Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 27
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Blood Cells Mol Dis ; 79: 102346, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31352162

RESUMO

Excessive red cell dehydration contributes to the pathophysiology of sickle cell disease (SCD). The densest fraction of sickle red cells (with the highest corpuscular hemoglobin concentration) undergoes the most rapid polymerization of deoxy-hemoglobin S, leading to accelerated cell sickling and increased susceptibility to endothelial activation, red cell adhesion, and vaso-occlusion. Increasing red cell volume in order to decrease red cell density can thus serve as an adjunct therapeutic goal in SCD. Regulation of circulating mouse red cell volume and density is mediated largely by the Gardos channel, KCNN4, and the K-Cl cotransporters, KCC3 and KCC1. Whereas inhibition of the Gardos channel in subjects with sickle cell disease increased red cell volume, decreased red cell density, and improved other hematological indices in subjects with SCD, specific KCC inhibitors have not been available for testing. We therefore investigated the effect of genetic inactivation of KCC3 and KCC1 in the SAD mouse model of sickle red cell dehydration, finding decreased red cell density and improved hematological indices. We describe here generation of mice genetically deficient in the three major red cell volume regulatory gene products, KCNN4, KCC3, and KCC1 in C57BL6 non-sickle and SAD sickle backgrounds. We show that combined loss-of-function of all three gene products in SAD mice leads to incrementally increased MCV, decreased CHCM and % hyperchromic cells, decreased red cell density (phthalate method), increased resistance to hypo-osmotic lysis, and increased cell K content. The data show that combined genetic deletion of the Gardos channel and K-Cl cotransporters in a mouse SCD model decreases red cell density and improves several hematological parameters, supporting the strategy of combined pharmacological inhibition of these ion transport pathways in the adjunct treatment of human SCD.


Assuntos
Anemia Falciforme/sangue , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Animais , Tamanho Celular/efeitos dos fármacos , Desidratação/tratamento farmacológico , Modelos Animais de Doenças , Eritrócitos/patologia , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/deficiência , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/genética , Camundongos , Simportadores/deficiência , Simportadores/genética , Cotransportadores de K e Cl-
2.
Sci Rep ; 8(1): 9320, 2018 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-29915289

RESUMO

Nearly 70% of cystic fibrosis (CF) patients bear the phenylalanine-508 deletion but disease severity differs greatly, and is not explained by the existence of different mutations in compound heterozygous. Studies demonstrated that genes other than CFTR relate to intestinal disease in humans and CF-mouse. Kcnn4, the gene encoding the calcium-activated potassium channel KCa3.1, important for intestinal secretion, is present in a locus linked with occurrence of intestinal CF-disease in mice and humans. We reasoned that it might be a CF-modifier gene and bred a CF-mouse with Kcnn4 silencing, finding that lethality was almost abolished. Silencing of Kcnn4 did not improve intestinal secretory functions, but rather corrected increased circulating TNF-α level and reduced intestinal mast cell increase. Given the importance of mast cells in intestinal disease additional double mutant CF-animals were tested, one lacking mast cells (C-kitW-sh/W-sh) and Stat6-/- to block IgE production. While mast cell depletion had no effect, silencing Stat6 significantly reduced lethality. Our results show that Kcnn4 is an intestinal CF modifier gene partially acting through a STAT6-dependent mechanism.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/genética , Genes Modificadores , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/genética , Enteropatias/genética , Animais , Citocinas/metabolismo , Imunoglobulina E/metabolismo , Mediadores da Inflamação/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/deficiência , Mucosa Intestinal/patologia , Ativação do Canal Iônico , Mastócitos/metabolismo , Camundongos Endogâmicos C57BL , Mutação/genética , Fenótipo , Fator de Transcrição STAT6/metabolismo , Análise de Sobrevida , Aumento de Peso
3.
Cell ; 173(2): 443-455.e12, 2018 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-29576450

RESUMO

Hereditary xerocytosis is thought to be a rare genetic condition characterized by red blood cell (RBC) dehydration with mild hemolysis. RBC dehydration is linked to reduced Plasmodium infection in vitro; however, the role of RBC dehydration in protection against malaria in vivo is unknown. Most cases of hereditary xerocytosis are associated with gain-of-function mutations in PIEZO1, a mechanically activated ion channel. We engineered a mouse model of hereditary xerocytosis and show that Plasmodium infection fails to cause experimental cerebral malaria in these mice due to the action of Piezo1 in RBCs and in T cells. Remarkably, we identified a novel human gain-of-function PIEZO1 allele, E756del, present in a third of the African population. RBCs from individuals carrying this allele are dehydrated and display reduced Plasmodium infection in vitro. The existence of a gain-of-function PIEZO1 at such high frequencies is surprising and suggests an association with malaria resistance.


Assuntos
Anemia Hemolítica Congênita/patologia , População Negra/genética , Hidropisia Fetal/patologia , Canais Iônicos/genética , Malária/patologia , Alelos , Anemia Hemolítica Congênita/genética , Animais , Desidratação , Modelos Animais de Doenças , Eritrócitos/citologia , Eritrócitos/metabolismo , Deleção de Genes , Genótipo , Humanos , Hidropisia Fetal/genética , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/deficiência , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/genética , Canais Iônicos/química , Malária/genética , Malária/parasitologia , Malária/prevenção & controle , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Plasmodium berghei/crescimento & desenvolvimento , Plasmodium berghei/patogenicidade , Linfócitos T/citologia , Linfócitos T/metabolismo
4.
J Neuroinflammation ; 14(1): 203, 2017 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-29037241

RESUMO

BACKGROUND: Reactive astrogliosis is one of the significantly pathological features in ischemic stroke accompanied with changes in gene expression, morphology, and proliferation. KCa3.1 was involved in TGF-ß-induced astrogliosis in vitro and also contributed to astrogliosis-mediated neuroinflammation in neurodegeneration disease. METHODS: Wild type mice and KCa3.1-/- mice were subjected to permanent middle cerebral artery occlusion (pMCAO) to evaluate the infarct areas by 2,3,5-triphenyltetrazolium hydrochloride staining and neurological deficit. KCa3.1 channels expression and cell localization in the brain of pMCAO mice model were measured by immunoblotting and immunostaining. Glia activation and neuron loss was measured by immunostaining. DiBAC4 (3) and Fluo-4AM were used to measure membrane potential and cytosolic Ca2+ level in oxygen-glucose deprivation induced reactive astrocytes in vitro. RESULTS: Immunohistochemistry on pMCAO mice infarcts showed strong upregulation of KCa3.1 immunoreactivity in reactive astrogliosis. KCa3.1-/- mice exhibited significantly smaller infarct areas on pMCAO and improved neurological deficit. Both activated gliosis and neuronal loss were attenuated in KCa3.1-/- pMCAO mice. In the primary cultured astrocytes, the expressions of KCa3.1 and TRPV4 were increased associated with upregulation of astrogliosis marker GFAP induced by oxygen-glucose deprivation. The activation of KCa3.1 hyperpolarized membrane potential and, by promoting the driving force for calcium, induced calcium entry through TRPV4, a cation channel of the transient receptor potential family. Double-labeled staining showed that KCa3.1 and TRPV4 channels co-localized in astrocytes. Blockade of KCa3.1 or TRPV4 inhibited the phenotype switch of reactive astrogliosis. CONCLUSIONS: Our data suggested that KCa3.1 inhibition might represent a promising therapeutic strategy for ischemia stroke.


Assuntos
Isquemia Encefálica/metabolismo , Sistemas de Liberação de Medicamentos , Gliose/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/deficiência , Bloqueadores dos Canais de Potássio/administração & dosagem , Acidente Vascular Cerebral/metabolismo , Animais , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/patologia , Células Cultivadas , Sistemas de Liberação de Medicamentos/métodos , Gliose/tratamento farmacológico , Gliose/patologia , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/antagonistas & inibidores , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Acidente Vascular Cerebral/tratamento farmacológico , Acidente Vascular Cerebral/patologia
5.
Neuropharmacology ; 125: 386-395, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28823609

RESUMO

Intermediate conductance calcium-activated potassium channels (KCa3.1) have been recently implicated in pain processing. However, the functional role and localization of KCa3.1 in the nociceptive system are largely unknown. We here characterized the behavior of mice lacking KCa3.1 (KCa3.1-/-) in various pain models and analyzed the expression pattern of KCa3.1 in dorsal root ganglia (DRG) and the spinal cord. KCa3.1-/- mice demonstrated normal behavioral responses in models of acute nociceptive, persistent inflammatory, and persistent neuropathic pain. However, their behavioral responses to noxious chemical stimuli such as formalin and capsaicin were increased. Accordingly, formalin-induced nociceptive behavior was increased in wild-type mice after administration of the KCa3.1 inhibitor TRAM-34. In situ hybridization experiments detected KCa3.1 in most DRG satellite glial cells, in a minority of DRG neurons, and in ependymal cells lining the central canal of the spinal cord. Together, our data point to a specific inhibitory role of KCa3.1 for the processing of noxious chemical stimuli.


Assuntos
Gânglios Espinais/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Neuralgia/metabolismo , Dor Nociceptiva/metabolismo , Animais , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Células Cultivadas , Epêndima/efeitos dos fármacos , Epêndima/metabolismo , Epêndima/patologia , Feminino , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/patologia , Inflamação/metabolismo , Inflamação/patologia , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/antagonistas & inibidores , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/deficiência , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neuralgia/patologia , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Neuroglia/patologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Dor Nociceptiva/patologia , Limiar da Dor/efeitos dos fármacos , Limiar da Dor/fisiologia , Bloqueadores dos Canais de Potássio/farmacologia , Pirazóis/farmacologia , Nervo Isquiático/lesões , Fármacos do Sistema Sensorial , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Medula Espinal/patologia
6.
Nat Commun ; 8: 14644, 2017 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-28248292

RESUMO

Voltage-gated Kv1.3 and Ca2+-dependent KCa3.1 are the most prevalent K+ channels expressed by human and rat T cells. Despite the preferential upregulation of Kv1.3 over KCa3.1 on autoantigen-experienced effector memory T cells, whether Kv1.3 is required for their induction and function is unclear. Here we show, using Kv1.3-deficient rats, that Kv1.3 is involved in the development of chronically activated antigen-specific T cells. Several immune responses are normal in Kv1.3 knockout (KO) rats, suggesting that KCa3.1 can compensate for the absence of Kv1.3 under these specific settings. However, experiments with Kv1.3 KO rats and Kv1.3 siRNA knockdown or channel-specific inhibition of human T cells show that maximal T-cell responses against autoantigen or repeated tetanus toxoid stimulations require both Kv1.3 and KCa3.1. Finally, our data also suggest that T-cell dependency on Kv1.3 or KCa3.1 might be irreversibly modulated by antigen exposure.


Assuntos
Epitopos/imunologia , Memória Imunológica , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Canal de Potássio Kv1.3/metabolismo , Linfócitos T/metabolismo , Animais , Técnicas de Silenciamento de Genes , Humanos , Imunidade/efeitos dos fármacos , Memória Imunológica/efeitos dos fármacos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/deficiência , Ativação Linfocitária/imunologia , Fenótipo , Bloqueadores dos Canais de Potássio/farmacologia , RNA Interferente Pequeno/metabolismo , Ratos , Linfócitos T/efeitos dos fármacos
7.
Arch Toxicol ; 90(9): 2249-2260, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26438401

RESUMO

Tubular cell apoptosis significantly contributes to cisplatin-induced acute kidney injury (AKI) pathogenesis. Although KCa3.1, a calcium-activated potassium channel, participates in apoptosis, its involvement in cisplatin-induced AKI is unknown. Here, we found that cisplatin treatment triggered an early induction of KCa3.1 expression associated with HK-2 cell apoptosis, the development of renal tubular damage, and apoptosis in mice. Treatment with the highly selective KCa3.1 blocker TRAM-34 suppressed cisplatin-induced HK-2 cell apoptosis. We further assessed whether KCa3.1 mediated cisplatin-induced AKI in genetic knockout and pharmacological blockade mouse models. KCa3.1 deficiency reduced renal function loss, renal tubular damage, and the induction of the apoptotic marker caspase-3 in the kidneys of cisplatin-treated KCa3.1 (-/-) mice. Pharmacological blockade of KCa3.1 by TRAM-34 similarly attenuated cisplatin-induced AKI in mice. Furthermore, we dissected the mechanisms underlying cisplatin-induced apoptosis reduction via KCa3.1 blockade. We found that KCa3.1 blockade attenuated cytochrome c release and the increase in the intrinsic apoptotic mediators Bax, Bak, and caspase-9 after cisplatin treatment. KCa3.1 blocking inhibited the cisplatin-induced activation of the endoplasmic reticulum (ER) stress mediator caspase-12, which is independent of calcium-dependent protease m-calpain activation. Taken together, KCa3.1 blockade protects against cisplatin-induced AKI through the attenuation of apoptosis by interference with intrinsic apoptotic and ER stress-related mediators, providing a potential target for the prevention of cisplatin-induced AKI.


Assuntos
Injúria Renal Aguda/prevenção & controle , Cisplatino/toxicidade , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/antagonistas & inibidores , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Túbulos Renais Proximais/efeitos dos fármacos , Bloqueadores dos Canais de Potássio/farmacologia , Pirazóis/toxicidade , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/genética , Injúria Renal Aguda/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular , Citoproteção , Modelos Animais de Doenças , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/deficiência , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/genética , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo
8.
Arterioscler Thromb Vasc Biol ; 35(8): 1852-61, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26088577

RESUMO

OBJECTIVE: Vascular smooth muscle cells (VSMC) proliferation is a hallmark of atherosclerosis and vascular restenosis. The intermediate conductance Ca(2+)-activated K(+) (SK4) channel is required for pathological VSMC proliferation. In T lymphocytes, nucleoside diphosphate kinase B (NDPKB) has been implicated in SK4 channel activation. We thus investigated the role of NDPKB in the regulation of SK4 currents (ISK4) in proliferating VSMC and neointima formation. APPROACH AND RESULTS: Function and expression of SK4 channels in VSMC from injured mouse carotid arteries were assessed by patch-clamping and real-time polymerase chain reaction. ISK4 was detectable in VSMC from injured but not from uninjured arteries correlating with the occurrence of the proliferative phenotype. Direct application of NDPKB to the membrane of inside-out patches increased ISK4, whereas NDPKB did not alter currents in VSMC obtained from injured vessels of SK4-deficient mice. The NDPKB-induced increase in ISK4 was prevented by protein histidine phosphatase 1, but not an inactive protein histidine phosphatase 1 mutant indicating that ISK4 is regulated via histidine phosphorylation in proliferating VSMC; moreover, genetic NDPKB ablation reduced ISK4 by 50% suggesting a constitutive activation of ISK4 in proliferating VSMC. In line, neointima formation after wire injury of the carotid artery was substantially reduced in mice deficient in SK4 channels or NDPKB. CONCLUSIONS: NDPKB to SK4 signaling is required for neointima formation. Constitutive activation of SK4 by NDPKB in proliferating VSMC suggests that targeting this interaction via, for example, activation of protein histidine phosphatase 1 may provide clinically meaningful effects in vasculoproliferative diseases such as atherosclerosis and post angioplasty restenosis.


Assuntos
Lesões das Artérias Carótidas/enzimologia , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Músculo Liso Vascular/enzimologia , Miócitos de Músculo Liso/enzimologia , Nucleosídeo NM23 Difosfato Quinases/metabolismo , Neointima , Animais , Artérias Carótidas/enzimologia , Artérias Carótidas/patologia , Lesões das Artérias Carótidas/genética , Lesões das Artérias Carótidas/patologia , Proliferação de Células , Células Cultivadas , Modelos Animais de Doenças , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/deficiência , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/genética , Potenciais da Membrana , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Nucleosídeo NM23 Difosfato Quinases/deficiência , Nucleosídeo NM23 Difosfato Quinases/genética , Transdução de Sinais
9.
Acta Physiol (Oxf) ; 213(3): 628-41, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25545736

RESUMO

AIMS: Nucleotides are important paracrine regulators of vascular tone. We previously demonstrated that activation of P2Y2 receptors causes an acute, NO-independent decrease in blood pressure, indicating this signalling pathway requires an endothelial-derived hyperpolarization (EDH) response. To define the mechanisms by which activation of P2Y2 receptors initiates EDH and vasodilation, we studied intermediate-conductance (KCa3.1, expressed in endothelial cells) and big-conductance potassium channels (KCa1.1, expressed in smooth muscle cells) as well as components of the myoendothelial gap junction, connexins 37 and 40 (Cx37, Cx40), all hypothesized to be part of the EDH response. METHODS: We compared the effects of a P2Y2/4 receptor agonist in wild-type (WT) mice and in mice lacking KCa3.1, KCa1.1, Cx37 or Cx40 under anaesthesia, while monitoring intra-arterial blood pressure and heart rate. RESULTS: Acute activation of P2Y2/4 receptors (0.01-3 mg kg(-1) body weight i.v.) caused a biphasic blood pressure response characterized by a dose-dependent and rapid decrease in blood pressure in WT (maximal response % of baseline at 3 mg kg(-1) : -38 ± 1%) followed by a consecutive increase in blood pressure (+44 ± 11%). The maximal responses in KCa3.1(-/-) and Cx37(-/-) were impaired (-13 ± 5, +17 ± 7 and -27 ± 1, +13 ± 3% respectively), whereas the maximal blood pressure decrease in response to acetylcholine at 3 µg kg(-1) was not significantly different (WT: -53 ± 3%; KCa3.1(-/-) : -52 ± 3; Cx37(-/-) : -53 ± 3%). KCa1.1(-/-) and Cx40(-/-) showed an identical biphasic response to P2Y2/4 receptor activation compared to WT. CONCLUSIONS: The data suggest that the P2Y2/4 receptor activation elicits blood pressure responses via distinct mechanisms involving KCa3.1 and Cx37.


Assuntos
Pressão Sanguínea/efeitos dos fármacos , Conexinas/metabolismo , Inosina/análogos & derivados , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Receptores Purinérgicos P2Y2/efeitos dos fármacos , Uridina Trifosfato/análogos & derivados , Animais , Conexinas/deficiência , Conexinas/genética , Relação Dose-Resposta a Droga , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Frequência Cardíaca/efeitos dos fármacos , Inosina/farmacologia , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/deficiência , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/genética , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Masculino , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Agonistas do Receptor Purinérgico P2Y , Receptores Purinérgicos P2Y2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Uridina Trifosfato/farmacologia , Vasodilatação/efeitos dos fármacos , Proteína alfa-4 de Junções Comunicantes
10.
PLoS One ; 9(5): e97687, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24858807

RESUMO

OBJECTIVE: In vascular biology, endothelial KCa2.3 and KCa3.1 channels contribute to arterial blood pressure regulation by producing membrane hyperpolarization and smooth muscle relaxation. The role of KCa2.3 and KCa3.1 channels in the pulmonary circulation is not fully established. Using mice with genetically encoded deficit of KCa2.3 and KCa3.1 channels, this study investigated the effect of loss of the channels in hypoxia-induced pulmonary hypertension. APPROACH AND RESULT: Male wild type and KCa3.1-/-/KCa2.3T/T(+DOX) mice were exposed to chronic hypoxia for four weeks to induce pulmonary hypertension. The degree of pulmonary hypertension was evaluated by right ventricular pressure and assessment of right ventricular hypertrophy. Segments of pulmonary arteries were mounted in a wire myograph for functional studies and morphometric studies were performed on lung sections. Chronic hypoxia induced pulmonary hypertension, right ventricular hypertrophy, increased lung weight, and increased hematocrit levels in either genotype. The KCa3.1-/-/KCa2.3T/T(+DOX) mice developed structural alterations in the heart with increased right ventricular wall thickness as well as in pulmonary vessels with increased lumen size in partially- and fully-muscularized vessels and decreased wall area, not seen in wild type mice. Exposure to chronic hypoxia up-regulated the gene expression of the KCa2.3 channel by twofold in wild type mice and increased by 2.5-fold the relaxation evoked by the KCa2.3 and KCa3.1 channel activator NS309, whereas the acetylcholine-induced relaxation - sensitive to the combination of KCa2.3 and KCa3.1 channel blockers, apamin and charybdotoxin - was reduced by 2.5-fold in chronic hypoxic mice of either genotype. CONCLUSION: Despite the deficits of the KCa2.3 and KCa3.1 channels failed to change hypoxia-induced pulmonary hypertension, the up-regulation of KCa2.3-gene expression and increased NS309-induced relaxation in wild-type mice point to a novel mechanism to counteract pulmonary hypertension and to a potential therapeutic utility of KCa2.3/KCa3.1 activators for the treatment of pulmonary hypertension.


Assuntos
Hipertensão Pulmonar/genética , Hipertensão Pulmonar/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/genética , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Baixa/genética , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismo , Animais , Doxiciclina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Hemodinâmica/efeitos dos fármacos , Hipertensão Pulmonar/etiologia , Hipertensão Pulmonar/fisiopatologia , Hipertrofia Ventricular Direita/complicações , Hipóxia/complicações , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/deficiência , Masculino , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Baixa/deficiência , Vasodilatação/efeitos dos fármacos
11.
PLoS One ; 9(4): e95173, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24733189

RESUMO

BACKGROUND: Inflammation plays a key role in the development and progression of diabetic nephropathy (DN). KCa3.1, a calcium activated potassium channel protein, is associated with vascular inflammation, atherogenesis, and proliferation of endothelial cells, macrophages, and fibroblasts. We have previously demonstrated that the KCa3.1 channel is activated by TGF-ß1 and blockade of KCa3.1 ameliorates renal fibrotic responses in DN through inhibition of the TGF-ß1 pathway. The present study aimed to identify the role of KCa3.1 in the inflammatory responses inherent in DN. METHODS: Human proximal tubular cells (HK2 cells) were exposed to high glucose (HG) in the presence or absence of the KCa3.1 inhibitor TRAM34 for 6 days. The proinflammatory cytokine chemokine (C-C motif) ligand 20 (CCL20) expression was examined by real-time PCR and enzyme-linked immunosorbent assay (ELISA). The activity of nuclear factor-κB (NF-κB) was measured by nuclear extraction and electrophoretic mobility shift assay (EMSA). In vivo, the expression of CCL20, the activity of NF-κB and macrophage infiltration (CD68 positive cells) were examined by real-time PCR and/or immunohistochemistry staining in kidneys from diabetic or KCa3.1-/- mice, and in eNOS-/- diabetic mice treated with the KCa3.1 channel inhibitor TRAM34. RESULTS: In vitro data showed that TRAM34 inhibited CCL20 expression and NF-κB activation induced by HG in HK2 cells. Both mRNA and protein levels of CCL20 significantly decreased in kidneys of diabetic KCa3.1-/- mice compared to diabetic wild type mice. Similarly, TRAM34 reduced CCL20 expression and NF-κB activation in diabetic eNOS-/- mice compared to diabetic controls. Blocking the KCa3.1 channel in both animal models led to a reduction in phosphorylated NF-κB. CONCLUSIONS: Overexpression of CCL20 in human proximal tubular cells is inhibited by blockade of KCa3.1 under diabetic conditions through inhibition of the NF-κB pathway.


Assuntos
Quimiocina CCL20/metabolismo , Glucose/farmacologia , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Animais , Linhagem Celular , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/antagonistas & inibidores , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/deficiência , Túbulos Renais Proximais/patologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Pirazóis/farmacologia , Regulação para Cima/efeitos dos fármacos
12.
J Neurochem ; 130(1): 41-49, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24606313

RESUMO

Reactive astrogliosis, characterized by cellular hypertrophy and various alterations in gene expression and proliferative phenotypes, is considered to contribute to brain injuries and diseases as diverse as trauma, neurodegeneration, and ischemia. KCa3.1 (intermediate-conductance calcium-activated potassium channel), a potassium channel protein, has been reported to be up-regulated in reactive astrocytes after spinal cord injury in vivo. However, little is known regarding the exact role of KCa3.1 in reactive astrogliosis. To elucidate the role of KCa3.1 in regulating reactive astrogliosis, we investigated the effects of either blocking or knockout of KCa3.1 channels on the production of astrogliosis and astrocytic proliferation in response to transforming growth factor (TGF)-ß in primary cultures of mouse astrocytes. We found that TGF-ß increased KCa3.1 protein expression in astrocytes, with a concomitant marked increase in the expression of reactive astrogliosis, including glial fibrillary acidic protein and chondroitin sulfate proteoglycans. These changes were significantly attenuated by the KCa3.1 inhibitor 1-((2-chlorophenyl) (diphenyl)methyl)-1H-pyrazole (TRAM-34). Similarly, the increase in glial fibrillary acidic protein and chondroitin sulfate proteoglycans in response to TGF-ß was attenuated in KCa3.1(-/-) astrocytes. TRAM-34 also suppressed astrocytic proliferation. In addition, the TGF-ß-induced phosphorylation of Smad2 and Smad3 proteins was reduced with either inhibition of KCa3.1 with TRAM-34 or in KCa3.1(-/-) astrocytes. These findings highlight a novel role for the KCa3.1 channel in reactive astrogliosis phenotypic modulation and provide a potential target for therapeutic intervention for brain injuries. Reactive astrogliosis is characterized by the expression of glial fibrillary acidic protein and chondroitin sulfate proteoglycans. We demonstrate that either pharmacological blockade or knockout of KCa3.1 channels reduces reactive gliosis in cultured astrocytes caused by TGF-ß, and also reduces TGF-ß-induced phosphorylation of Smad2/3.


Assuntos
Gliose/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/antagonistas & inibidores , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/deficiência , Transdução de Sinais/fisiologia , Proteína Smad2/fisiologia , Proteína Smad3/fisiologia , Fator de Crescimento Transformador beta/fisiologia , Animais , Animais Recém-Nascidos , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Células Cultivadas , Marcação de Genes/métodos , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pirazóis/administração & dosagem , Transdução de Sinais/efeitos dos fármacos
13.
Microbiol Immunol ; 58(1): 61-7, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24236732

RESUMO

Migration of dendritic cells (DCs) plays an important role in T-cell-mediated adaptive immune responses. Lipopolysaccharide (LPS) sensed by Toll-like receptor 4 (TLR4) serves as a signal for DC migration. We analyzed LPS-induced DC volume changes preceding the directed movement towards chemoattractants. Treatment with LPS resulted in rapid, prolonged cell swelling in wild-type (WT), but not in TLR4(-/-) bone marrow-derived (BM) DCs indicating that TLR4 signaling is essential for LPS-induced swelling. As a consequence, LPS-treatment enhanced the migratory activity along a chemokine (CCL21)-gradient in WT, but not in TLR4-deficient BMDCs suggesting that the LPS/TLR4-induced swelling response facilitates DC migration. Moreover, the role of calcium-activated potassium channels (K(Ca) 3.1) as putative regulators of immune cell volume regulation and migration was analyzed in LPS-challenged BMDCs. We found that the LPS-induced swelling of K(Ca) 3.1-deficient DCs was impaired when compared to WT DCs. Accordingly, the LPS-induced increase in [Ca(2+)](i) detected in WT DCs was reduced in K(Ca) 3.1-deficient DCs. Finally, directed migration of LPS-challenged K(Ca) 3.1-deficient DCs was low compared to WT DCs indicating that activation of K(Ca) 3.1 is involved in LPS-induced DC migration. These findings suggest that both TLR4 and K(Ca) 3.1 contribute to the migration of LPS-activated DCs as an important feature of the adaptive immune response.


Assuntos
Movimento Celular/imunologia , Células Dendríticas/citologia , Células Dendríticas/imunologia , Animais , Cálcio/metabolismo , Movimento Celular/genética , Tamanho Celular/efeitos dos fármacos , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Feminino , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/deficiência , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/genética , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Espaço Intracelular/metabolismo , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Knockout , Transdução de Sinais , Receptor 4 Toll-Like/metabolismo
14.
Arterioscler Thromb Vasc Biol ; 34(1): 127-35, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24177326

RESUMO

OBJECTIVE: Intermediate and small conductance KCa channels IK1 (KCa3.1) and SK3 (KCa2.3) are primary targets of endothelial Ca(2+) signals in the arterial vasculature, and their ablation results in increased arterial tone and hypertension. Activation of IK1 channels by local Ca(2+) transients from internal stores or plasma membrane channels promotes arterial hyperpolarization and vasodilation. Here, we assess arteries from genetically altered IK1 knockout mice (IK1(-/-)) to determine whether IK1 channels exert a positive feedback influence on endothelial Ca(2+) dynamics. APPROACH AND RESULTS: Using confocal imaging and custom data analysis software, we found that although the occurrence of basal endothelial Ca(2+) dynamics was not different between IK1(-/-) and wild-type mice (P>0.05), the frequency of acetylcholine-stimulated (2 µmol/L) Ca(2+) dynamics was greatly decreased in IK1(-/-) endothelium (515±153 versus 1860±319 events; P<0.01). In IK1(-/-)/SK3(T/T) mice, ancillary suppression (+Dox) or overexpression (-Dox) of SK3 channels had little additional effect on the occurrence of events under basal or acetylcholine-stimulated conditions. However, SK3 overexpression did restore the decreased event amplitudes. Removal of extracellular Ca(2+) reduced acetylcholine-induced Ca(2+) dynamics to the same level in wild-type and IK1(-/-) arteries. Blockade of IK1 and SK3 with the combination of charybdotoxin (0.1 µmol/L) and apamin (0.5 µmol/L) or transient receptor potential vanilloid 4 channels with HC-067047 (1 µmol/L) reduced acetylcholine Ca(2+) dynamics in wild-type arteries to the level of IK1(-/-)/SK3(T/T)+Dox arteries. These drug effects were not additive. CONCLUSIONS: IK1, and to some extent SK3, channels exert a substantial positive feedback influence on endothelial Ca(2+) dynamics.


Assuntos
Acetilcolina/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/agonistas , Artérias Mesentéricas/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia , Animais , Retroalimentação Fisiológica , Feminino , Processamento de Imagem Assistida por Computador , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/deficiência , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/genética , Cinética , Masculino , Artérias Mesentéricas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio Ativados por Cálcio de Condutância Baixa/agonistas , Canais de Potássio Ativados por Cálcio de Condutância Baixa/deficiência , Canais de Potássio Ativados por Cálcio de Condutância Baixa/genética , Software , Canais de Cátion TRPV/antagonistas & inibidores , Canais de Cátion TRPV/metabolismo
15.
Transplantation ; 95(2): 285-92, 2013 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-23325003

RESUMO

BACKGROUND: The calcium-activated potassium channel KCa3.1 is critically involved in T-cell activation as well as in the proliferation of smooth muscle cells and fibroblasts. We sought to investigate whether KCa3.1 contributes to the pathogenesis of obliterative airway disease (OAD) and whether knockout or pharmacologic blockade would prevent the development of OAD. METHODS: Tracheas from CBA donors were heterotopically transplanted into the omentum of C57Bl/6J wild-type or KCa3.1 mice. C57Bl/6J recipients were either left untreated or received the KCa3.1 blocker TRAM-34 (120 mg/kg/day). Histopathology and immunologic assays were performed on postoperative day 5 or 28. RESULTS: Subepithelial T-cell and macrophage infiltration on postoperative day 5, as seen in untreated allografts, was significantly reduced in the KCa3.1 and TRAM-34 groups. Also, systemic Th1 activation was significantly and Th2 mildly reduced by KCa3.1 knockout or blockade. After 28 days, luminal obliteration of tracheal allografts was reduced from 89%±21% in untreated recipients to 53%±26% (P=0.010) and 59%±33% (P=0.032) in KCa3.1 and TRAM-34-treated animals, respectively. The airway epithelium was mostly preserved in syngeneic grafts, mostly destroyed in the KCa3.1 and TRAM-34 groups, and absent in untreated allografts. Allografts triggered an antibody response in untreated recipients, which was significantly reduced in KCa3.1 animals. KCa3.1 was detected in T cells, airway epithelial cells, and myofibroblasts. TRAM-34 dose-dependently suppressed proliferation of wild-type C57B/6J splenocytes but did not show any effect on KCa3.1 splenocytes. CONCLUSIONS: Our findings suggest that KCa3.1 channels are involved in the pathogenesis of OAD and that KCa3.1 blockade holds promise to reduce OAD development.


Assuntos
Bronquiolite Obliterante/prevenção & controle , Terapia Genética , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/antagonistas & inibidores , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Bloqueadores dos Canais de Potássio/farmacologia , Pirazóis/farmacologia , Traqueia/efeitos dos fármacos , Animais , Bronquiolite Obliterante/genética , Bronquiolite Obliterante/imunologia , Bronquiolite Obliterante/metabolismo , Bronquiolite Obliterante/patologia , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , ELISPOT , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/deficiência , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/genética , Isoanticorpos/sangue , Ativação Linfocitária/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Knockout , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/metabolismo , Mucosa Respiratória/patologia , Células Th1/efeitos dos fármacos , Células Th1/imunologia , Células Th1/metabolismo , Células Th2/efeitos dos fármacos , Células Th2/imunologia , Células Th2/metabolismo , Fatores de Tempo , Traqueia/imunologia , Traqueia/metabolismo , Traqueia/patologia , Traqueia/transplante
16.
PLoS One ; 7(10): e47744, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23077667

RESUMO

BACKGROUND: The calmodulin/calcium-activated K(+) channel KCa3.1 is expressed in red and white blood cells, epithelia and endothelia, and possibly central and peripheral neurons. However, our knowledge about its contribution to neurological functions and behavior is incomplete. Here, we investigated whether genetic deficiency or pharmacological activation of KCa3.1 change behavior and cerebral monoamine levels in mice. METHODOLOGY/PRINCIPAL FINDINGS: In the open field test, KCa3.1-deficiency increased horizontal activity, as KCa3.1(-/-) mice travelled longer distances (≈145% of KCa3.1(+/+)) and at higher speed (≈1.5-fold of KCa3.1(+/+)). Working memory in the Y-maze was reduced by KCa3.1-deficiency. Motor coordination on the rotarod and neuromuscular functions were unchanged. In KCa3.1(-/-) mice, HPLC analysis revealed that turn-over rates of serotonin were reduced in frontal cortex, striatum and brain stem, while noradrenalin turn-over rates were increased in the frontal cortex. Dopamine turn-over rates were unaltered. Plasma catecholamine and corticosterone levels were unaltered. Intraperitoneal injections of 10 mg/kg of the KCa3.1/KCa2-activator SKA-31 reduced rearing and turning behavior in KCa3.1(+/+) but not in KCa3.1(-/-) mice, while 30 mg/kg SKA-31 caused strong sedation in 50% of the animals of either genotypes. KCa3.1(-/-) mice were hyperactive (≈+60%) in their home cage and SKA-31-administration reduced nocturnal physical activity in KCa3.1(+/+) but not in KCa3.1(-/-) mice. CONCLUSIONS/SIGNIFICANCE: KCa3.1-deficiency causes locomotor hyperactivity and altered monoamine levels in selected brain regions, suggesting a so far unknown functional link of KCa3.1 channels to behavior and monoaminergic neurotransmission in mice. The tranquilizing effects of low-dose SKA-31 raise the possibility to use KCa3.1/KCa2 channels as novel pharmacological targets for the treatment of neuropsychiatric hyperactivity disorders.


Assuntos
Comportamento Animal , Córtex Cerebral , Canais de Potássio Ativados por Cálcio de Condutância Intermediária , Atividade Motora , Animais , Comportamento Animal/efeitos dos fármacos , Comportamento Animal/fisiologia , Benzotiazóis/administração & dosagem , Catecolaminas/sangue , Córtex Cerebral/metabolismo , Córtex Cerebral/fisiologia , Corticosterona/sangue , Dopamina/metabolismo , Hipercinese/genética , Hipercinese/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/deficiência , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/genética , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/fisiologia , Atividade Motora/genética , Atividade Motora/fisiologia , Norepinefrina/metabolismo , Serotonina/metabolismo
17.
J Physiol ; 589(Pt 24): 5965-86, 2011 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-22041182

RESUMO

The anterior pituitary corticotroph is a major control point for the regulation of the hypothalamic-pituitary-adrenal (HPA) axis and the neuroendocrine response to stress. Although corticotrophs are known to be electrically excitable, ion channels controlling the electrical properties of corticotrophs are poorly understood. Here, we exploited a lentiviral transduction system to allow the unequivocal identification of live murine corticotrophs in culture. We demonstrate that corticotrophs display highly heterogeneous spontaneous action-potential firing patterns and their resting membrane potential is modulated by a background sodium conductance. Physiological concentrations of corticotrophin-releasing hormone (CRH) and arginine vasopressin (AVP) cause a depolarization of corticotrophs, leading to a sustained increase in action potential firing. A major component of the outward potassium conductance was mediated via intermediate conductance calcium-activated (SK4) potassium channels. Inhibition of SK4 channels with TRAM-34 resulted in an increase in corticotroph excitability and exaggerated CRH/AVP-stimulated ACTH secretion in vitro. In accordance with a physiological role for SK4 channels in vivo, restraint stress-induced plasma ACTH and corticosterone concentrations were significantly enhanced in gene-targeted mice lacking SK4 channels (Kcnn4(-/-)). In addition, Kcnn4(-/-) mutant mice displayed enhanced hypothalamic c-fos and nur77 mRNA expression following restraint, suggesting increased neuronal activation. Thus, stress hyperresponsiveness observed in Kcnn4(-/-) mice results from enhanced secretagogue-induced ACTH output from anterior pituitary corticotrophs and may also involve increased hypothalamic drive, thereby suggesting an important role for SK4 channels in HPA axis function.


Assuntos
Sistema Hipotálamo-Hipofisário/fisiologia , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/fisiologia , Sistema Hipófise-Suprarrenal/fisiologia , Estresse Fisiológico/fisiologia , Hormônio Adrenocorticotrópico/sangue , Hormônio Adrenocorticotrópico/fisiologia , Animais , Células Cultivadas , Feminino , Células HEK293 , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/deficiência , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/genética , Lentivirus/genética , Potenciais da Membrana , Camundongos , Camundongos Knockout , RNA Mensageiro/genética , Restrição Física/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução Genética
18.
PLoS One ; 5(8): e12304, 2010 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-20808839

RESUMO

Gliomas are morbid brain tumors that are extremely resistant to available chemotherapy and radiology treatments. Some studies have suggested that calcium-activated potassium channels contribute to the high proliferative potential of tumor cells, including gliomas. However, other publications demonstrated no role for these channels or even assigned them antitumorogenic properties. In this work we characterized the expression and functional contribution to proliferation of Ca(2+)-activated K(+) channels in human glioblastoma cells. Quantitative RT-PCR detected transcripts for the big conductance (BK), intermediate conductance (IK1), and small conductance (SK2) K(+) channels in two glioblastoma-derived cell lines and a surgical sample of glioblastoma multiforme. Functional expression of BK and IK1 in U251 and U87 glioma cell lines and primary glioma cultures was verified using whole-cell electrophysiological recordings. Inhibitors of BK (paxilline and penitrem A) and IK1 channels (clotrimazole and TRAM-34) reduced U251 and U87 proliferation in an additive fashion, while the selective blocker of SK channels UCL1848 had no effect. However, the antiproliferative properties of BK and IK1 inhibitors were seen at concentrations that were higher than those necessary to inhibit channel activity. To verify specificity of pharmacological agents, we downregulated BK and IK1 channels in U251 cells using gene-specific siRNAs. Although siRNA knockdowns caused strong reductions in the BK and IK1 current densities, neither single nor double gene silencing significantly affected rates of proliferation. Taken together, these results suggest that Ca(2+)-activated K(+) channels do not play a critical role in proliferation of glioma cells and that the effects of pharmacological inhibitors occur through their off-target actions.


Assuntos
Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Glioblastoma/patologia , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/genética , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Alta/genética , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Sequência de Aminoácidos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo/genética , Condutividade Elétrica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Glioblastoma/cirurgia , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/antagonistas & inibidores , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/deficiência , Canais de Potássio Ativados por Cálcio de Condutância Alta/antagonistas & inibidores , Canais de Potássio Ativados por Cálcio de Condutância Alta/deficiência , Dados de Sequência Molecular , Potássio/metabolismo , Bloqueadores dos Canais de Potássio/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Canais de Potássio Ativados por Cálcio de Condutância Baixa/genética
19.
Pflugers Arch ; 460(6): 1029-44, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20857305

RESUMO

Increased cytosolic Ca(2+) concentrations activate Gardos K(+) channels in human erythrocytes with membrane hyperpolarization, efflux of K(+), Cl⁻, and osmotically obliged H2O resulting in cell shrinkage, a phenomenon referred to as Gardos effect. We tested whether the Gardos effect delays colloid osmotic hemolysis of injured erythrocytes from mice lacking the Ca(2+)-activated K(+) channel K(Ca)3.1. To this end, we applied patch clamp and flow cytometry and determined in vitro as well as in vivo hemolysis. As a result, erythrocytes from K(Ca)3.1-deficient (K(Ca)3.1(-/-)) mice lacked Gardos channel activity and the Gardos effect. Blood parameters, reticulocyte count, or osmotic erythrocyte resistance, however, did not differ between K(Ca)3.1(-/-) mice and their wild-type littermates, suggesting low or absent Gardos channel activity in unstressed erythrocytes. Oxidative stress-induced Ca(2+) entry and phospholipid scrambling were significantly less pronounced in K(Ca)3.1(-/-) than in wild-type erythrocytes. Moreover, in vitro treatment with α-toxin from Staphylococcus aureus, which forms pores in the cellular membrane, resulted in significantly stronger hemolysis of K(Ca)3.1(-/-) than of wild-type erythrocytes. Intravenous injection of α-toxin induced more profound hemolysis in K(Ca)3.1(-/-) than in wild-type mice. Similarly, intra-peritoneal application of the redox-active substance phenylhydrazine, an agent for the induction of hemolytic anemia, was followed by a significantly stronger decrease of hematocrit in K(Ca)3.1(-/-) than in wild-type mice. Finally, malaria infection triggered the activation of K(Ca)3.1 and transient shrinkage of the infected erythrocytes. In conclusion, K(Ca)3.1 channel activity and Gardos effect counteract hemolysis of injured erythrocytes, thus decreasing hemoglobin release into circulating blood.


Assuntos
Eritrócitos/fisiologia , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/fisiologia , Anemia Hemolítica/induzido quimicamente , Animais , Toxinas Bacterianas/farmacologia , Cálcio/sangue , Eritrócitos/efeitos dos fármacos , Feminino , Proteínas Hemolisinas/farmacologia , Hemólise/efeitos dos fármacos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/deficiência , Malária/sangue , Malária/patologia , Masculino , Camundongos , Fenil-Hidrazinas/farmacologia , Plasmodium berghei/patogenicidade , Staphylococcus aureus
20.
Proc Natl Acad Sci U S A ; 107(4): 1541-6, 2010 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-20080610

RESUMO

The calcium-activated K(+) channel KCa3.1 plays an important role in T lymphocyte Ca(2+) signaling by helping to maintain a negative membrane potential, which provides an electrochemical gradient to drive Ca(2+) influx. To assess the role of KCa3.1 channels in lymphocyte activation in vivo, we studied T cell function in KCa3.1(-/-) mice. CD4 T helper (i.e., Th0) cells isolated from KCa3.1(-/-) mice lacked KCa3.1 channel activity, which resulted in decreased T cell receptor-stimulated Ca(2+) influx and IL-2 production. Although loss of KCa3.1 did not interfere with CD4 T cell differentiation, both Ca(2+) influx and cytokine production were impaired in KCa3.1(-/-) Th1 and Th2 CD4 T cells, whereas T-regulatory and Th17 function were normal. We found that inhibition of KCa3.1(-/-) protected mice from developing severe colitis in two mouse models of inflammatory bowel disease, which were induced by (i) the adoptive transfer of mouse naïve CD4 T cells into rag2(-/-) recipients and (ii) trinitrobenzene sulfonic acid. Pharmacologic inhibitors of KCa3.1 have already been shown to be safe in humans. Thus, if these preclinical studies continue to show efficacy, it may be possible to rapidly test whether KCa3.1 inhibitors are efficacious in patients with inflammatory bowel diseases such as Crohn's disease and ulcerative colitis.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Colite/imunologia , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/imunologia , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Animais , Linfócitos B/citologia , Linfócitos B/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Diferenciação Celular , Células Cultivadas , Colite/genética , Colite/metabolismo , Colite/patologia , Citocinas/biossíntese , Citocinas/imunologia , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/imunologia , Modelos Animais de Doenças , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T Auxiliares-Indutores/citologia , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...