adult neurogenesis. Elevation in NLRP3, caspase-1, and IL-1ß levels was observed during HH from day 1. A notable increase in the COX-1/EP1R pathway in activated glial cells in DG was evident during HH. COX-1 inhibitor and EP1R antagonist mitigated the detrimental effects of HH on social memory, adult neurogenesis via blunting NLRP3-mediated inflammation. Our data showed induction of the COX-1/EP1R pathway in the glial cells, which is detrimental to neurogenesis and social memory, opening up the possibility that the COX-1/EP1R pathway is a plausible target for inflammasome-related neurogenesis impairments.
Assuntos
Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Animais , Proteínas de Transporte , Ciclo-Oxigenase 1/metabolismo , Hipóxia/metabolismo , Inflamassomos/metabolismo , Inflamação/metabolismo , Masculino , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Neurogênese , Oxigênio/metabolismo , Ratos , Ratos Sprague-Dawley , Interação SocialRESUMO
INTRODUCTION: Boron (B) is thought to play key role in proper bone growth and development as well as have some role in regulation of minerals such as calcium (Ca), phosphorus (P) and magnesium (Mg) which act synergistically with vitamin D. OBJECTIVE: Present study was planned in two phases to assess the effect of optimum and supranutritional levels of (B) in the form of boric acid (BA) supplementation on bone health of growing cross bred calves. METHOD: During Phase-1, twenty four male crossbred calves were blocked into four groups (nâ¯=â¯6) on the basis of their body weight (154.83⯱â¯8.5â¯kg), age (7-9 months) and were supplemented with 0 (C), 2.6 (T-1), 5.4 (T-2) and 10.7 (T-3) g BA for appropriate B (0.175 adjustment factor to calculate B form BA) consumption i.e. 0, 100, 200 and 400â¯ppm in each group respectively, for 90 days. During phase 2, twenty-one male crossbred calves were divided into 3 groups (nâ¯=â¯7) on the basis of their body weight (103.76⯱â¯4.34â¯kg) and age (5-8 months). All the groups were on similar dietary regimen with additional supplementation of boric acid as 0â¯g (control); 3.6â¯g (200â¯ppm B; T-1) and 10.8â¯g (600â¯ppm B; T-2), respectively for a period of 120 d. RESULTS: From the first experiment it is reported that plasma levels of bovine alkaline phosphatase (BALP), type I collagen cross-linked N-telopeptide (NTx) and Ca were significantly (Pâ¯<â¯0.05) affected in T-2 and T-3 groups as compared to T-1 and control groups. Whereas, plasma osteocalcin (OCN) concentration was found to be higher in T-2 and T-3 groups as compared to control group. However, plasma concentrations (ng/mL) of tartrate resistant acid phosphatase (TRAP) remained unaltered due to dietary treatments. Based on the results, another experiment was conducted to validate the above findings and further to determine the effect of still higher i.e supranutritional levels of BA supplementation on bone health of calves. Results revealed that supplementation of BA in T-2â¯group had no beneficial effect on bone health as the plasma concentration of BALP, OCN, NTx, 25 (OH) vitamin D and Ca as compared to T-1â¯group in phase 2. Other possible attributes of bone health i.e. plasma concentration of Mg, P, parathyroid hormone (PTH), and calcitonin were not affected by BA supplementation at any levels. CONCLUSION: Overall from present study it can be concluded that supplementation of boric acid 3.6â¯g/d (equivalent to 200â¯ppm B) in the diet of growing animals has positive effect on bone health related biomarkers (OCN, NTx and BALP) and supplementation of supranutritional level of BA i.e. 10.8â¯g (equivalent to 600â¯ppm B) level had neither additional beneficial nor harmful effect on bone health of calves.
Assuntos
Osso e Ossos/efeitos dos fármacos , Ácidos Bóricos/farmacologia , Animais , Biomarcadores/sangue , Peso Corporal/efeitos dos fármacos , Densidade Óssea/efeitos dos fármacos , Osso e Ossos/metabolismo , Ácidos Bóricos/administração & dosagem , Bovinos , Suplementos Nutricionais , Masculino , Clima TropicalRESUMO
AIMS: Evidence suggests that hypobaric hypoxia (HH) exposure causes biochemical and molecular level perturbations in brain resulting in associated cognitive dysfunction. However, the possible effect of HH on amygdala and the associated limbic regions based functions remains elusive. Regulated fear expression is essential for quick adaptations and optimal behavioral response. Therefore, the present study aims to investigate the effect of HH on biochemical and molecular mechanisms in amygdala involved in fear memory regulation along with the hippocampus and prefrontal cortex based fear memory. MATERIALS AND METHODS: Adult male Sprague Dawley rats were subjected to cued and contextual fear memory assessment following simulated HH exposure (25,000 ft) for 3 and 7 days. Plasma and limbic tissue (Prefrontal cortex, hippocampus and amygdala) samples were collected for biochemical and molecular studies. KEY FINDINGS: Results revealed a decrease in contextual and cued fear memory retrieval, indicating fear memory dysregulation under HH exposure. Increased level of norepinephrine, dopamine, corticosterone and glutamate along with a decline in serotonin and GABA level was observed in plasma and limbic tissue after 3 and 7 days of HH exposure. Dysregulation of neuromodulation, neuronal survival and synaptic homeostasis was also evident from observed decline in tryptophan hydroxylase, BDNF, synaptophysin, synapsin1, PSD95 and an increase in tyrosine hydroxylase immunoreactivity in limbic region under HH exposure. SIGNIFICANCE: Dysregulation of limbic region signaling molecules associated with survival and maintenance of synaptic plasticity (Synaptophysin, synapsin1 and PSD95), neurotrophic factor (BDNF) and shift in monoamines, corticosterone, glutamate and GABA levels may contribute to the HH induced fear memory impairment.
Assuntos
Medo , Hipóxia/fisiopatologia , Memória , Plasticidade Neuronal , Animais , Condicionamento Operante , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Regulated fear and extinction memory is essential for balanced behavioral response. Limbic brain regions are susceptible to hypobaric hypoxia (HH) and are putative target for fear extinction deficit and dysregulation. The present study aimed to examine the effect of HH and Ginkgo biloba extract (GBE) on fear and extinction memory with the underlying mechanism. Adult male Sprague-Dawley rats were evaluated for fear extinction and anxious behavior following GBE administration during HH exposure. Blood and tissue (PFC, hippocampus and amygdala) samples were collected for biochemical, morphological and molecular studies. Results revealed deficit in contextual and cued fear extinction following 3 days of HH exposure. Increased corticosterone, glutamate with decreased GABA level was found with marked pyknosis, decrease in apical dendritic length and number of functional spines. Decline in mRNA expression level of synaptic plasticity genes and immunoreactivity of BDNF, synaptophysin, PSD95, spinophilin was observed following HH exposure. GBE administration during HH exposure improved fear and extinction memory along with decline in anxious behavior. It restored corticosterone, glutamate and GABA levels with an increase in apical dendritic length and number of functional spines with a reduction in pyknosis. It also improved mRNA expression level and immunoreactivity of neurotrophic and synaptic proteins. The present study is the first which demonstrates fear extinction deficit and anxious behavior following HH exposure. GBE administration ameliorated fear and extinction memory dysregulation by restoration of neurotransmitter levels, neuronal pyknosis and synaptic connections along with improved neurotrophic and synaptic protein expressions.
Assuntos
Encéfalo/fisiopatologia , Extinção Psicológica/fisiologia , Medo/fisiologia , Hipóxia/fisiopatologia , Hipóxia/psicologia , Transtornos da Memória/fisiopatologia , Extratos Vegetais/administração & dosagem , Animais , Encéfalo/efeitos dos fármacos , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Condicionamento Clássico/efeitos dos fármacos , Condicionamento Clássico/fisiologia , Extinção Psicológica/efeitos dos fármacos , Medo/efeitos dos fármacos , Ginkgo biloba , Hipóxia/complicações , Masculino , Transtornos da Memória/etiologia , Transtornos da Memória/prevenção & controle , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Ratos Sprague-DawleyRESUMO
Agonist stimulation of G-protein-coupled receptors (GPCRs) typically results in phosphorylation and activation of ERK (Extracellular-signal Regulated Kinase) which is a member of MAP kinase (Mitogen-Activated Protein kinase) family. Detection of phosphorylated ERK1/2 MAP kinase has been widely used as readout of GPCR signaling in heterologous cells, primary cells, tissues and even in animal studies. ERK1/2 phosphorylation downstream of GPCRs is now well established to arise from the activation of both, the heterotrimeric G-proteins and ß-arrestins (ßarrs) with distinct spatio-temporal components. Here, we present a step-by-step protocol for measuring agonist-induced ERK1/2 MAP kinase activation downstream of GPCRs using standard Western blotting assay. Note: ERK1/2 is also referred to as p44/42 MAP kinase. ERK1 and ERK2 are same as Mitogen-Activated Protein Kinase 3 (MAP3) and Mitogen-Activated Protein Kinase 1 (MAP1), respectively.
Assuntos
Técnicas Citológicas/métodos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Proliferação de Células , Células HEK293 , Humanos , Fosforilação , Polietilenoimina/químicaRESUMO
High-altitude hypoxia (HH) causes a spectrum of pathophysiological effects, including headaches, gliovascular dysfunction, and cognitive slowing. Previous studies have shown arachidonic acid (AA) metabolism due to cyclooxygenase (COX) activity before clinical manifestations in many diseases. AA metabolites, including COXs and prostaglandin E2 (PGE2), are well known immunomodulators. However, the relative contribution of COX-2 and COX-1 isoforms in the downstream proinflammatory responses and cognitive deficit in HH remains unknown. In the present study, AA metabolism via the COX pathway was investigated in Sprague Dawley rats after 0, 1, 3, and 7 days of HH exposure. Furthermore, we investigated the inflammatory response and cell-type-specific induction of both COXs. Our data revealed that AA metabolites peaked on day 3 of HH exposure. Interestingly, we observed endothelial and microglial activation on day 1, accompanied by an increase in the levels of proinflammatory cytokines, followed by astrocyte activation on day 3. We showed that the increase in COX activity during HH culminated in a significant increase in hippocampal inflammation, concomitant with spatial memory impairment and neuronal injury at day 7 of HH. We showed HH induced distinct COX-1 expression in endothelial and microglial cells, whereas it induced COX-2 expression predominantly in neurons, endothelial cells, and astrocytes. Notably, our data showed that the inhibition of COX-1 using valeryl salicylate had a prominent role in containing hippocampal inflammation by reducing microglial activation. COX-2 inhibition using celecoxib, along with COX-1 inhibition, ameliorated spatial memory impairment, astrocyte activation, and neurodegeneration after HH exposure.
Assuntos
Doença da Altitude/metabolismo , Disfunção Cognitiva/metabolismo , Ciclo-Oxigenase 1/metabolismo , Ciclo-Oxigenase 2/metabolismo , Hipóxia/metabolismo , Inflamação/metabolismo , Animais , Apoptose/fisiologia , Ácido Araquidônico/metabolismo , Astrócitos/metabolismo , Dinoprostona/metabolismo , Hipocampo/metabolismo , Masculino , Microglia/patologia , Ratos Sprague-Dawley , Aprendizagem Espacial , Memória EspacialRESUMO
Hypobaric hypoxia (HH) is an environmental stress encountered at high altitude. It has been shown that HH resulted in spine atrophy and working memory deficits. Kalirin-7, a postsynaptic density protein, plays an important and key role in regulating spine dynamics and its plasticity. Spine atrophy is implicated in HH induced memory deficits but role of Kalirin-7 in this phenomenon is not studied. Present study is therefore designed to investigate the effect of chronic HH exposure on Kalirin-7 expression in hippocampus and its role in spatial working memory deficits. Adult rats (n = 12, 3 months old) were exposed to a simulated altitude of 25,000 feet for 7 days. Following HH exposure, spatial working memory was assessed with Radial arm maze and T maze. Hippocampal expression of Kalrin-7 was estimated at mRNA and protein levels. Results of behavioural experiments showed that HH causes significant decrease in the spatial working memory. There was a significant reduction in the protein expression of Kalirin-7 in the hippocampus of hypoxia exposed rats (43.89 ± 7.43) as compared to the control (69.54 ± 10.99). The mRNA expression of Kalrin-7 also exhibits significant reduction (0.59 ± 0.05) in the exposed group as compared to the control (0.98 ± 0.07). Immunohistochemistry showed that Kalirin-7 is decreased significantly in CA1, CA3 and DG regions of the hippocampus. Moreover, memory deficits are significantly correlated with decreased immunoreactivity of the hippocampal Kalirin-7. In conclusion, it can be said therefore, that change in Kalirin-7 expression in the hippocampus is associated with HH induced working memory deficit.
Assuntos
Doença da Altitude/metabolismo , Fatores de Troca do Nucleotídeo Guanina/biossíntese , Memória de Curto Prazo/fisiologia , Altitude , Animais , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Hipocampo/metabolismo , Hipocampo/patologia , Hipóxia/metabolismo , Hipóxia/fisiopatologia , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Transtornos da Memória/etiologia , Ratos , Ratos Sprague-DawleyRESUMO
Desensitization, signaling, and trafficking of G-protein-coupled receptors (GPCRs) are critically regulated by multifunctional adaptor proteins, ß-arrestins (ßarrs). The two isoforms of ßarrs (ßarr1 and 2) share a high degree of sequence and structural similarity; still, however, they often mediate distinct functional outcomes in the context of GPCR signaling and regulation. A mechanistic basis for such a functional divergence of ßarr isoforms is still lacking. By using a set of complementary approaches, including antibody-fragment-based conformational sensors, we discover structural differences between ßarr1 and 2 upon their interaction with activated and phosphorylated receptors. Interestingly, domain-swapped chimeras of ßarrs display robust complementation in functional assays, thereby linking the structural differences between receptor-bound ßarr1 and 2 with their divergent functional outcomes. Our findings reveal important insights into the ability of ßarr isoforms to drive distinct functional outcomes and underscore the importance of integrating this aspect in the current framework of biased agonism.
Assuntos
beta-Arrestinas/química , Células HEK293 , Humanos , Simulação de Dinâmica Molecular , Domínios Proteicos , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de Sinais , beta-Arrestinas/genética , beta-Arrestinas/metabolismoRESUMO
The effect of multiple/repeated impacts on a repaired composite was investigated using a low-velocity impact test. The composite samples were fabricated through a vacuum resin infusion method (VARI) and repaired by a scarf repair technique. Later, a repeated low-velocity impact test was performed on the original and repaired composites samples. Performance of the multi-impacted repaired and original samples was evaluated and compared by measuring maximum contact force, maximum displacement, maximum time duration, absorbed energy and damage area. Photographs of the post-impacted samples were taken to observe the multi-impact damage progression through visual inspection. The results showed that each repeated impact subjected the samples to more damage. Tensile tests revealed that the scarf repair restored 81.23% strength. It was also observed that the sample obtained the highest damage dent in the low-velocity impact test that failed early during a tensile test and carried the lowest ultimate load.