RESUMO
The cerebellum, the site where protein kinase C (PKC) was first discovered, contains the highest amount of PKC in the central nervous system, with PKCγ being the major isoform. Systemic PKCγ-knockout (KO) mice showed impaired motor coordination and deficient pruning of surplus climbing fibers (CFs) from developing cerebellar Purkinje cells (PCs). However, the physiological significance of PKCγ in the mature cerebellum and the cause of motor incoordination remain unknown. Using adeno-associated virus vectors targeting PCs, we showed that impaired motor coordination was restored by re-expression of PKCγ in mature PKCγ-KO mouse PCs in a kinase activity-dependent manner, while normal motor coordination in mature Prkcgfl/fl mice was impaired by the Cre-dependent removal of PKCγ from PCs. Notably, the rescue or removal of PKCγ from mature PKCγ-KO or Prkcgfl/fl mice, respectively, did not affect the CF innervation profile of PCs, suggesting the presence of a mechanism distinct from multiple CF innervation of PCs for the motor defects in PKCγ-deficient mice. We found marked potentiation of Ca2+-activated large-conductance K+ (BK) channel currents in PKCγ-deficient mice, as compared to wild-type mice, which decreased the membrane resistance, resulting in attenuation of the electrical signal during the propagation and significant alterations of the complex spike waveform. These changes in PKCγ-deficient mice were restored by the rescue of PKCγ or pharmacological suppression of BK channels. Our results suggest that PKCγ is a critical regulator that negatively modulates BK currents in PCs, which significantly influences PC output from the cerebellar cortex and, eventually, motor coordination.
Assuntos
Terapia Genética , Atividade Motora/genética , Canais de Potássio Cálcio-Ativados/metabolismo , Proteína Quinase C/metabolismo , Células de Purkinje/enzimologia , Animais , Sinalização do Cálcio , Deleção de Genes , Camundongos , Camundongos Knockout , Atividade Motora/fisiologia , Canais de Potássio Cálcio-Ativados/genética , Proteína Quinase C/genética , Potenciais SinápticosRESUMO
Postsynaptic NMDARs at spinal synapses are required for postsynaptic long-term potentiation and chronic pain. However, how presynaptic NMDARs (PreNMDARs) in spinal nociceptor terminals control presynaptic plasticity and pain hypersensitivity has remained unclear. Here we report that PreNMDARs in spinal nociceptor terminals modulate synaptic transmission in a nociceptive tone-dependent manner. PreNMDARs depresses presynaptic transmission in basal state, while paradoxically causing presynaptic potentiation upon injury. This state-dependent modulation is dependent on Ca2+ influx via PreNMDARs. Small conductance Ca2+-activated K+ (SK) channels are responsible for PreNMDARs-mediated synaptic depression. Rather, tissue inflammation induces PreNMDARs-PKG-I-dependent BDNF secretion from spinal nociceptor terminals, leading to SK channels downregulation, which in turn converts presynaptic depression to potentiation. Our findings shed light on the state-dependent characteristics of PreNMDARs in spinal nociceptor terminals on modulating nociceptive transmission and revealed a mechanism underlying state-dependent transition. Moreover, we identify PreNMDARs in spinal nociceptor terminals as key constituents of activity-dependent pain sensitization.
Assuntos
Dor Crônica/fisiopatologia , Nociceptores/metabolismo , Terminações Pré-Sinápticas/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Cálcio/metabolismo , Dor Crônica/genética , Dor Crônica/metabolismo , Proteína Quinase Dependente de GMP Cíclico Tipo I/genética , Proteína Quinase Dependente de GMP Cíclico Tipo I/metabolismo , Gânglios Espinais/citologia , Gânglios Espinais/fisiologia , Inflamação , Potenciação de Longa Duração , Depressão Sináptica de Longo Prazo , Camundongos , Camundongos Transgênicos , Substância Cinzenta Periaquedutal/citologia , Substância Cinzenta Periaquedutal/fisiologia , Canais de Potássio Cálcio-Ativados/genética , Canais de Potássio Cálcio-Ativados/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Transmissão SinápticaRESUMO
Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death worldwide. Its high metastasis rate is significantly correlated with poor patient prognosis. Elucidating the molecular mechanism underlying HCC metastasis is essential for HCC treatment. Owing to their high conductance, large-conductance calcium-activated potassium channels (BK channels) play a critical role in the control of membrane potential and have repeatedly been proposed as potential targets for cancer therapy. Emerging evidence suggests that BK channels are involved in the progression of cancer malignancies. The present study investigated the role of BK channels in mediating the hypoxia-stimulated migration of HCC cells both in vitro and in vivo in the absence and presence of various BK channels modulators. We found that BK channels were functionally expressed on the membranes of the SMMC-7721 and Huh7 HCC cell lines. Furthermore, blockage or activation of BK channels on the surface of HCC cells correspondingly inhibited or promoted HCC cell proliferation, migration and invasion in hypoxia conditions, with altered expression and distribution of cell-cell adhesion molecule E-cadherin and typical marker of mesenchymal cells, Vimentin, but not N-cadherin. Hypoxia conditions did not alter BK channels expression but increased its open probability. Moreover, BK channels blocker IbTX significantly inhibited HCC cell remote colonization in HCC cell xenografted mice. In conclusion, the results of this study suggest that blocking BK channels offers an attractive strategy for treating HCC.
Assuntos
Movimento Celular/genética , Canais de Potássio Cálcio-Ativados/genética , Canais de Potássio Cálcio-Ativados/metabolismo , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Fenômenos Eletrofisiológicos , Xenoenxertos , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Camundongos , Bloqueadores dos Canais de Potássio/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We have shown that calcium-activated potassium (KCa)-channels regulate fundamental progenitor-cell functions, including proliferation, but their contribution to cell-therapy effectiveness is unknown. Here, we test the participation of KCa-channels in human heart explant-derived cell (EDC) physiology and therapeutic potential. TRAM34-sensitive KCa3.1-channels, encoded by the KCNN4 gene, are exclusively expressed in therapeutically bioactive EDC subfractions and maintain a strongly polarized resting potential; whereas therapeutically inert EDCs lack KCa3.1 channels and exhibit depolarized resting potentials. Somatic gene transfer of KCNN4 results in membrane hyperpolarization and increases intracellular [Ca2+], which boosts cell-proliferation and the production of pro-healing cytokines/nanoparticles. Intramyocardial injection of EDCs after KCNN4-gene overexpression markedly increases the salutary effects of EDCs on cardiac function, viable myocardium and peri-infarct neovascularization in a well-established murine model of ischemic cardiomyopathy. Thus, electrophysiological engineering provides a potentially valuable strategy to improve the therapeutic value of progenitor cells for cardioprotection and possibly other indications.
Assuntos
Cálcio/metabolismo , Terapia Baseada em Transplante de Células e Tecidos/métodos , Fenômenos Eletrofisiológicos , Coração , Canais de Potássio Cálcio-Ativados/metabolismo , Potássio/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Citocinas , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Regulação da Expressão Gênica , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/genética , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Isquemia , Potenciais da Membrana/fisiologia , Camundongos , Miocárdio/metabolismo , Nanopartículas , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio Cálcio-Ativados/genética , Células-TroncoRESUMO
Visceral Leishmaniasis is a major neglected tropical disease with increasing incidences of drug resistance. This has led to the search for a suitable drug target for chemotherapeutic intervention. Potassium channels are a family of membrane proteins which play a vital role in homeostasis and any perturbation in them alters cell survival which makes them an attractive target. To characterize a calcium-activated potassium channel from Leishmania donovani (LdKCa), a putative ion-channel like protein which showed sequence similarity with other Trypanosoma cruzi putative potassium channels was selected. It was cloned and expressed with a histidine tag. MALDI confirmed that it is a potassium channel. Homology model of LdKCa was generated by I-TASSER. It is a transmembrane protein localized in the plasma membrane as predicted by DeepLoc tool. In silico analyses of the protein showed that it is a small conductance calcium activated potassium channel. Point mutation in conserved signature domain 'TXGYGD' affects the protein function as predicted by heat map analysis. The LdKCa model predicted amino acids S207, T208 and M236 as ligand-binding sites. The sequence HSLRGRSARVIQLAWRLRKARKVGPHAPSLKQKVYTLVLSWLLT was the highest scoring B-cell epitope. The highest scoring T-cell epitope was RLYSVIVYL. This study may provide new insights into antigenicity features of leishmanial calcium-activated potassium channels.
Assuntos
Leishmaniose Visceral/imunologia , Canais de Potássio Cálcio-Ativados/imunologia , Animais , Sítios de Ligação , Cálcio/metabolismo , Cálcio/uso terapêutico , Membrana Celular , Simulação por Computador , Humanos , Leishmania donovani , Canais de Potássio Cálcio-Ativados/genética , Mapas de Interação de Proteínas , Linfócitos T/imunologiaRESUMO
BACKGROUND: Calcium-activated potassium channels (KCa) are a specific type of potassium channel activated by intracellular calcium concentration changes. This group of potassium channels plays fundamental roles ranging from regulating neuronal excitability to immune cell activation. Many human diseases such as schizophrenia, hypertension, epilepsy, and cancers have been linked to mutations in this group of potassium channels. Although the KCa channels have been extensively studied electrophysiologically and pharmacologically, their spatiotemporal gene expression during embryogenesis remains mostly unknown. RESULTS: Using zebrafish as a model, we identified and renamed 14 KCa genes. We further performed phylogenetic and syntenic analyses on vertebrate KCa genes. Our data revealed that the number of KCa genes in zebrafish was increased, most likely due to teleost-specific whole-genome duplication. Moreover, we examined zebrafish KCa gene expression during early embryogenesis. The duplicated ohnologous genes show distinct and overlapped gene expression. Furthermore, we found that zebrafish KCa genes are expressed in various tissues and organs (somites, fins, olfactory regions, eye, kidney, and so on) and neuronal tissues, suggesting that they may play important roles during zebrafish embryogenesis. CONCLUSIONS: Our phylogenetic and developmental analyses shed light on the potential functions of the KCa genes during embryogenesis related to congenital diseases and human channelopathies.
Assuntos
Desenvolvimento Embrionário/fisiologia , Filogenia , Canais de Potássio Cálcio-Ativados/metabolismo , Peixe-Zebra/metabolismo , Animais , Regulação da Expressão Gênica no Desenvolvimento , Canais de Potássio Cálcio-Ativados/genética , Somitos/metabolismo , Peixe-Zebra/genéticaRESUMO
Tacrolimus (TAC, also called FK506), a common immunosuppressive drug used to prevent allograft rejection in transplant patients, is well known to alter the functions of blood vessels. In this study, we sought to determine whether chronic treatment of TAC could inhibit the activity of big-conductance Ca2+-activated K+ (BK) channels in vascular smooth muscle cells (SMCs), leading to hypertension. Our data reveal that the activity of BK channels was inhibited in cerebral artery SMCs (CASMCs) from mice after intraperitoneal injection of TAC once a day for 4 weeks. The voltage sensitivity, Ca2+ sensitivity, and open time of single BK channels were all decreased. In support, BK channel ß1-, but not α-subunit protein expression was significantly decreased in cerebral arteries. In TAC-treated mice, application of norepinephrine induced stronger vasoconstriction in both cerebral and mesenteric arteries as well as a larger [Ca2+]i in CASMCs. Chronic treatment of TAC, similar to BK channel ß1-subunit knockout (KO), resulted in hypertension in mice, but did not cause a further increase in blood pressure in BK channel ß1-subunit KO mice. Moreover, BK channel activity in CASMCs was negatively correlated with blood pressure. Our findings provide novel evidence that TAC inhibits BK channels by reducing the channel ß1-subunit expression and functions in vascular SMCs, leading to enhanced vasoconstriction and hypertension.
Assuntos
Artérias Cerebrais/efeitos dos fármacos , Hipertensão/induzido quimicamente , Músculo Liso/efeitos dos fármacos , Canais de Potássio Cálcio-Ativados/metabolismo , Tacrolimo/toxicidade , Animais , Cálcio/metabolismo , Imunossupressores/toxicidade , Camundongos , Norepinefrina/farmacologia , Canais de Potássio Cálcio-Ativados/genética , Vasoconstrição/efeitos dos fármacosRESUMO
Physiological response to thermal stimuli in mammals is mediated by a structurally diverse class of ion channels, many of which exhibit polymodal behavior. To probe the diversity of biophysical mechanisms of temperature-sensitivity, we characterized the temperature-dependent activation of MthK, a two transmembrane calcium-activated potassium channel from thermophilic archaebacteria. Our functional complementation studies show that these channels are more efficient at rescuing K+ transport at 37°C than at 24°C. Electrophysiological activity of the purified MthK is extremely sensitive (Q10 >100) to heating particularly at low-calcium concentrations whereas channels lacking the calcium-sensing RCK domain are practically insensitive. By analyzing single-channel activities at limiting calcium concentrations, we find that temperature alters the coupling between the cytoplasmic RCK domains and the pore domain. These findings reveal a hitherto unexplored mechanism of temperature-dependent regulation of ion channel gating and shed light on ancient origins of temperature-sensitivity.
Assuntos
Proteínas Arqueais/metabolismo , Regulação da Expressão Gênica em Archaea/fisiologia , Methanobacterium/metabolismo , Canais de Potássio Cálcio-Ativados/metabolismo , Proteínas Arqueais/genética , Clonagem Molecular , Escherichia coli/metabolismo , Teste de Complementação Genética , Methanobacterium/genética , Modelos Genéticos , Canais de Potássio Cálcio-Ativados/genética , Domínios Proteicos , TemperaturaRESUMO
A causative agent of Alzheimer's disease (AD) is a short amphipathic peptide called amyloid beta (Aß). Aß monomers undergo structural changes leading to their oligomerization or fibrillization. The monomers as well as all aggregated forms of Aß, i.e., oligomers, and fibrils, can bind to biological membranes, thereby modulating membrane mechanical properties. It is also known that some isoforms of the large-conductance calcium-activated potassium (BKCa) channel, including the mitochondrial BKCa (mitoBKCa) channel, respond to mechanical changes in the membrane. Here, using the patch-clamp technique, we investigated the impact of full-length Aß (Aß1-42) and its fragment, Aß25-35, on the activity of mitoBKCa channels. We found that all forms of Aß inhibited the activity of the mitoBKCa channel in a concentration-dependent manner. Since monomers, oligomers, and fibrils of Aß exhibit different molecular characteristics and structures, we hypothesized that the inhibition was not due to direct peptide-protein interactions but rather to membrane-binding of the Aß peptides. Our findings supported this hypothesis by showing that Aß peptides block mitoBKCa channels irrespective of the side of the membrane to which they are applied. In addition, we found that the enantiomeric peptide, D-Aß1-42, demonstrated similar inhibitory activity towards mitoBKCa channels. As a result, we proposed a general model in which all Aß forms i.e., monomers, oligomers, and amyloid fibrils, contribute to the progression of AD by exerting a modulatory effect on mechanosensitive membrane components.
Assuntos
Doença de Alzheimer/genética , Peptídeos beta-Amiloides/química , Amiloide/química , Canais de Potássio Cálcio-Ativados/economia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Amiloide/genética , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/farmacologia , Cálcio/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/genética , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Técnicas de Patch-Clamp , Canais de Potássio Cálcio-Ativados/antagonistas & inibidores , Canais de Potássio Cálcio-Ativados/genéticaRESUMO
Recent studies have suggested that calciumactivated potassium channel (KCa) agonists increase the proportion of mouse embryonic stem cellderived cardiomyocytes and promote the differentiation of pacemaker cells. In the present study, it was hypothesized that adiposederived stem cells (ADSCs) can differentiate into pacemakerlike cells via overexpression of the SK4 gene. ADSCs were transduced with a recombinant adenovirus vector carrying the mouse SK4 gene, whereas the control group was transduced with GFP vector. ADSCs transduced with SK4 vector were implanted into the rat left ventricular free wall. Complete atrioventricular block (AVB) was established in isolated perfused rat hearts after 2 weeks. SK4 was successfully and stably expressed in ADSCs following transduction. The mRNA levels of the pluripotent markers Oct4 and Sox2 declined and that of the transcription factor Shox2 was upregulated following SK4 transduction. The expression of αactinin and hyperpolarizationactivated cyclic nucleotidegated potassium channel 4 (HCN4) increased in the SK4 group. The hyperpolarizing activated pacemaker current If (8/20 cells) was detected in ADSCs transduced with SK4, but not in the GFP group. Furthermore, SK4 transduction induced the expression of pERK1/2 and pp38 MAPK. In the ex vivo experiments, the heart rate of the SK4 group following AVB establishment was significantly higher compared with that in the GFP group. Immunofluorescence revealed that the transduced ADSCs were successfully implanted and expressed HCN4 in the SK4 group. In conclusion, SK4 induced ADSCs to differentiate into cardiomyocytelike and pacemakerlike cells via activation of the extracellular signalregulated kinase 1/2 and p38 mitogenactivated protein kinase pathways. Therefore, ADSCs transduced with SK4 may be used to generate biological pacemakers in ex vivo rat hearts.
Assuntos
Relógios Biológicos/genética , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/genética , Miocárdio/metabolismo , Canais de Potássio Cálcio-Ativados/genética , Animais , Diferenciação Celular/genética , Regulação da Expressão Gênica no Desenvolvimento , Defeitos dos Septos Cardíacos/genética , Proteínas de Homeodomínio/genética , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Sistema de Sinalização das MAP Quinases/genética , Células-Tronco Mesenquimais/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Técnicas de Cultura de Órgãos , Ratos , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
Impaired bone formation is one of the major causes of low bone mass and skeletal fragility that occurs in osteoporosis. However, the mechanisms underlying the defects in bone formation are not well understood. Here, we report that big conductance calcium-activated potassium channels (BKs) are required for bone formation and osteoblast function both in vivo and in vitro. By 15 weeks of age, BK knockout (BKO) mice exhibited a decline in bone mineral density and trabecular bone volume of the tibiae and lumbar vertebrae, which were associated with impaired bone formation and osteoblast activity. Mechanistically, BK ablation in bone and bone marrow mesenchymal stem cells (BMSCs) of BKO mice inhibited integrin signaling. Furthermore, the binding of α subunit of BK with integrin ß1 protein in osteoblasts was confirmed, and FAK-ERK1/2 signaling was proved to be involved by genetic modification of KCNMA1 (which encodes the α subunit of BK) in ROS17/2.8 osteoblast cells. These findings indicated that BK regulates bone formation by promoting osteoblast differentiation via integrin pathway, which provided novel insight into ion transporter crosstalk with the extracellular matrix in osteoblast regulation and revealed a new potential strategy for intervention in correcting bone formation defects.
Assuntos
Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Osteogênese/genética , Osteoporose/genética , Canais de Potássio Cálcio-Ativados/genética , Animais , Diferenciação Celular/genética , Quinase 1 de Adesão Focal/genética , Integrinas/genética , Vértebras Lombares/crescimento & desenvolvimento , Vértebras Lombares/patologia , Sistema de Sinalização das MAP Quinases/genética , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos Knockout , Osteoblastos/metabolismo , Osteoblastos/patologia , Osteoporose/fisiopatologiaRESUMO
Diabetes mellitus is one of the major risk factors for cardiovascular disease and is an important health issue worldwide. Long-term diabetes causes endothelial dysfunction, which in turn leads to diabetic vascular complications. Endothelium-derived nitric oxide is a major vasodilator in large-size vessels, and the hyperpolarization of vascular smooth muscle cells mediated by the endothelium plays a central role in agonist-mediated and flow-mediated vasodilation in resistance-size vessels. Although the mechanisms underlying diabetic vascular complications are multifactorial and complex, impairment of endothelium-dependent hyperpolarization (EDH) of vascular smooth muscle cells would contribute at least partly to the initiation and progression of microvascular complications of diabetes. In this review, we present the current knowledge about the pathophysiology and underlying mechanisms of impaired EDH in diabetes in animals and humans. We also discuss potential therapeutic approaches aimed at the prevention and restoration of EDH in diabetes.
Assuntos
Fatores Biológicos/genética , Doenças Cardiovasculares/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Endotélio Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Canais de Potássio Cálcio-Ativados/genética , Animais , Fatores Biológicos/metabolismo , Doenças Cardiovasculares/tratamento farmacológico , Doenças Cardiovasculares/etiologia , Doenças Cardiovasculares/genética , Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus Tipo 1/tratamento farmacológico , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/genética , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/patologia , Junções Comunicantes/efeitos dos fármacos , Junções Comunicantes/metabolismo , Junções Comunicantes/patologia , Regulação da Expressão Gênica , Humanos , Hipoglicemiantes/uso terapêutico , Resistência à Insulina , Potenciais da Membrana/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/patologia , Canais de Potássio Cálcio-Ativados/metabolismo , Fatores de Risco , Transdução de Sinais , Vasodilatação/efeitos dos fármacosRESUMO
Elevated plasma aldosterone (Aldo) levels are associated with greater risk of cardiac ischemic events and cardiovascular mortality. Adenosine-mediated coronary vasodilation is a critical cardioprotective mechanism during ischemia; however, whether this response is impaired by increased Aldo is unclear. We hypothesized that chronic Aldo impairs coronary adenosine-mediated vasodilation via downregulation of vascular K+ channels. Male C57BL/6J mice were treated with vehicle (Con) or subpressor Aldo for 4 wk. Coronary artery function, assessed by wire myography, revealed Aldo-induced reductions in vasodilation to adenosine and the endothelium-dependent vasodilator acetylcholine but not to the nitric oxide donor sodium nitroprusside. Coronary vasoconstriction to endothelin-1 and the thromboxane A2 mimetic U-46619 was unchanged by Aldo. Additional mechanistic studies revealed impaired adenosine A2A, not A2B, receptor-dependent vasodilation by Aldo with a tendency for Aldo-induced reduction of coronary A2A gene expression. Adenylate cyclase inhibition attenuated coronary adenosine dilation but did not eliminate group differences, and adenosine-stimulated vascular cAMP production was similar between Con and Aldo mice. Similarly, blockade of inward rectifier K+ channels reduced but did not eliminate group differences in adenosine dilation whereas group differences were eliminated by blockade of Ca2+-activated K+ (KCa) channels that blunted and abrogated adenosine and A2A-dependent dilation, respectively. Gene expression of several coronary KCa channels was reduced by Aldo. Together, these data demonstrate Aldo-induced impairment of adenosine-mediated coronary vasodilation involving blunted A2A-KCa-dependent vasodilation, independent of blood pressure, providing important insights into the link between plasma Aldo and cardiac mortality and rationale for aldosterone antagonist use to preserve coronary microvascular function.NEW & NOTEWORTHY Increased plasma aldosterone levels are associated with worsened cardiac outcomes in diverse patient groups by unclear mechanisms. We identified that, in male mice, elevated aldosterone impairs coronary adenosine-mediated vasodilation, an important cardioprotective mechanism. This aldosterone-induced impairment involves reduced adenosine A2A, not A2B, receptor-dependent vasodilation associated with downregulation of coronary KCa channels and does not involve altered adenylate cyclase/cAMP signaling. Importantly, this effect of aldosterone occurred independent of changes in coronary vasoconstrictor responsiveness and blood pressure.
Assuntos
Adenosina/farmacologia , Aldosterona/farmacologia , Vasos Coronários/efeitos dos fármacos , Canais de Potássio Cálcio-Ativados/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia , Animais , Vasos Coronários/metabolismo , AMP Cíclico/metabolismo , Regulação para Baixo , Masculino , Camundongos Endogâmicos C57BL , Canais de Potássio Cálcio-Ativados/genética , Canais de Potássio Cálcio-Ativados/metabolismo , Receptor A2A de Adenosina/genética , Receptor A2A de Adenosina/metabolismo , Transdução de SinaisRESUMO
Although leaves of Anchietea salutaris are used in Brazilian traditional medicine, there is no available data in the literature proving its efficacy and safety. Thus, the aim of the study was to perform a meticulous botanical, phytochemical, toxicological, and pharmacological investigation of A. salutaris in Wistar rats. At first, a morphoanatomical characterization of Anchietea pyrifolia leaves and stems was performed. Then, a purified infusion (ethanol-soluble fraction obtained from A. pyrifolia [ESAP]) was obtained followed by its chemical profile elucidation. Furthermore, an acute toxicity test was performed, and the acute and prolonged diuretic and hypotensive effects were also evaluated in Wistar rats. Finally, the vasodilatory responses of ESAP in mesenteric vascular beds were investigated. The main secondary metabolites identified from ESAP were O-glycosylated flavonoids, chlorogenic acids, and phenylpropanoic acid derivatives. ESAP did not promote any toxic effects in female rats nor increased urinary excretion in male rats after a single exposure. However, ESAP significantly reduced renal elimination of sodium, potassium, and chloride after prolonged treatment. An ESAP highest dose promoted significant acute hypotension without affecting blood pressure levels after prolonged use. Furthermore, its cardiovascular effects seem to be related with the calcium-activated potassium channel activation in resistance vessels.
Assuntos
Anti-Hipertensivos/administração & dosagem , Hipertensão/tratamento farmacológico , Extratos Vegetais/administração & dosagem , Violaceae/química , Animais , Anti-Hipertensivos/efeitos adversos , Anti-Hipertensivos/química , Pressão Sanguínea/efeitos dos fármacos , Brasil , Diuréticos/administração & dosagem , Diuréticos/efeitos adversos , Diuréticos/química , Feminino , Humanos , Hipertensão/genética , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Masculino , Extratos Vegetais/efeitos adversos , Extratos Vegetais/química , Folhas de Planta/química , Canais de Potássio Cálcio-Ativados/genética , Canais de Potássio Cálcio-Ativados/metabolismo , Ratos WistarRESUMO
Trypanosoma cruzi develops in environments where nutrient availability, osmolarity, ionic concentrations, and pH undergo significant changes. The ability to adapt and respond to such conditions determines the survival and successful transmission of T. cruzi. Ion channels play fundamental roles in controlling physiological parameters that ensure cell homeostasis by rapidly triggering compensatory mechanisms. Combining molecular, cellular and electrophysiological approaches we have identified and characterized the expression and function of a novel calcium-activated potassium channel (TcCAKC). This channel resides in the plasma membrane of all 3 life stages of T. cruzi and shares structural features with other potassium channels. We expressed TcCAKC in Xenopus laevis oocytes and established its biophysical properties by two-electrode voltage clamp. Oocytes expressing TcCAKC showed a significant increase in inward currents after addition of calcium ionophore ionomycin or thapsigargin. These responses were abolished by EGTA suggesting that TcCAKC activation is dependent of extracellular calcium. This activation causes an increase in current and a negative shift in reversal potential that is blocked by barium. As predicted, a single point mutation in the selectivity filter (Y313A) completely abolished the activity of the channels, confirming its potassium selective nature. We have generated knockout parasites deleting one or both alleles of TcCAKC. These parasite strains showed impaired growth, decreased production of trypomastigotes and slower intracellular replication, pointing to an important role of TcCAKC in regulating infectivity. To understand the cellular mechanisms underlying these phenotypic defects, we used fluorescent probes to evaluate intracellular membrane potential, pH, and intracellular calcium. Epimastigotes lacking the channel had significantly lower cytosolic calcium, hyperpolarization, changes in intracellular pH, and increased rate of proton extrusion. These results are in agreement with previous reports indicating that, in trypanosomatids, membrane potential and intracellular pH maintenance are linked. Our work shows TcCAKC is a novel potassium channel that contributes to homeostatic regulation of important physiological processes in T. cruzi and provides new avenues to explore the potential of ion channels as targets for drug development against protozoan parasites.
Assuntos
Citoplasma/metabolismo , Potenciais da Membrana/fisiologia , Canais de Potássio Cálcio-Ativados/metabolismo , Trypanosoma cruzi/metabolismo , Cálcio/metabolismo , Membrana Celular/metabolismo , Doença de Chagas , Clonagem Molecular , Citosol/metabolismo , Fenômenos Eletrofisiológicos , Eletrofisiologia , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Concentração de Íons de Hidrogênio , Mutagênese Sítio-Dirigida , Potássio/metabolismo , Canais de Potássio/metabolismo , Canais de Potássio Cálcio-Ativados/química , Canais de Potássio Cálcio-Ativados/genética , Análise de Sequência , Trypanosoma cruzi/genéticaRESUMO
Temporal lobe epilepsy (TLE) is the most common epileptic syndrome in adults and often presents with seizures that prove intractable with currently available anticonvulsants. Thus, there is still a need for new anti-seizure drugs in this condition. Recently, we found that the casein kinase 2 inhibitor 4,5,6,7-tetrabromotriazole (TBB) prevented the emergence of spontaneous epileptic discharges in an acute in vitro epilepsy model. This prompted us to study the anti-seizure effects of TBB in the pilocarpine model of chronic epilepsy in vivo. To this end, we performed long-term video-EEG monitoring lasting 78-167 days of nine chronically epileptic rats and obtained a baseline seizure rate of 3.3 ± 1.3 per day (baseline of 27-80 days). We found a significant age effect with more pronounced seizure rates in older animals as compared to younger ones. However, the seizure rate increased to 6.3 ± 2.2 per day during the oral TBB administration (treatment period of 21-50 days), and following discontinuation of TBB, this rate remained stable with 5.2 ± 1.4 seizures per day (follow-up of 30-55 days). After completing the video-EEG during the follow-up the hippocampal tissue was prepared and studied for the expression of the Ca2+-activated K+ channel KCa2.2. We found a significant up-regulation of KCa2.2 in the epileptic CA1 region and in the neocortex, but in no other hippocampal subfield. Hence, our findings indicate that oral administration of TBB leads to persistent up-regulation of KCa2.2 in the epileptic CA1 subfield and in the neocortex, but lacks anti-seizure efficacy in the pilocarpine epilepsy model.
Assuntos
Anticonvulsivantes/uso terapêutico , Região CA1 Hipocampal/efeitos dos fármacos , Hidrocarbonetos Bromados/uso terapêutico , Canais de Potássio Cálcio-Ativados/metabolismo , Estado Epiléptico/tratamento farmacológico , Estado Epiléptico/patologia , Triazóis/uso terapêutico , Regulação para Cima/efeitos dos fármacos , Administração Oral , Animais , Região CA1 Hipocampal/metabolismo , Caseína Quinase II/metabolismo , Modelos Animais de Doenças , Estimulação Elétrica , Eletroencefalografia , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Agonistas Muscarínicos/toxicidade , Neurotransmissores/metabolismo , Pilocarpina/toxicidade , Canais de Potássio Cálcio-Ativados/genética , Ratos , Ratos Wistar , Estado Epiléptico/induzido quimicamente , Regulação para Cima/fisiologia , Gravação em VídeoRESUMO
Life-threatening malignant arrhythmias in pathophysiological conditions can increase the mortality and morbidity of patients with cardiovascular diseases. Cardiac electrical activity depends on the coordinated propagation of excitatory stimuli and the generation of action potentials in cardiomyocytes. Action potential formation results from the opening and closing of ion channels. Recent studies have indicated that small-conductance calcium-activated potassium (SK) channels play a critical role in cardiac repolarization in pathophysiological but not normal physiological conditions. The aim of this review is to describe the role of SK channels in healthy and diseased hearts, to suggest cardiovascular pathophysiologic targets for intervention, and to discuss studies of agents that target SK channels for the treatment of cardiovascular diseases.
Assuntos
Doenças Cardiovasculares/etiologia , Doenças Cardiovasculares/metabolismo , Miocárdio/metabolismo , Canais de Potássio Cálcio-Ativados/metabolismo , Animais , Doenças Cardiovasculares/fisiopatologia , Diabetes Mellitus/etiologia , Diabetes Mellitus/metabolismo , Humanos , Mutação , Canais de Potássio Cálcio-Ativados/química , Canais de Potássio Cálcio-Ativados/genética , Relação Estrutura-AtividadeRESUMO
PURPOSE: To explore the influence of intermediate-conductance-Ca2+-activated K+ channels. (IKCal) on HeLa cell proliferation. MATERIALS AND METHODS: An IKCal blocking agent (clotrimazole (CLT)) and small hairpin ribonucleic acid interference (RNAi) was used to block IKCal in HeLa cells; subsequently, cell growth was observed. Furthermore, the messenger ribonucleic acid (mRNA) expression of IKCal was detected by reverse transcriptase polymerase chain reaction (RT-PCR) after IKCal-blocking. RESULTS: The obvious morphological changes in HeLa cells were observed 48 h after CLT-blocking. The PCR results indicated that CLT reduced the mRNA expression of IKCal in HeLa cells. HeLa cells were transfected with pGenesil via RNAi; the HeLa cells transfected with pGenesil-IK displayed obvious morphological changes 48 h after transfection. In addition, RT-PCR further demonstrated the reduced mRNA expression of IKCal in the pGenesil group. CONCLUSION: CLT and blocking of IKCal gene expression effectively inhibits HeLa cell proliferation; therefore, the use of a blocking agent and RNAi both effectively downregulated the mRNA expression of IKCal, which in turn mediated the proliferation of HeLa cells, producing an antitumor effect.
Assuntos
Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio Cálcio-Ativados/antagonistas & inibidores , Canais de Potássio Cálcio-Ativados/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Células HeLa , Humanos , Microscopia de Fluorescência , RNA Mensageiro , TransfecçãoRESUMO
The currently available antihypertensive agents have undesirable adverse effects due to systemically altering target activity including receptors, channels, and enzymes. These effects, such as loss of potassium ions induced by diuretics, bronchospasm by beta-blockers, constipation by Ca2+ channel blockers, and dry cough by ACEI, lead to non-compliance with therapies (Moser, 1990). Here, based on new hypertension mechanisms, we explored a new antihypertensive approach. We report that transient receptor potential vanilloid 4 (TRPV4) interacts with Ca2+-activated potassium channel 3 (KCa2.3) in endothelial cells (ECs) from small resistance arteries of normotensive humans, while ECs from hypertensive patients show a reduced interaction between TRPV4 and KCa2.3. Murine hypertension models, induced by high-salt diet, N(G)-nitro-l-arginine intake, or angiotensin II delivery, showed decreased TRPV4-KCa2.3 interaction in ECs. Perturbation of the TRPV4-KCa2.3 interaction in mouse ECs by overexpressing full-length KCa2.3 or defective KCa2.3 had hypotensive or hypertensive effects, respectively. Next, we developed a small-molecule drug, JNc-440, which showed affinity for both TRPV4 and KCa2.3. JNc-440 significantly strengthened the TRPV4-KCa2.3 interaction in ECs, enhanced vasodilation, and exerted antihypertensive effects in mice. Importantly, JNc-440 specifically targeted the impaired TRPV4-KCa2.3 interaction in ECs but did not systemically activate TRPV4 and KCa2.3. Together, our data highlight the importance of impaired endothelial TRPV4-KCa2.3 coupling in the progression of hypertension and suggest a novel approach for antihypertensive drug development.