RESUMO
Cancer invasion and metastasis are known to be potentiated by the expression of aquaporins (AQPs). Likewise, the expression levels of AQPs have been shown to be prognostic for survival in patients and have a role in tumor growth, edema, angiogenesis, and tumor cell migration. Thus, AQPs are key players in cancer biology and potential targets for drug development. Here, we present the single-particle cryo-EM structure of human AQP7 at 3.2-Å resolution in complex with the specific inhibitor compound Z433927330. The structure in combination with MD simulations shows that the inhibitor binds to the endofacial side of AQP7. In addition, cancer cells treated with Z433927330 show reduced proliferation. The data presented here serve as a framework for the development of AQP inhibitors.
Assuntos
Aquaporinas , Neoplasias , Humanos , Aquaporinas/metabolismo , Aquaporina 1/metabolismoRESUMO
The aquaglyceroporins are a subfamily of aquaporins that conduct both water and glycerol. Aquaporin-3 (AQP3) has an important physiological function in renal water reabsorption, and AQP3-mediated hydrogen peroxide (H2O2) permeability can enhance cytokine signaling in several cell types. The related aquaglyceroporin AQP7 is required for dendritic cell chemokine responses and antigen uptake. Selective small-molecule inhibitors are desirable tools for investigating the biological and pathological roles of these and other AQP isoforms. Here, using a calcein fluorescence quenching assay, we screened a library of 7360 drug-like small molecules for inhibition of mouse AQP3 water permeability. Hit confirmation and expansion with commercially available substances identified the ortho-chloride-containing compound DFP00173, which inhibited mouse and human AQP3 with an IC50 of â¼0.1-0.4 µm but had low efficacy toward mouse AQP7 and AQP9. Surprisingly, inhibitor specificity testing revealed that the methylurea-linked compound Z433927330, a partial AQP3 inhibitor (IC50, â¼0.7-0.9 µm), is a potent and efficacious inhibitor of mouse AQP7 water permeability (IC50, â¼0.2 µm). Stopped-flow light scattering measurements confirmed that DFP00173 and Z433927330 inhibit AQP3 glycerol permeability in human erythrocytes. Moreover, DFP00173, Z433927330, and the previously identified AQP9 inhibitor RF03176 blocked aquaglyceroporin H2O2 permeability. Molecular docking to AQP3, AQP7, and AQP9 homology models suggested interactions between these inhibitors and aquaglyceroporins at similar binding sites. DFP00173 and Z433927330 constitute selective and potent AQP3 and AQP7 inhibitors, respectively, and contribute to a set of isoform-specific aquaglyceroporin inhibitors that will facilitate the evaluation of these AQP isoforms as drug targets.
Assuntos
Aquaporina 3/antagonistas & inibidores , Aquaporinas/antagonistas & inibidores , Tiofenos/farmacologia , Animais , Células CHO , Permeabilidade da Membrana Celular , Cricetulus , Eritrócitos/metabolismo , Glicerol/metabolismo , Humanos , Camundongos , Simulação de Acoplamento Molecular , Relação Estrutura-Atividade , Tiofenos/química , Água/metabolismoRESUMO
Aquaporin 11 (AQP11) is a channel protein with unknown biological function that is expressed in multiple tissues, including the kidney proximal tubule (PT) epithelium. Constitutive deletion of Aqp11 in mice (Aqp11-/-) results in early postnatal vacuolization in the PT and development of apparent cysts at 2 wk of age. Electron microscopy of adult Aqp11-/- mouse PT cells revealed a dilated rough endoplasmic reticulum. These changes may cause renal failure and premature death. This study examined 1) whether postnatal deletion of Aqp11 affects PT injury and cyst formation, 2) the temporal role of Aqp11 deletion on cyst development, and 3) the nature of apparent cysts. Tamoxifen-inducible Aqp11-/- mice were generated (Ti-Aqp11-/-). Deletion of Aqp11 at postnatal days (P) P2, P4, P6, P8, and P12 was investigated. Deranged renal development, especially in kidney cortex, PT cell vacuolization, and apparent tubular cysts developed only in mice where Aqp11 gene disruption was induced until P8. Aqp11 gene deletion from P12 onward did not result in a clear deficiency in renal development, PT injury, or cyst formation. Intraperitoneal injection of biotinylated-dextran (10 kDa) into adult mice resulted in extensive endocytic dextran uptake in both cystic Aqp11-/- and control PT epithelium, respectively. This suggests that apparent cysts are not membrane-enclosed structures but represent PT dilations. We conclude that Aqp11-/- mice develop cyst-like dilated proximal tubules without documented cysts at time of death.
Assuntos
Aquaporinas/metabolismo , Túbulos Renais Proximais/metabolismo , Rim/metabolismo , Doenças Renais Policísticas/genética , Animais , Aquaporinas/genética , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/patologia , Rim/patologia , Túbulos Renais Proximais/patologia , Camundongos , Camundongos Knockout , Doenças Renais Policísticas/metabolismo , Doenças Renais Policísticas/patologia , Índice de Gravidade de DoençaRESUMO
Aquaporins (AQPs) are water channel proteins robustly expressed in the central nervous system (CNS). A number of previous studies described the cellular expression sites and investigated their major roles and function in the brain and spinal cord. Among thirteen different mammalian AQPs, AQP1 and AQP4 have been mainly studied in the CNS and evidence has been presented that they play important roles in the pathogenesis of CNS injury, edema and multiple diseases such as multiple sclerosis, neuromyelitis optica spectrum disorders, amyotrophic lateral sclerosis, glioblastoma multiforme, Alzheimer's disease and Parkinson's disease. The objective of this review is to highlight the current knowledge about AQPs in the spinal cord and their proposed roles in pathophysiology and pathogenesis related to spinal cord lesions and injury.
Assuntos
Aquaporinas/metabolismo , Medula Espinal/metabolismo , Animais , Aquaporina 4/metabolismo , Astrócitos/metabolismo , Sistema Nervoso Central/metabolismo , HumanosRESUMO
In the kidney, the actions of vasopressin on its type-2 receptor (V2R) induce increased water reabsorption alongside polyphosphorylation and membrane targeting of the water channel aquaporin-2 (AQP2). Loss-of-function mutations in the V2R cause X-linked nephrogenic diabetes insipidus. Treatment of this condition would require bypassing the V2R to increase AQP2 membrane targeting, but currently no specific pharmacological therapy is available. The present study examined specific E-prostanoid receptors for this purpose. In vitro, prostaglandin E2 (PGE2) and selective agonists for the E-prostanoid receptors EP2 (butaprost) or EP4 (CAY10580) all increased trafficking and ser-264 phosphorylation of AQP2 in Madin-Darby canine kidney cells. Only PGE2 and butaprost increased cAMP and ser-269 phosphorylation of AQP2. Ex vivo, PGE2, butaprost, or CAY10580 increased AQP2 phosphorylation in isolated cortical tubules, whereas PGE2 and butaprost selectively increased AQP2 membrane accumulation in kidney slices. In vivo, a V2R antagonist caused a severe urinary concentrating defect in rats, which was greatly alleviated by treatment with butaprost. In conclusion, EP2 and EP4 agonists increase AQP2 phosphorylation and trafficking, likely through different signaling pathways. Furthermore, EP2 selective agonists can partially compensate for a nonfunctional V2R, providing a rationale for new treatment strategies for hereditary nephrogenic diabetes insipidus.
Assuntos
Aquaporina 2/metabolismo , Diabetes Insípido Nefrogênico/metabolismo , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Alprostadil/análogos & derivados , Alprostadil/farmacologia , Animais , Antagonistas dos Receptores de Hormônios Antidiuréticos , Aquaporina 2/genética , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , AMP Cíclico/metabolismo , Diabetes Insípido Nefrogênico/genética , Diabetes Insípido Nefrogênico/prevenção & controle , Dinoprostona/análogos & derivados , Dinoprostona/farmacologia , Cães , Relação Dose-Resposta a Droga , Immunoblotting , Rim/efeitos dos fármacos , Rim/metabolismo , Masculino , Microscopia Confocal , Fosforilação/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Pirrolidinonas/farmacologia , Ratos , Ratos Wistar , Receptores de Prostaglandina E Subtipo EP2/agonistas , Receptores de Prostaglandina E Subtipo EP2/genética , Receptores de Prostaglandina E Subtipo EP4/agonistas , Receptores de Prostaglandina E Subtipo EP4/genética , Receptores de Vasopressinas/genética , Receptores de Vasopressinas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Vasopressinas/metabolismo , Vasopressinas/farmacologiaRESUMO
BACKGROUND: The involvement of aquaporin (AQP) water and small solute channels in the etiology of several diseases, including cancer, neuromyelitis optica and body fluid imbalance disorders, has been suggested previously. Furthermore, results obtained in a mouse model suggested that AQP9 function contributes to hyperglycemia in type-2 diabetes. In addition, the physiological role of several AQP family members remains poorly understood. Small molecule inhibitors of AQPs are therefore desirable to further study AQP physiological and pathophysiological functions. METHODS: The binding of recently established AQP9 inhibitors to a homology model of AQP9 was investigated by molecular dynamics simulations and molecular docking. Putative inhibitor binding sites identified with this procedure were modified by site-directed mutagenesis. Active compounds were measured in a mammalian cell water permeability assay of mutated AQP9 isoforms and tested for changes in inhibitory effects. CONTROLS: Three independent cell lines were established for each mutated AQP9 isoform and functionality of mutant isoforms was established. PRINCIPAL FINDINGS: We have identified putative binding sites of recently established AQP9 inhibitors. This information facilitated successful identification of novel AQP9 inhibitors with low micromolar IC50 values in a cell based assay by in silico screening of a compound library targeting specifically this binding site. SIGNIFICANCE: We have established a successful strategy for AQP small molecule inhibitor identification. AQP inhibitors may be relevant as experimental tools, to enhance our understanding of AQP function, and in the treatment of various diseases.
Assuntos
Aquaporinas/antagonistas & inibidores , Aquaporinas/química , Modelos Moleculares , Bibliotecas de Moléculas Pequenas/química , Animais , Aquaporinas/genética , Aquaporinas/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Humanos , Camundongos , Mutagênese Sítio-Dirigida , Bibliotecas de Moléculas Pequenas/metabolismo , Homologia Estrutural de ProteínaRESUMO
Inflammation, apoptosis and oxidative stress play crucial roles in the deterioration of severe acute pancreatitis-associated acute respiratory distress syndrome (SAP-ARDS). Unfortunately, despite a high mortality rate of 45 %[1], there are limited treatment options available for ARDS outside of last resort options such as mechanical ventilation and extracorporeal support strategies[2]. This study investigated the potential therapeutic role and mechanisms of AQP9 inhibitor RG100204 in two animal models of severe acute pancreatitis, inducing acute respiratory distress syndrome: 1) a sodium-taurocholate induced rat model, and 2) and Cerulein and lipopolysaccharide induced mouse model. RG100204 treatment led to a profound reduction in inflammatory cytokine expression in pancreatic, and lung tissue, in both models. In addition, infiltration of CD68 + and CD11b + cells into these tissues were reduced in RG100204 treated SAP animals, and edema and SAP associated tissue damage were improved. Moreover, we demonstrate that RG100204 reduced apoptosis in the lungs of rat SAP animals, and reduces NF-κB signaling, NLRP3, expression, while profoundly increasing the Nrf2-dependent anti oxidative stress response. We conclude that AQP9 inhibition is a promising strategy for the treatment of pancreatitis and its systemic complications, such as ARDS.
Assuntos
Fator 2 Relacionado a NF-E2 , Proteína 3 que Contém Domínio de Pirina da Família NLR , Pancreatite , Síndrome do Desconforto Respiratório , Transdução de Sinais , Animais , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Pancreatite/tratamento farmacológico , Fator 2 Relacionado a NF-E2/metabolismo , Masculino , Transdução de Sinais/efeitos dos fármacos , Camundongos , Ratos , Síndrome do Desconforto Respiratório/tratamento farmacológico , Síndrome do Desconforto Respiratório/metabolismo , Aquaporinas/metabolismo , Aquaporinas/antagonistas & inibidores , Modelos Animais de Doenças , Ratos Sprague-Dawley , Pulmão/patologia , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Lipopolissacarídeos , Camundongos Endogâmicos C57BL , Ácido Taurocólico , Lesão Pulmonar/tratamento farmacológico , Lesão Pulmonar/metabolismo , Lesão Pulmonar/patologia , Pâncreas/patologia , Pâncreas/efeitos dos fármacos , Pâncreas/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Ceruletídeo , Humanos , Heme Oxigenase (Desciclizante)/metabolismoRESUMO
The water channel aquaporin-2 (AQP2) is essential for urine concentration. Vasopressin regulates phosphorylation of AQP2 at four conserved serine residues at the COOH-terminal tail (S256, S261, S264, and S269). We used numerous stably transfected Madin-Darby canine kidney cell models, replacing serine residues with either alanine (A), which prevents phosphorylation, or aspartic acid (D), which mimics the charged state of phosphorylated AQP2, to address whether phosphorylation is involved in regulation of (i) apical plasma membrane abundance of AQP2, (ii) internalization of AQP2, (iii) AQP2 protein-protein interactions, and (iv) degradation of AQP2. Under control conditions, S256D- and 269D-AQP2 mutants had significantly greater apical plasma membrane abundance compared to wild type (WT)-AQP2. Activation of adenylate cyclase significantly increased the apical plasma membrane abundance of all S-A or S-D AQP2 mutants with the exception of 256D-AQP2, although 256A-, 261A-, and 269A-AQP2 mutants increased to a lesser extent than WT-AQP2. Biotin internalization assays and confocal microscopy demonstrated that the internalization of 256D- and 269D-AQP2 from the plasma membrane was slower than WT-AQP2. The slower internalization corresponded with reduced interaction of S256D- and 269D-AQP2 with several proteins involved in endocytosis, including Hsp70, Hsc70, dynamin, and clathrin heavy chain. The mutants with the slowest rate of internalization, 256D- and 269D-AQP2, had a greater protein half-life (t(1/2) = 5.1 h and t(1/2) = 4.4 h, respectively) compared to WT-AQP2 (t(1/2) = 2.9 h). Our results suggest that vasopressin-mediated membrane accumulation of AQP2 can be controlled via regulated exocytosis and endocytosis in a process that is dependent on COOH terminal phosphorylation and subsequent protein-protein interactions.
Assuntos
Aquaporina 2/metabolismo , Endocitose/fisiologia , Animais , Aquaporina 2/genética , Biotina/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Cães , Exocitose/fisiologia , Camundongos , Mutagênese Sítio-Dirigida , Fosforilação , Ligação Proteica , Transfecção , Vasopressinas/metabolismoRESUMO
It has been hypothesized that aquaporin-9 (AQP9) is part of the unknown route of hepatocyte glycerol uptake. In a previous study, leptin receptor-deficient wild-type mice became diabetic and suffered from fasting hyperglycemia whereas isogenic AQP9(-/-) knock-out mice remained normoglycemic. The reason for this improvement in AQP9(-/-) mice was not established before. Here, we show increased glucose output (by 123% ± 36% S.E.) in primary hepatocyte culture when 0.5 mM extracellular glycerol was added. This increase depended on AQP9 because it was absent in AQP9(-/-) cells. Likewise, the increase was abolished by 25 µM HTS13286 (IC(50) ~ 2 µM), a novel AQP9 inhibitor, which we identified in a small molecule library screen. Similarly, AQP9 deletion or chemical inhibition eliminated glycerol-enhanced glucose output in perfused liver preparations. The following control experiments suggested inhibitor specificity to AQP9: (i) HTS13286 affected solute permeability in cell lines expressing AQP9, but not in cell lines expressing AQPs 3, 7, or 8. (ii) HTS13286 did not influence lactate- and pyruvate-dependent hepatocyte glucose output. (iii) HTS13286 did not affect glycerol kinase activity. Our experiments establish AQP9 as the primary route of hepatocyte glycerol uptake for gluconeogenesis and thereby explain the previously observed, alleviated diabetes in leptin receptor-deficient AQP9(-/-) mice.
Assuntos
Aquaporinas/metabolismo , Gluconeogênese/fisiologia , Glucose/metabolismo , Glicerol/metabolismo , Hepatócitos/metabolismo , Animais , Aquaporinas/genética , Células CHO , Cricetinae , Cricetulus , Crioprotetores/metabolismo , Crioprotetores/farmacologia , Diabetes Mellitus/genética , Diabetes Mellitus/metabolismo , Gluconeogênese/efeitos dos fármacos , Glucose/genética , Glicerol/farmacologia , Hepatócitos/citologia , Ácido Láctico/metabolismo , Camundongos , Camundongos Knockout , Ácido Pirúvico/metabolismo , Receptores para Leptina/genética , Receptores para Leptina/metabolismoRESUMO
In mammals, the majority of nitrogen from protein degradation is disposed of as urea. Several studies have partly characterized expression of urea transporters (UTs) in hepatocytes, where urea is produced. Nevertheless, the contribution of these proteins to hepatocyte urea permeability (P(urea)) and their role in liver physiology remains unknown. The purpose of this study was to biophysically examine hepatocyte urea transport. We hypothesized that the water, glycerol, and urea channel aquaporin-9 (AQP9) is involved in hepatocyte urea release. Stopped-flow light-scattering measurements determined that the urea channel inhibitors phloretin and dimethylurea reduced urea permeability of hepatocyte basolateral membranes by 70 and 40%, respectively. In basolateral membranes isolated from AQP9(-/-) and UT-A1/3(-/-) single-knockout and AQP9(-/-):UT-A1/3(-/-) double-knockout mice, P(urea) was decreased by 30, 40, and 76%, respectively, compared with AQP9(+/-):UT-A1/3(+/-) mice. However, expression analysis by RT-PCR did not identify known UT-A transcripts in liver. High-protein diet followed by 24-h fasting affected the concentrations of urea and ammonium ions in AQP9(-/-) mouse liver and plasma without generating an apparent tissue-to-plasma urea gradient. We conclude that AQP9 and unidentified UT-A urea channels constitute primary but redundant urea facilitators in murine hepatocytes.
Assuntos
Aquaporinas/deficiência , Hepatócitos/metabolismo , Proteínas de Membrana Transportadoras/genética , Ureia/metabolismo , Animais , Proteínas Alimentares/administração & dosagem , Deleção de Genes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transportadores de UreiaRESUMO
Aquaporin-9 (AQP9) is a facilitator of glycerol and other small neutral solute transmembrane diffusion. Identification of specific inhibitors for aquaporin family proteins has been difficult, due to high sequence similarity between the 13 human isoforms, and due to the limited channel surface areas that permit inhibitor binding. The few AQP9 inhibitor molecules described to date were not suitable for in vivo experiments. We now describe the characterization of a new small molecule AQP9 inhibitor, RG100204 in cell-based calcein-quenching assays, and by stopped-flow light-scattering recordings of AQP9 permeability in proteoliposomes. Moreover, we investigated the effects of RG100204 on glycerol metabolism in mice. In cell-based assays, RG100204 blocked AQP9 water permeability and glycerol permeability with similar, high potency (~5 × 10-8 M). AQP9 channel blocking by RG100204 was confirmed in proteoliposomes. After oral gavage of db/db mice with RG100204, a dose-dependent elevation of plasma glycerol was observed. A blood glucose-lowering effect was not statistically significant. These experiments establish RG100204 as a direct blocker of the AQP9 channel, and suggest its use as an experimental tool for in vivo experiments on AQP9 function.
Assuntos
Aquaporinas , Glicerol , Animais , Humanos , Camundongos , Aquaporinas/metabolismo , Glicemia/metabolismo , Glicerol/metabolismo , Glicerol/farmacologia , Fígado/metabolismo , Camundongos Endogâmicos , Água/metabolismoRESUMO
Sepsis is caused by systemic infection and is a major health concern as it is the primary cause of death from infection. It is the leading cause of mortality worldwide and there are no specific effective treatments for sepsis. Gene deletion of the neutral solute channel Aquaporin 9 (AQP9) normalizes oxidative stress and improves survival in a bacterial endotoxin induced mouse model of sepsis. In this study we described the initial characterization and effects of a novel small molecule AQP9 inhibitor, RG100204, in a cecal ligation and puncture (CLP) induced model of polymicrobial infection. In vitro, RG100204 blocked mouse AQP9 H2O2 permeability in an ectopic CHO cell expression system and abolished the LPS induced increase in superoxide anion and nitric oxide in FaO hepatoma cells. Pre-treatment of CLP-mice with RG100204 (25 mg/kg p.o. before CLP and then again at 8 h after CLP) attenuated the hypothermia, cardiac dysfunction (systolic and diastolic), renal dysfunction and hepatocellular injury caused by CLP-induced sepsis. Post-treatment of CLP-mice with RG100204 also attenuated the cardiac dysfunction (systolic and diastolic), the renal dysfunction caused by CLP-induced sepsis, but did not significantly reduce the liver injury or hypothermia. The most striking finding was that oral administration of RG100204 as late as 3 h after the onset of polymicrobial sepsis attenuated the cardiac and renal dysfunction caused by severe sepsis. Immunoblot quantification demonstrated that RG100204 reduced activation of the NLRP3 inflammasome pathway. Moreover, myeloperoxidase activity in RG100204 treated lung tissue was reduced. Together these results indicate that AQP9 may be a novel drug target in polymicrobial sepsis.
Assuntos
Aquaporinas , Cardiomiopatias , Cardiopatias , Hipotermia , Nefropatias , Sepse , Animais , Aquaporinas/genética , Peróxido de Hidrogênio/metabolismo , Camundongos , Insuficiência de Múltiplos Órgãos , Sepse/complicações , Sepse/tratamento farmacológicoRESUMO
Septic shock is the most severe complication of sepsis, being characterized by a systemic inflammatory response following bacterial infection, leading to multiple organ failure and dramatically high mortality. Aquaporin-9 (AQP9), a membrane channel protein mainly expressed in hepatocytes and leukocytes, has been recently associated with inflammatory and infectious responses, thus triggering strong interest as a potential target for reducing septic shock-dependent mortality. Here, we evaluated whether AQP9 contributes to murine systemic inflammation during endotoxic shock. Wild type (Aqp9+/+; WT) and Aqp9 gene knockout (Aqp9-/-; KO) male mice were submitted to endotoxic shock by i.p. injection of lipopolysaccharide (LPS; 40 mg/kg) and the related survival times were followed during 72 h. The electronic paramagnetic resonance and confocal microscopy were employed to analyze the nitric oxide (NO) and superoxide anion (O2-) production, and the expression of inducible NO-synthase (iNOS) and cyclooxigenase-2 (COX-2), respectively, in the liver, kidney, aorta, heart and lung of the mouse specimens. LPS-treated KO mice survived significantly longer than corresponding WT mice, and 25% of the KO mice fully recovered from the endotoxin treatment. The LPS-injected KO mice showed lower inflammatory NO and O2- productions and reduced iNOS and COX-2 levels through impaired NF-κB p65 activation in the liver, kidney, aorta, and heart as compared to the LPS-treated WT mice. Consistent with these results, the treatment of FaO cells, a rodent hepatoma cell line, with the AQP9 blocker HTS13268 prevented the LPS-induced increase of inflammatory NO and O2-. A role for AQP9 is suggested in the early acute phase of LPS-induced endotoxic shock involving NF-κB signaling. The modulation of AQP9 expression/function may reveal to be useful in developing novel endotoxemia therapeutics.
Assuntos
Aquaporinas/deficiência , Endotoxemia/imunologia , Inflamação/imunologia , Animais , Aquaporinas/genética , Aquaporinas/imunologia , Modelos Animais de Doenças , Endotoxemia/genética , Endotoxemia/patologia , Inflamação/genética , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Camundongos Knockout , Choque Séptico/genética , Choque Séptico/imunologiaRESUMO
BACKGROUND: Many species of mosquitoes, including the major malaria vector Anopheles gambiae, utilize carbon dioxide (CO(2)) and 1-octen-3-ol as olfactory cues in host-seeking behaviors that underlie their vectorial capacity. However, the molecular and cellular basis of such olfactory responses remains largely unknown. RESULTS: Here, we use molecular and physiological approaches coupled with systematic functional analyses to define the complete olfactory sensory map of the An. gambiae maxillary palp, an olfactory appendage that mediates the detection of these compounds. In doing so, we identify three olfactory receptor neurons (ORNs) that are organized in stereotyped triads within the maxillary-palp capitate-peg-sensillum population. One ORN is CO(2)-responsive and characterized by the coexpression of three receptors that confer CO(2) responses, whereas the other ORNs express characteristic odorant receptors (AgORs) that are responsible for their in vivo olfactory responses. CONCLUSIONS: Our results describe a complete and highly concordant map of both the molecular and cellular olfactory components on the maxillary palp of the adult female An. gambiae mosquito. These results also facilitate the understanding of how An. gambiae mosquitoes sense olfactory cues that might be exploited to compromise their ability to transmit malaria.
Assuntos
Anopheles/fisiologia , Insetos Vetores/fisiologia , Neurônios Receptores Olfatórios/fisiologia , Olfato/fisiologia , Animais , Anopheles/efeitos dos fármacos , Anopheles/ultraestrutura , Dióxido de Carbono/farmacologia , Condutividade Elétrica , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Insetos Vetores/efeitos dos fármacos , Insetos Vetores/ultraestrutura , Malária/transmissão , Octanóis/farmacologia , Odorantes , Neurônios Receptores Olfatórios/efeitos dos fármacos , Olfato/genéticaRESUMO
Stopped-Flow Light Scattering (SFLS) is a method devised to analyze the kinetics of fast chemical reactions that result in a significant change of the average molecular weight and/or in the shape of the reaction substrates. Several modifications of the original stopped-flow system have been made leading to a significant extension of its technical applications. One of these modifications allows the biophysical characterization of the water and solute permeability of biological and artificial membranes. Here, we describe a protocol of SFLS to measure the glycerol permeability of isolated human red blood cells (RBCs) and evaluate the pharmacokinetics properties (selectivity and potency) of isoform-specific inhibitors of AQP3, AQP7 and AQP9, three mammalian aquaglyceroporins allowing transport of glycerol across membranes. Suspensions of RBCs (1% hematocrit) are exposed to an inwardly directed gradient of 100 mM glycerol in a SFLS apparatus at 20 °C and the resulting changes in scattered light intensity are recorded at a monochromatic wavelength of 530 nm for 120 s. The SFLS apparatus is set up to have a dead time of 1.6-ms and 99% mixing efficiency in less than 1 ms. Data are fitted to a single exponential function and the related time constant (τ, seconds) of the cell-swelling phase of light scattering corresponding to the osmotic movement of water that accompanies the entry of glycerol into erythrocytes is measured. The coefficient of glycerol permeability ( Pgly , cm/s) of RBCs is calculated with the following equation: Pgly = 1/[(S/V)τ] where τ (s) is the fitted exponential time constant and S/V is the surface-to-volume ratio (cm-1) of the analyzed RBC specimen. Pharmacokinetics of the isoform-specific inhibitors of AQP3, AQP7 and AQP9 are assessed by evaluating the extent of RBC Pgly values resulting after the exposure to serial concentrations of the blockers.
RESUMO
The aquaporin protein family performs fundamental tasks in the physiology of several organs in the human body. Their roles in several disorders known to involve water movement make them attractive targets for the development of novel drug therapies.This chapter describes assays commonly used to study the water permeability across AQPs. It also describes the effect of some known inhibitors of aquaporins on water permeability, such as mercury, gold, silver, copper, phloretin, tetraethyl ammonium salts and acetazolamide compounds.
Assuntos
Aquaporinas/antagonistas & inibidores , Permeabilidade da Membrana Celular/efeitos dos fármacos , Desenho de Fármacos , Água/metabolismo , Animais , Aquaporinas/química , Aquaporinas/metabolismo , Bioensaio/métodos , Corantes Fluorescentes , Humanos , Técnicas de Diluição do Indicador , Metais/farmacologia , Estrutura Molecular , Floretina/farmacologia , Conformação Proteica , Compostos de Amônio Quaternário/farmacologia , Relação Estrutura-Atividade , Sulfonamidas/farmacologiaRESUMO
To initiate a comprehensive investigation of chemosensory signal transduction downstream of odorant receptors, we identified and characterized the complete set of genes that encode G-protein alpha subunits in the genome of the malaria vector mosquito An. gambiae. Data are provided on the tissue-specific expression patterns of 10 corresponding aga-transcripts in adult mosquitoes and pre-imago developmental stages. Specific immunoreactivity in chemosensory hairs of female antennae provides evidence in support of the participation of a subset of AGalphaq isoforms in olfactory signal transduction in this mosquito. In contrast, AGalphao is localized along the flagellar axon bundle but is absent from chemosensory sensilla, which suggests that this G-protein alpha subunit does not participate in olfactory signal transduction.
Assuntos
Anopheles/fisiologia , Encéfalo/metabolismo , Subunidades alfa de Proteínas de Ligação ao GTP/fisiologia , Expressão Gênica , Mecanotransdução Celular/fisiologia , Vibrissas/metabolismo , Sequência de Aminoácidos , Animais , Western Blotting , Drosophila melanogaster/genética , Eletroforese em Gel de Poliacrilamida , Feminino , Perfilação da Expressão Gênica , Imuno-Histoquímica , Hibridização In Situ , Dados de Sequência Molecular , Filogenia , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Receptores Odorantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
AQP9 is known to facilitate hepatocyte glycerol uptake. Murine AQP9 protein expression has been verified in liver, skin, epididymis, epidermis and neuronal cells using knockout mice. Further expression sites have been reported in humans. We aimed to verify AQP9 expression in a large set of human normal organs, different cancer types, rheumatoid arthritis synovial biopsies as well as in cell lines and primary cells. Combining standardized immunohistochemistry with high-throughput mRNA sequencing, we found that AQP9 expression in normal tissues was limited, with high membranous expression only in hepatocytes. In cancer tissues, AQP9 expression was mainly found in hepatocellular carcinomas, suggesting no general contribution of AQP9 to carcinogenesis. AQP9 expression in a subset of rheumatoid arthritis synovial tissue samples was affected by Humira, thereby supporting a suggested role of TNFα in AQP9 regulation in this disease. Among cell lines and primary cells, LP-1 myeloma cells expressed high levels of AQP9, whereas low expression was observed in a few other lymphoid cell lines. AQP9 mRNA and protein expression was absent in HepG2 hepatocellular carcinoma cells. Overall, AQP9 expression in human tissues appears to be more selective than in mice.
Assuntos
Aquaporinas/metabolismo , Artrite Reumatoide/metabolismo , Neoplasias/metabolismo , Aquaporinas/genética , Artrite Reumatoide/patologia , Linhagem Celular , Linhagem Celular Tumoral , Feminino , Humanos , Masculino , Neoplasias/patologia , Especificidade de Órgãos , RNA Mensageiro/metabolismoRESUMO
Cellular metabolism provides various sources of hydrogen peroxide (H2O2) in different organelles and compartments. The suitability of H2O2 as an intracellular signaling molecule therefore also depends on its ability to pass cellular membranes. The propensity of the membranous boundary of the endoplasmic reticulum (ER) to let pass H2O2 has been discussed controversially. In this essay, we challenge the recent proposal that the ER membrane constitutes a simple barrier for H2O2 diffusion and support earlier data showing that (i) ample H2O2 permeability of the ER membrane is a prerequisite for signal transduction, (ii) aquaporin channels are crucially involved in the facilitation of H2O2 permeation, and (iii) a proper experimental framework not prone to artifacts is necessary to further unravel the role of H2O2 permeation in signal transduction and organelle biology.
Assuntos
Permeabilidade da Membrana Celular , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Peróxido de Hidrogênio/metabolismo , Membrana Celular/química , Difusão , Retículo Endoplasmático/química , Peróxido de Hidrogênio/química , Espécies Reativas de Oxigênio/metabolismo , Transdução de SinaisRESUMO
Deletion of the glycerol channel aquaporin-9 (Aqp9) reduces postprandial blood glucose levels in leptin receptor-deficient (db/db) obese mice on a C57BL/6 × C57BLKS mixed genetic background. Furthermore, shRNA-mediated reduction of Aqp9 expression reduces liver triacylglycerol (TAG) accumulation in a diet-induced rat model of obesity. The aim of this study was to investigate metabolic effects of Aqp9 deletion in coisogenic db/db mice of the C57BL/6 background. Aqp9(wt) db/db and Aqp9(-/-) db/db mice did not differ in body weight and liver TAG contents. On the C57BL/6 genetic background, we observed elevated plasma glucose in Aqp9(-/-) db/db mice (+1.1 mmol/L, life-time average), while plasma insulin concentration was reduced at the time of death. Glucose levels changed similarly in pentobarbital anesthetized, glucagon challenged Aqp9(wt) db/db and Aqp9(-/-) db/db mice. Liver transcriptional profiling did not detect differential gene expression between genotypes. Metabolite profiling revealed a sex independent increase in plasma glycerol (+55%) and glucose (+24%), and reduction in threonate (all at q < 0.1) in Aqp9(-/-) db/db mice compared to controls. Metabolite profiling thus confirms a role of AQP9 in glycerol metabolism of obese C57BL/6 db/db mice. In this animal model of obesity Aqp9 gene deletion elevates plasma glucose and does not alleviate hepatosteatosis.