RESUMO
Aminoglycoside antibiotics are essential for treating life-threatening bacterial infections, despite the risk of lifelong hearing loss. Infections induce inflammation and up-regulate expression of candidate aminoglycoside-permeant cation channels, including transient receptor potential vanilloid-1 (TRPV1). Heterologous expression of TRPV1 facilitated cellular uptake of (fluorescently tagged) gentamicin that was enhanced by agonists, and diminished by antagonists, of TRPV1. Cochlear TRPV1 was immunolocalized near the apical membranes of sensory hair cells, adjacent supporting cells, and marginal cells in the stria vascularis. Exposure to immunostimulatory lipopolysaccharides, to simulate of bacterial infections, increased cochlear expression of TRPV1 and hair cell uptake of gentamicin. Lipopolysaccharide exposure exacerbated aminoglycoside-induced auditory threshold shifts and loss of cochlear hair cells in wild-type, but not in heterozygous Trpv1+/- or Trpv1 knockout, mice. Thus, TRPV1 facilitates cochlear uptake of aminoglycosides, and bacteriogenic stimulation upregulates TRPV1 expression to exacerbate cochleotoxicity. Furthermore, loss-of-function polymorphisms in Trpv1 can protect against immunogenic exacerbation of aminoglycoside-induced cochleotoxicity.
Assuntos
Aminoglicosídeos/efeitos adversos , Células Ciliadas Auditivas/efeitos dos fármacos , Células Ciliadas Auditivas/metabolismo , Perda Auditiva/etiologia , Inflamação/complicações , Inflamação/genética , Canais de Cátion TRPV/genética , Animais , Cálcio/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Gentamicinas/efeitos adversos , Células Ciliadas Auditivas/ultraestrutura , Perda Auditiva/metabolismo , Perda Auditiva/fisiopatologia , Ativação do Canal Iônico , Camundongos , Camundongos Knockout , Receptor 4 Toll-Like/metabolismoRESUMO
Aminoglycoside antibiotics are used as prophylaxis, or urgent treatment, for many life-threatening bacterial infections, including tuberculosis, sepsis, respiratory infections in cystic fibrosis, complex urinary tract infections and endocarditis. Although aminoglycosides are clinically-essential antibiotics, the mechanisms underlying their selective toxicity to the kidney and inner ear continue to be unraveled despite more than 70 years of investigation. The following mechanisms each contribute to aminoglycoside-induced toxicity after systemic administration: (1) drug trafficking across endothelial and epithelial barrier layers; (2) sensory cell uptake of these drugs; and (3) disruption of intracellular physiological pathways. Specific factors can increase the risk of drug-induced toxicity, including sustained exposure to higher levels of ambient sound, and selected therapeutic agents such as loop diuretics and glycopeptides. Serious bacterial infections (requiring life-saving aminoglycoside treatment) induce systemic inflammatory responses that also potentiate the degree of ototoxicity and permanent hearing loss. We discuss prospective clinical strategies to protect auditory and vestibular function from aminoglycoside ototoxicity, including reduced cochlear or sensory cell uptake of aminoglycosides, and otoprotection by ameliorating intracellular cytotoxicity.
RESUMO
Cisplatin is one of the most widely-used drugs to treat cancers. However, its nephrotoxic and ototoxic side-effects remain major clinical limitations. Recent studies have improved our understanding of the molecular mechanisms of cisplatin-induced nephrotoxicity and ototoxicity. While cisplatin binding to DNA is the major cytotoxic mechanism in proliferating (cancer) cells, nephrotoxicity and ototoxicity appear to result from toxic levels of reactive oxygen species and protein dysregulation within various cellular compartments. In this review, we discuss molecular mechanisms of cisplatin-induced nephrotoxicity and ototoxicity. We also discuss potential clinical strategies to prevent nephrotoxicity and ototoxicity and their current limitations.
Assuntos
Antineoplásicos/efeitos adversos , Cisplatino/efeitos adversos , Nefropatias/patologia , Doenças Vestibulares/patologia , Humanos , Nefropatias/induzido quimicamente , Espécies Reativas de Oxigênio/metabolismo , Doenças Vestibulares/induzido quimicamenteRESUMO
The resting membrane potential (RP) of vascular smooth muscle cells (VSMCs) is a major determinant of cytosolic calcium concentration and vascular tone. The heterogeneity of RPs and its underlying mechanism among different vascular beds remain poorly understood. We compared the RPs and vasomotion properties between the guinea pig spiral modiolar artery (SMA), brain arterioles (BA) and mesenteric arteries (MA). We found: 1) RPs showed a robust bimodal distribution peaked at -76 and -40 mV evenly in the SMA, unevenly at -77 and -51 mV in the BA and ~-71 and -52 mV in the MA. Ba(2+) 0.1 mM eliminated their high RP peaks ~-75 mV. 2) Cells with low RP (~-45 mV) hyperpolarized in response to 10 mM extracellular K(+), while cells with a high RP depolarized, and cells with intermediate RP (~-58 mV) displayed an initial hyperpolarization followed by prolonged depolarization. Moderate high K(+) typically induced dilation, constriction and a dilation followed by constriction in the SMA, MA and BA, respectively. 3) Boltzmann-fit analysis of the Ba(2+)-sensitive inward rectifier K(+) (Kir) whole-cell current showed that the maximum Kir conductance density significantly differed among the vessels, and the half-activation voltage was significantly more negative in the MA. 4) Corresponding to the whole-cell data, computational modeling simulated the three RP distribution patterns and the dynamics of RP changes obtained experimentally, including the regenerative swift shifts between the two RP levels after reaching a threshold. 5) Molecular works revealed strong Kir2.1 and Kir2.2 transcripts and Kir2.1 immunolabeling in all 3 vessels, while Kir2.3 and Kir2.4 transcript levels varied. We conclude that a dense expression of functional Kir2.X channels underlies the more negative RPs in endothelial cells and a subset of VSMC in these arterioles, and the heterogeneous Kir function is primarily responsible for the distinct bimodal RPs among these arterioles. The fast Kir-based regenerative shifts between two RP states could form a critical mechanism for conduction/spread of vasomotion along the arteriole axis.
Assuntos
Arteríolas/fisiologia , Expressão Gênica , Potenciais da Membrana , Canais de Potássio Corretores do Fluxo de Internalização/genética , Algoritmos , Animais , Bário/metabolismo , Simulação por Computador , Espaço Extracelular/metabolismo , Cobaias , Artérias Mesentéricas/fisiologia , Modelos Biológicos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Técnicas de Patch-Clamp , Potássio/metabolismoRESUMO
Striatal-enriched protein tyrosine phosphatase (STEP) is a brain-specific tyrosine phosphatase that plays a major role in the development of synaptic plasticity. Recent findings have implicated STEP in several psychiatric and neurological disorders, including Alzheimer's disease, schizophrenia, fragile X syndrome, Huntington's disease, stroke/ischemia, and stress-related psychiatric disorders. In these disorders, STEP protein expression levels and activity are dysregulated, contributing to the cognitive deficits that are present. In this review, we focus on the most recent findings on STEP, discuss how STEP expression and activity are maintained during normal cognitive function, and how disruptions in STEP activity contribute to a number of illnesses.
Assuntos
Encéfalo/enzimologia , Transtornos Mentais/enzimologia , Plasticidade Neuronal , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Doença de Alzheimer/enzimologia , Animais , Isquemia Encefálica/enzimologia , Cognição/fisiologia , Síndrome do Cromossomo X Frágil/enzimologia , Humanos , Doença de Huntington/enzimologia , Fosforilação , Esquizofrenia/enzimologia , Estresse Psicológico/enzimologia , Acidente Vascular Cerebral/enzimologiaRESUMO
Aminoglycoside antibiotics, like gentamicin, continue to be clinically essential worldwide to treat life-threatening bacterial infections. Yet, the ototoxic and nephrotoxic side-effects of these drugs remain serious complications. A major site of gentamicin uptake and toxicity resides within kidney proximal tubules that also heavily express electrogenic sodium-glucose transporter-2 (SGLT2; SLC5A2) in vivo. We hypothesized that SGLT2 traffics gentamicin, and promotes cellular toxicity. We confirmed in vitro expression of SGLT2 in proximal tubule-derived KPT2 cells, and absence in distal tubule-derived KDT3 cells. D-glucose competitively decreased the uptake of 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG), a fluorescent analog of glucose, and fluorescently-tagged gentamicin (GTTR) by KPT2 cells. Phlorizin, an SGLT2 antagonist, strongly inhibited uptake of 2-NBDG and GTTR by KPT2 cells in a dose- and time-dependent manner. GTTR uptake was elevated in KDT3 cells transfected with SGLT2 (compared to controls); and this enhanced uptake was attenuated by phlorizin. Knock-down of SGLT2 expression by siRNA reduced gentamicin-induced cytotoxicity. In vivo, SGLT2 was robustly expressed in kidney proximal tubule cells of heterozygous, but not null, mice. Phlorizin decreased GTTR uptake by kidney proximal tubule cells in Sglt2+/- mice, but not in Sglt2-/- mice. However, serum GTTR levels were elevated in Sglt2-/- mice compared to Sglt2+/- mice, and in phlorizin-treated Sglt2+/- mice compared to vehicle-treated Sglt2+/- mice. Loss of SGLT2 function by antagonism or by gene deletion did not affect gentamicin cochlear loading or auditory function. Phlorizin did not protect wild-type mice from kanamycin-induced ototoxicity. We conclude that SGLT2 can traffic gentamicin and contribute to gentamicin-induced cytotoxicity.
Assuntos
Antibacterianos/metabolismo , Gentamicinas/metabolismo , Canamicina/metabolismo , Túbulos Renais Distais/efeitos dos fármacos , Túbulos Renais Proximais/efeitos dos fármacos , Transportador 2 de Glucose-Sódio/metabolismo , 4-Cloro-7-nitrobenzofurazano/análogos & derivados , Animais , Antibacterianos/toxicidade , Transporte Biológico , Linhagem Celular , Cóclea/efeitos dos fármacos , Cóclea/fisiologia , Desoxiglucose/análogos & derivados , Feminino , Corantes Fluorescentes , Expressão Gênica , Gentamicinas/toxicidade , Testes Auditivos , Canamicina/toxicidade , Túbulos Renais Distais/citologia , Túbulos Renais Distais/metabolismo , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/metabolismo , Cinética , Masculino , Camundongos , Camundongos Transgênicos , Especificidade de Órgãos , Florizina/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Sódio/metabolismo , Transportador 2 de Glucose-Sódio/genética , Inibidores do Transportador 2 de Sódio-GlicoseRESUMO
Cisplatin is widely used as an antineoplastic drug, but its ototoxic and nephrotoxic side-effects, as well as the inherent or acquired resistance of some cancers to cisplatin, remain significant clinical problems. Cisplatin's selectivity in killing rapidly proliferating cancer cells is largely dependent on covalent binding to DNA via cisplatin's chloride sites that had been aquated. We hypothesized that cisplatin's toxicity in slowly proliferating or terminally differentiated cells is primarily due to drug-protein interactions, instead of drug-DNA binding. To identify proteins that bind to cisplatin, we synthesized two different platinum-agarose conjugates, one with two amino groups and another with two chlorides attached to platinum that are available for protein binding, and conducted pull-down assays using cochlear and kidney cells. Mass spectrometric analysis on protein bands after gel electrophoresis and Coomassie blue staining identified several proteins, including myosin IIA, glucose-regulated protein 94 (GRP94), heat shock protein 90 (HSP90), calreticulin, valosin containing protein (VCP), and ribosomal protein L5, as cisplatin-binding proteins. Future studies on the interaction of these proteins with cisplatin will elucidate whether these drug-protein interactions are involved in ototoxicity and nephrotoxicity, or contribute to tumor sensitivity or resistance to cisplatin treatment.
Assuntos
Antineoplásicos/química , Extratos Celulares/química , Cisplatino/química , Glicoconjugados/química , Sefarose/química , Adenosina Trifosfatases/química , Adenosina Trifosfatases/isolamento & purificação , Animais , Antineoplásicos/síntese química , Calreticulina/química , Calreticulina/isolamento & purificação , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/isolamento & purificação , Linhagem Celular , Cisplatino/análogos & derivados , Cisplatino/síntese química , Células Epiteliais/química , Células Epiteliais/citologia , Glicoconjugados/síntese química , Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/isolamento & purificação , Túbulos Renais Proximais/química , Túbulos Renais Proximais/citologia , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/isolamento & purificação , Camundongos , Miosina não Muscular Tipo IIA/química , Miosina não Muscular Tipo IIA/isolamento & purificação , Órgão Espiral/química , Órgão Espiral/citologia , Ligação Proteica , Proteínas Ribossômicas/química , Proteínas Ribossômicas/isolamento & purificação , Proteína com ValosinaRESUMO
Loop diuretics such as bumetanide and furosemide enhance aminoglycoside ototoxicity when co-administered to patients and animal models. The underlying mechanism(s) is poorly understood. We investigated the effect of these diuretics on cellular uptake of aminoglycosides, using Texas Red-tagged gentamicin (GTTR), and intracellular/whole-cell recordings of Madin-Darby canine kidney (MDCK) cells. We found that bumetanide and furosemide dose-dependently enhanced cytoplasmic GTTR fluorescence by ~60 %. This enhancement was suppressed by La(3+), a non-selective cation channel (NSCC) blocker, and by K(+) channel blockers Ba(2+) and clotrimazole, but not by tetraethylammonium (TEA), 4-aminopyridine (4-AP) or glipizide, nor by Cl(-) channel blockers diphenylamine-2-carboxylic acid (DPC), niflumic acid (NFA), and CFTRinh-172. Bumetanide and furosemide hyperpolarized MDCK cells by ~14 mV, increased whole-cell I/V slope conductance; the bumetanide-induced net current I/V showed a reversal potential (V r) ~-80 mV. Bumetanide-induced hyperpolarization and I/V change was suppressed by Ba(2+) or clotrimazole, and absent in elevated [Ca(2+)]i, but was not affected by apamin, 4-AP, TEA, glipizide, DPC, NFA, or CFTRinh-172. Bumetanide and furosemide stimulated a surge of Fluo-4-indicated cytosolic Ca(2+). Ba(2+) and clotrimazole alone depolarized cells by ~18 mV and reduced I/V slope with a net current V r near -85 mV, and reduced GTTR uptake by ~20 %. La(3+) alone hyperpolarized the cells by ~-14 mV, reduced the I/V slope with a net current V r near -10 mV, and inhibited GTTR uptake by ~50 %. In the presence of La(3+), bumetanide-caused negligible change in potential or I/V. We conclude that NSCCs constitute a major cell entry pathway for cationic aminoglycosides; bumetanide enhances aminoglycoside uptake by hyperpolarizing cells that increases the cation influx driving force; and bumetanide-induced hyperpolarization is caused by elevating intracellular Ca(2+) and thus facilitating activation of the intermediate conductance Ca(2+)-activated K(+) channels.
Assuntos
Bumetanida/farmacologia , Cálcio/metabolismo , Polaridade Celular/efeitos dos fármacos , Diuréticos/farmacologia , Gentamicinas/metabolismo , Canais de Potássio Cálcio-Ativados/metabolismo , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Cães , Regulação da Expressão Gênica , Gentamicinas/química , Cinética , Células Madin Darby de Rim Canino , Potenciais da Membrana/efeitos dos fármacos , Técnicas de Patch-Clamp , Simportadores de Cloreto de Sódio-Potássio/genética , Simportadores de Cloreto de Sódio-Potássio/metabolismo , ATPase Trocadora de Sódio-Potássio/genética , ATPase Trocadora de Sódio-Potássio/metabolismo , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo , Xantenos/químicaRESUMO
Since introduction into clinical practice over 60 years ago, aminoglycoside antibiotics remain important drugs in the treatment of bacterial infections, cystic fibrosis and tuberculosis. However, the ototoxic and nephrotoxic properties of these drugs are still a major clinical problem. Recent advances in molecular biology and biochemistry have begun to uncover the intracellular actions of aminoglycosides that lead to cytotoxicity. In this review, we discuss intracellular binding targets of aminoglycosides, highlighting specific aminoglycoside-binding proteins (HSP73, calreticulin and CLIMP-63) and their potential for triggering caspases and Bcl-2 signalling cascades that are involved in aminoglycoside-induced cytotoxicity. We also discuss potential strategies to reduce aminoglycoside cytotoxicity, which are necessary for greater bactericidal efficacy during aminoglycoside pharmacotherapy.
Assuntos
Aminoglicosídeos/toxicidade , Antibacterianos/toxicidade , Aminoglicosídeos/química , Aminoglicosídeos/metabolismo , Animais , Antibacterianos/química , Antibacterianos/metabolismo , Infecções Bacterianas/tratamento farmacológico , Caspases/metabolismo , Morte Celular/efeitos dos fármacos , Células Ciliadas Auditivas/efeitos dos fármacos , Humanos , Ferro/metabolismo , Rim/efeitos dos fármacos , Modelos Biológicos , Fosfatidilinositóis/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Aminoglycosides like gentamicin are among the most commonly used antibiotics in clinical practice and are essential for treating life-threatening tuberculosis and Gram-negative bacterial infections. However, aminoglycosides are also nephrotoxic and ototoxic. Although a number of mechanisms have been proposed, it is still unclear how aminoglycosides induce cell death in auditory sensory epithelia and subsequent deafness. Aminoglycosides bind to various intracellular molecules, such as RNA and phosphoinositides. We hypothesized that aminoglycosides, based on their tissue-specific susceptibility, also bind to intracellular proteins that play a role in drug-induced ototoxicity. By conjugating an aminoglycoside, gentamicin, to agarose beads and conducting a gentamicin-agarose pull-down assay, we have isolated gentamicin-binding proteins (GBPs) from immortalized cells of mouse organ of Corti, HEI-OC1. Mass spectrometry identified calreticulin (CRT) as a GBP. Immunofluorescence revealed that CRT expression is concentrated in strial marginal cells and hair cell stereocilia, primary locations of drug uptake and cytotoxicity in the cochlea. In HEI-OC1 cells treated with gentamicin, reduction of CRT expression using small interfering RNA (siRNA) reduced intracellular drug levels. CRT-deficient mouse embryonic fibroblast (MEF) cells as well as CRT siRNA-transfected wild-type MEFs also had reduced cell viability after gentamicin treatment. A pull-down assay using deletion mutants of CRT determined that the carboxyl C-domain of CRT binds to gentamicin. HeLa cells transfected with CRT C-domain deletion mutant construct were more susceptible to gentamicin-induced cytotoxicity compared with cells transfected with full-length CRT or other deletion mutants. Therefore, we conclude that CRT binding to gentamicin is protective against gentamicin-induced cytotoxicity.
Assuntos
Antibacterianos/toxicidade , Calreticulina/metabolismo , Fibroblastos/efeitos dos fármacos , Gentamicinas/toxicidade , Perda Auditiva/prevenção & controle , Órgão Espiral/efeitos dos fármacos , Animais , Antibacterianos/metabolismo , Sítios de Ligação , Calreticulina/química , Calreticulina/genética , Sobrevivência Celular/efeitos dos fármacos , Fibroblastos/metabolismo , Gentamicinas/metabolismo , Proteínas de Fluorescência Verde/genética , Células HeLa , Perda Auditiva/induzido quimicamente , Perda Auditiva/metabolismo , Humanos , Camundongos , Microscopia Confocal , Órgão Espiral/citologia , Órgão Espiral/metabolismo , Plasmídeos , Ligação Proteica , RNA Interferente Pequeno/genética , Proteínas Recombinantes de Fusão/genética , TransfecçãoRESUMO
Cochlear sensory hair cells are pharmacologically sensitive to aminoglycoside antibiotics that are used for treating life-threatening bacterial sepsis. Cochlear tissues are compartmentalized behind an impermeable paracellular barrier called the blood-labyrinth barrier (BLB). Most macromolecules cannot cross the blood-labyrinth barrier; however, aminoglycosides can cross this barrier into the cochlear fluids and enter hair cells, inducing hair cell death and consequent permanent hearing loss or deafness. The trafficking routes and cellular mechanisms required for aminoglycoside trafficking across the blood-labyrinth barrier remain unknown.Aminoglycosides enter cochlear hair cells across their apical membranes that are bathed in endolymph, a hitherto unexpected trafficking route. The stria vascularis, a component of the blood- labyrinth barrier, preferentially loads with aminoglycosides. Our recent work demonstrates that the stria vascularis exhibits high expression of the cation-selective ion channel TRPV4, and that this channel is permeable to aminoglycosides. However, aminoglycosides must employ more than one cellular mechanism to cross the blood-labyrinth barrier into endolymph against the electrical gradient.
RESUMO
The cochlea and kidney are susceptible to aminoglycoside-induced toxicity. The non-selective cation channel TRPV4 is expressed in kidney distal tubule cells, and hair cells and the stria vascularis in the inner ear. To determine whether TRPV4 is involved in aminoglycoside trafficking, we generated a murine proximal-tubule cell line (KPT2) and a distal-tubule cell line (KDT3). TRPV4 expression was confirmed in KDT3 cells but not in KPT2 cells. Removal of extracellular Ca(2+) significantly enhanced gentamicin-Texas-Red (GTTR) uptake by KDT3, indicative of permeation through non-selective cation channels. To determine whether TRPV4 is permeable to GTTR, stable cell lines were generated that express TRPV4 in KPT2 (KPT2-TRPV4). KPT2-TRPV4 cells took up more GTTR than control cell lines (KPT2-pBabe) in the absence of extracellular Ca(2+). TRPV4-dependent GTTR uptake was abolished by a point mutation within the crucial pore region of the channel, suggesting that GTTR permeates the TRPV4 channel. In an endolymph-like extracellular environment, clearance of GTTR was attenuated from KPT2-TRPV4 cells in a TRPV4-dependent fashion. We propose that TRPV4 has a role in aminoglycoside uptake and retention in the cochlea.
Assuntos
Aminoglicosídeos/metabolismo , Antibacterianos/metabolismo , Canais de Cátion TRPV/metabolismo , Animais , Cálcio/farmacologia , Linhagem Celular , Cães , Orelha Interna/citologia , Orelha Interna/metabolismo , Endolinfa/efeitos dos fármacos , Endolinfa/metabolismo , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/metabolismo , Gentamicinas/metabolismo , Rim/citologia , Rim/metabolismo , Metionina/metabolismo , Camundongos , Ratos , Xantenos/metabolismoRESUMO
Frizzled has been known to function as a Wnt receptor. Although there have been a number of mammalian Frizzled members identified, their binding specificities with Wnt and functions in mammalian cells have been poorly understood. Here, we demonstrate that rat Frizzled-9 (Rfz9) functions in Wnt/beta-catenin signaling in 293T cells. Rfz9 overexpression induces the hyperphosphorylation and relocalization of mouse Dishevelled-1 (Dvl-1) from the cytoplasm to the cell membrane and the accumulation of cytosolic beta-catenin. Transfections of Rfz9 with each of several Wnt members show that only Wnt-2 activates Rfz9 in T cell factor (TCF)-dependent transcription. Deletion mutant analysis determines that there is a difference in Rfz9 C-terminal residues required for the modifications of Dvl-1 and those required for the inductions of beta-catenin stabilization and TCF transactivation. Deletion of the Wnt-binding domain does not abolish Rfz9 activity completely, although it causes the inactivation of Wnt-2-dependent TCF transcription. Rfz9 also relocalizes Axin from the cytoplasm to the plasma membrane in the presence of Dvl-1, suggesting that one of the consequences of Dvl-1 relocalization by Rfz9 is to bring Axin to the cell membrane.