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1.
Gene ; 718: 143989, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31326551

RESUMO

Our comparative studies seek to understand the structure and function of ion channels in cartilaginous fish that can detect very low voltage gradients in seawater. The principal channels of the electroreceptor include a calcium activated K channel whose α subunit is Kcnma1, and a voltage-dependent calcium channel, Cacna1d. It has also been suggested based on physiological and pharmacological evidence that a voltage-gated K channel is present in the basal membranes of the receptor cells which modulates synaptic transmitter release. Large conductance calcium-activated K channels (BK) are comprised of four α subunits, encoded by Kcnma1 and modulatory ß subunits of the Kcnmb class. We recently cloned and published the skate Kcnma1 gene and most of Kcnmb4 using purified mRNA of homogenized electroreceptors. Bellono et al. have recently performed RNA sequencing (RNA-seq) on purified mRNA from skate electroreceptors and found several ion channels including Kcnma1. We searched the Bellono et al. RNA-seq repository for additional channels and subunits. Our most significant findings are the presence of two Shaker type voltage dependent K channel sequences which are grouped together as isoforms in the data repository. The larger of these is a skate ortholog of the voltage dependent fast potassium channel Kv1.1, which is expressed at appreciable levels. The second ortholog is similar to Kv1.5 but has fewer N-terminal amino acids than other species. The sequence for Kv1.5 in the skate is very strongly aligned with the recently reported sequence for potassium channels in the electroreceptors of the cat shark, S. retifer, which also modulate synaptic transmission. The latter channel was designated as Kv1.3 in the initial report, but we suggest that these channels are actually orthologs of each other, and that Kv1.5 is the prevailing designation. We also found a beta subunit sequence (Kcnab2) which may co-assemble with one or both of the voltage gated channels. The new channels and subunits were verified by RT-PCR and the Kv1.1 sequence was confirmed by cloning. We also searched the RNA-seq repository for accessory subunits of Kcnma1, and found a computer-generated assembly that contained a complete sequence of its ß subunit, Kcnmb2. Skate Kcnmb2 has a total of 279 amino acids, with 51 novel amino acids at the N-terminus which may play a specific physiological role. This sequence was confirmed by PCR and cloning. However, skate Kcnmb2 is expressed at low levels in the electroreceptor compared to Kcnma1 and skate Kcnmb1 is absent. The evolutionary origin of the newly described K channels and their subunits was studied by alignments with mammalian sequences, including human, and also those in related fish: the whale shark (R. typus), the ghost shark (C.milii), and (S. retifer). There are also orthologous K channels of the lamprey, which has electroreceptors. Tree building and bootstrap programs were used to confirm phylogenetic inferences. Further research should focus on the subcellular locations of these channels, their gating behavior, and the effects of accessory subunits on gating.


Assuntos
Clonagem Molecular , Proteínas de Peixes/genética , Canal de Potássio Kv1.1/genética , Canal de Potássio Kv1.5/genética , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Filogenia , Raias/genética , Animais , Proteínas de Peixes/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Canal de Potássio Kv1.1/metabolismo , Canal de Potássio Kv1.5/metabolismo , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Raias/metabolismo , Especificidade da Espécie
2.
Life Sci ; 231: 116555, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31194991

RESUMO

AIMS: Caffeine is a methylxanthine with multiple actions in vascular smooth muscle cells (VSMCs), including the increase in the intracellular Ca2+ (iCa2+) concentration by the activation of ryanodine receptors (RyRs). The present study aimed at investigating the participation of Ca2+-influx through different Ca2+-channels on the transient contraction (TC) induced by caffeine in mice mesenteric arteries. MAIN METHODS: Second-order of mesenteric arteries was isolated from male Swiss mice. Vessels without functional endothelium were stimulated with caffeine (10 mM). The caffeine-induced TC was evaluated after the incubation of artery rings for 30 min with the following drugs: nifedipine (10 µM), a Cav1.2 blocker; 2-aminoethoxydiphenyl borate (2-APB; 10 µM) and ruthenium red (RuR; 10 µM), transient receptor potential (TRPs) channels blockers; capsazepine (10 µM) and HC067047 (10 µM), TRPV1 and TRPV4 antagonists, respectively; paxilline (1 µM), a selective BKCa blocker; and SKF-96365 (30 µM), an Orai blocker. Ca2+-fluorescence measurements were also performed on the investigated arteries. KEY FINDINGS: The TC induced by caffeine was partially dependent on Ca2+-influx. However, the blockage of Cav1.2 increased the TC while reduced the iCa2+ signal. Similar results were observed after the blockage of TRPs or BKCa. Therefore, caffeine promoted Ca2+-influx via TRPs and Cav1.2, and hyperpolarization through the activation of BKCa, inducing negative feedback of TC. SIGNIFICANCE: Our results indicate an alternative mechanism for the control of VSMCs contraction in resistance arteries. The evidence of the negative feedback of contraction via TRP-Cav1.2-BKCa provides a new perspective for understanding the mechanism involved in the vascular responses triggered by caffeine.


Assuntos
Cafeína/farmacologia , Canais de Cálcio Tipo L/metabolismo , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Artérias Mesentéricas/efeitos dos fármacos , Animais , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Masculino , Artérias Mesentéricas/metabolismo , Camundongos , Contração Muscular/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Canais de Cátion TRPV/metabolismo , Vasoconstrição/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia
3.
Eur J Pharmacol ; 854: 365-371, 2019 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-31051158

RESUMO

Tanshinone ⅡA Sodium Sulfonate (DS-201), a derivative of traditional Chinese medicinal herb Danshen, has been clinically used for various cardiovascular diseases. Previous studies showed that DS-201 induced vascular relaxation partly due to the activation of the large conductance Ca2+-activated potassium (BKCa) channels. However, the efficacy of DS-201 on the resistant vessels in hypertension remains unknown. Mesentery arteries obtained from spontaneously hypertensive rats (SHR) and hypertension patients were used in this study. The endothelium-denuded mesenteric arteries were prepared to measure the artery tension and evaluate the vasodilatory effect of DS-201. The results showed that DS-201 had a vasodilatory effect on the mesenteric artery rings pre-contracted with either phenylephrine (PE) or thromboxane mimetic U46619 in a concentration-dependent manner. However, the vasodilatory effect of DS-201 significantly decreased in hypertension than in control arteries due to a decrease in protein level of BKCa ß1subunit. A BKCa channel blocker IbTX (200 nM) significantly inhibited the relaxant effect of DS-201 on non-hypertensive arteries, whereas the BKCa channel specific agonist NS1619 rescued the vasodilating effects of DS-201 on hypertensive vessels. These results indicate that the vasodilating effect of DS-201 is BKCa-dependent. This study demonstrated that DS-201 alone may not be effective for treating hypertension, but it may be considered as therapy combined with BKCa-agonists or methods rescuing BKCa functions.


Assuntos
Hipertensão/fisiopatologia , Artérias Mesentéricas/efeitos dos fármacos , Artérias Mesentéricas/fisiopatologia , Fenantrenos/farmacologia , Vasodilatação/efeitos dos fármacos , Animais , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Hipertensão/metabolismo , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/antagonistas & inibidores , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Masculino , Ratos , Vasoconstrição/efeitos dos fármacos
4.
Toxicol Lett ; 312: 55-64, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-30974163

RESUMO

Formaldehyde (FA), a well-known toxic gas molecule similar to nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H2S), is widely produced endogenously via numerous biochemical pathways, and has a number of physiological roles in the biosystem. We attempted to investigate the vasorelaxant effects of FA and their underlying mechanisms. We found that FA induced vasorelaxant effects on rat aortic rings in a concentration-dependent manner. The NO/cyclic guanosine 5' monophosphate (cGMP) pathway was up-regulated when the rat aortas were treated with FA. The expression of large-conductance Ca2+-activated K+ (BKCa) channel subunits α and ß of the rat aortas was increased by FA. Similarly, the levels of ATP-sensitive K+ (KATP) channel subunits Kir6.1 and Kir6.2 were also up-regulated when the rat aortas were incubated with FA. In contrast, levels of the L-type Ca2+ channel (LTCC) subunits, Cav1.2 and Cav1.3, decreased dramatically with increasing concentrations of FA. We demonstrated that the regulation of FA on vascular contractility may be via the up-regulation of the NO/cGMP pathway and the modulation of ion channels, including the upregulated expression of the KATP and BKCa channels and the inhibited expression of LTCCs. Further study is needed to explore the in-depth mechanisms of FA induced vasorelaxation.


Assuntos
GMP Cíclico/metabolismo , Formaldeído/farmacologia , Óxido Nítrico/metabolismo , Transdução de Sinais/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos , Animais , Aorta Torácica , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo L/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Canais KATP/genética , Canais KATP/metabolismo , 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 , Ratos , Ratos Wistar
5.
Proc Natl Acad Sci U S A ; 115(40): 9923-9928, 2018 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-30224470

RESUMO

Structural symmetry is a hallmark of homomeric ion channels. Nonobligatory regulatory proteins can also critically define the precise functional role of such channels. For instance, the pore-forming subunit of the large conductance voltage and calcium-activated potassium (BK, Slo1, or KCa1.1) channels encoded by a single KCa1.1 gene assembles in a fourfold symmetric fashion. Functional diversity arises from two families of regulatory subunits, ß and γ, which help define the range of voltages over which BK channels in a given cell are activated, thereby defining physiological roles. A BK channel can contain zero to four ß subunits per channel, with each ß subunit incrementally influencing channel gating behavior, consistent with symmetry expectations. In contrast, a γ1 subunit (or single type of γ1 subunit complex) produces a functionally all-or-none effect, but the underlying stoichiometry of γ1 assembly and function remains unknown. Here we utilize two distinct and independent methods, a Forster resonance energy transfer-based optical approach and a functional reporter in single-channel recordings, to reveal that a BK channel can contain up to four γ1 subunits, but a single γ1 subunit suffices to induce the full gating shift. This requires that the asymmetric association of a single regulatory protein can act in a highly concerted fashion to allosterically influence conformational equilibria in an otherwise symmetric K+ channel.


Assuntos
Ativação do Canal Iônico/fisiologia , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Subunidades Proteicas/metabolismo , Animais , Transferência Ressonante de Energia de Fluorescência/métodos , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Camundongos , Subunidades Proteicas/genética , Xenopus laevis
6.
Bioelectrochemistry ; 124: 80-92, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30015269

RESUMO

The metastatic potential of cancer cells is related to their migratory ability, which is influenced by in vivo microenvironment possessing specific physiochemical factors including electric properties. In the present study, we isolated two different subsets of lung adenocarcinoma H1975 cells, as side population (SP) and main population (MP). SP cells were demonstrated to have cancer stem cell characteristics. Using a microscale device to provide physiological direct-current electric field (dcEF), we investigated the electrotactic responses of the SP and MP cells. The results showed that both SP and MP cells exhibited enhanced cathodal migration ability with actin reorganization and transient intracellular calcium ions ([Ca2+]i) increase under dcEF stimulation. For SP cells, the treatment of either stretch-activated cation channels (SACCs) inhibitor or the blockage of intracellular Ca2+ release could partially inhibited dcEF-activated [Ca2+]i increase, and the concomitant treatment led to a complete inhibition. For MP cells, SACCs activation was entirely responsible for EF-activated increase of [Ca2+]i. All these results suggested that that intracellular Ca2+ activation may be associated with cancer cell tumorigenicity and metastasis.


Assuntos
Adenocarcinoma/patologia , Cálcio/metabolismo , Eletricidade , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Neoplasias Pulmonares/patologia , Actinas/metabolismo , Adenocarcinoma de Pulmão , Linhagem Celular Tumoral , Eletrodos , Retículo Endoplasmático/metabolismo , Homeostase , Humanos , Células-Tronco Neoplásicas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Acta Biochim Biophys Sin (Shanghai) ; 50(6): 560-566, 2018 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-29697782

RESUMO

Autoantibodies against angiotensin II Type 1 receptor (AT1-AA) are routinely detected in the serum of preeclampsia patients, which results in an increase in vascular tone and an elevation in intracellular calcium concentration of rat vascular smooth muscle (VSM). The big conductance calcium-activated potassium channels (BKCa channels) account for the dominant outward currents in VSMCs, contributing to membrane hyperpolarization and vasodilation. In the present study, we investigated the effect of AT1-AA on BKCa channels. A preeclampsia model was established by passively immunizing healthy pregnant BALB/c mice with AT1-AA extracted from hybridoma culture supernatant. Blood pressure, serum AT1-AA levels, and urinary protein were measured in the immunized mice. BKCa channel expression was detected using qRT-PCR and immunohistochemical technique. The patch-clamp technique was used to record the single currents of BKCa channels in the HEK293T cells that had been transfected. AT1-AA immunized mice exhibited elevated AT1-AA and urinary protein levels compared with mice of the vehicle group. Systolic blood pressure was also increased in the immunized group. BKCa channel ß1-subunit expression was reduced in the mesenteric arteries of immunized mice. AT1-AA could inhibit the BKCa currents and the inhibitory effects were not completely reversed following the application of valsartan, an inhibitor of AT1 receptor. In conclusion, AT1-AA could decrease BKCa expression and inhibit BKCa activity independent of AT1R. These inhibitory effects are likely to be contributory factors in the promotion of increased vascular tone caused by AT1-AA in preeclampsia.


Assuntos
Autoanticorpos/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/antagonistas & inibidores , Receptor Tipo 1 de Angiotensina/imunologia , Animais , Autoanticorpos/administração & dosagem , Autoanticorpos/imunologia , Pressão Sanguínea/efeitos dos fármacos , Pressão Sanguínea/imunologia , Feminino , Células HEK293 , Humanos , Imunização , 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 , Artérias Mesentéricas/efeitos dos fármacos , Artérias Mesentéricas/metabolismo , Artérias Mesentéricas/fisiologia , Camundongos Endogâmicos BALB C , Pré-Eclâmpsia/genética , Pré-Eclâmpsia/imunologia , Pré-Eclâmpsia/metabolismo , Gravidez , Receptor Tipo 1 de Angiotensina/metabolismo
8.
Glia ; 66(7): 1507-1519, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29508439

RESUMO

Müller cell gliosis is a common response in many retinal pathological conditions. We previously demonstrated that downregulation of Kir channels contributes to Müller cell gliosis in a rat chronic ocular hypertension (COH) model. Here, the possible involvement of outward K+ currents in Müller cell gliosis was investigated. Outward K+ current densities in Müller cells isolated from COH rats, as compared with those in normal rats, showed a significant increase, which was mainly contributed by large-conductance Ca2+ -activated K+ (BKCa ) channels. The involvement of BKCa channels in Müller cell gliosis is suggested by the fact that glial fibrillary acidic protein (GFAP) levels were augmented in COH retinas when these channels were suppressed by intravitreal injections of iberiotoxin. In COH retinas an increase in dopamine (DA) D1 receptor (D1R) expression in Müller cells was revealed by both immunohistochemistry and Western blotting. Moreover, protein levels of tyrosine hydroxylase were also increased, and consistent to this, retinal DA contents were elevated. SKF81297, a selective D1R agonist, enhanced BKCa currents of normal Müller cells through intracellular cAMP-PKA signaling pathway. Furthermore, GFAP levels were increased by the D1R antagonist SCH23390 injected intravitreally through eliminating the BKCa current upregulation in COH retinas, but partially reduced by SKF81297. All these results strongly suggest that the DA-D1R system may be activated to a stronger extent in COH rat retinas, thus increasing BKCa currents of Müller cells. The upregulation of BKCa channels may antagonize the Kir channel inhibition-induced depolarization of Müller cells, thereby attenuating the gliosis of these cells.


Assuntos
Células Ependimogliais/metabolismo , Gliose/metabolismo , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Hipertensão Ocular/metabolismo , Receptores de Dopamina D1/metabolismo , Animais , Modelos Animais de Doenças , Células Ependimogliais/patologia , Proteína Glial Fibrilar Ácida/metabolismo , Gliose/patologia , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/antagonistas & inibidores , Masculino , Potenciais da Membrana/fisiologia , Hipertensão Ocular/patologia , Ratos Sprague-Dawley , Receptores de Dopamina D1/antagonistas & inibidores , Tirosina 3-Mono-Oxigenase/metabolismo , Corpo Vítreo/metabolismo , Corpo Vítreo/patologia
9.
Biol Pharm Bull ; 41(2): 272-276, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29386487

RESUMO

Methylglyoxal, a highly reactive dicarbonyl compound, is formed as a by-product of glycolysis and plays an important role in the pathogenesis of diabetic complications, including diabetic retinopathy. However, it remains to be determined how methylglyoxal affects the regulatory mechanisms of retinal blood flow. In this study, we examined the effects of methylglyoxal on ß2-adrenoceptor-mediated vasodilatory mechanisms in rat retinal arterioles. The retinal vasodilator responses were assessed by measuring the diameter of retinal arterioles in the fundus images. Intravitreal injection of methylglyoxal significantly diminished the vasodilation of retinal arterioles induced by the ß2-adrenoceptor agonist salbutamol. The vasodilator effect of BMS-191011, a large-conductance Ca2+-activated K+ (BKCa) channel opener, on retinal arterioles was also attenuated by methylglyoxal. In contrast, methylglyoxal had no significant effect on retinal vasodilator response to forskolin. Methylglyoxal attenuated retinal vasodilator response to salbutamol under blockade of BKCa channels with iberiotoxin, an inhibitor of the channels. These results suggest that methylglyoxal attenuates ß2-adrenoceptor-mediated retinal vasodilation by impairing the coupling of the ß2-adrenoceptor to the guanine nucleotide-binding protein (Gs protein) and the function of the BKCa channel. Increased methylglyoxal in the eyes may contribute to the impairment of regulatory mechanisms of retinal blood flow in patients with diabetic retinopathy.


Assuntos
Antagonistas de Receptores Adrenérgicos beta 2/metabolismo , Arteríolas/metabolismo , Aldeído Pirúvico/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Retina/metabolismo , Vasos Retinianos/metabolismo , Vasodilatação , Agonistas de Receptores Adrenérgicos beta 2/administração & dosagem , Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Antagonistas de Receptores Adrenérgicos beta 2/administração & dosagem , Albuterol/administração & dosagem , Albuterol/farmacologia , Animais , Arteríolas/efeitos dos fármacos , Agonistas dos Canais de Cálcio/administração & dosagem , Agonistas dos Canais de Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Colforsina/administração & dosagem , Colforsina/farmacologia , Relação Dose-Resposta a Droga , Subunidades alfa Gs de Proteínas de Ligação ao GTP/antagonistas & inibidores , Subunidades alfa Gs de Proteínas de Ligação ao GTP/química , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Injeções Intravenosas , Injeções Intravítreas , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/agonistas , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/antagonistas & inibidores , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Masculino , Oxidiazóis/administração & dosagem , Oxidiazóis/farmacologia , Peptídeos/farmacologia , Aldeído Pirúvico/administração & dosagem , Ratos Sprague-Dawley , Receptores Adrenérgicos beta 2/química , Retina/efeitos dos fármacos , Vasos Retinianos/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos , Vasodilatadores/administração & dosagem , Vasodilatadores/farmacologia
10.
Vascul Pharmacol ; 102: 44-55, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29355732

RESUMO

Endothelium-dependent component of cannabinoid-induced vasodilation has been postulated to require G-protein-coupled non-CB1/CB2 endothelial cannabinoid (eCB) receptor. GPR18 was proposed as a candidate for eCBR. To address the hypothesis that the effects attributed to eCBR are mediated by G-protein-coupled receptor (GPCR)-independent targets, we studied the electrical responses in endothelial cells, focusing on BKCa channels. In patches excised from endothelial-derived EA.hy926 cells, N-arachidonoyl glycine (NAGly) and abnormal cannabidiol (abn-cbd), prototypical agonists for eCB receptor, stimulate single BKCa activity in a concentration- and Ca2+-dependent manner. The postulated eCB receptor inhibitors rimonabant and AM251 were found to inhibit basal and stimulated by NAGly- and abn-cbd BKCa activity in cell-free patches. In isolated mice aortas, abn-cbd and NAGly produced endothelial cell hyperpolarization that was sensitive to paxilline, a selective BKCa inhibitor, but not to GPR18 antibody, and mimicked by NS1619, a direct BKCa opener. In excised patches from mice aortic endothelium, single channel activity with characteristics similar to BKCa was established by the addition of abn-cbd and NAGly. We conclude that the two cannabinoids abn-cbd and NAGly initiate a GPR18-independent activation of BKCa channels in mice aortic endothelial cells that might contribute to vasodilation to cannabinoids.


Assuntos
Aorta Torácica/efeitos dos fármacos , Ácidos Araquidônicos/farmacologia , Agonistas de Receptores de Canabinoides/farmacologia , Células Endoteliais/efeitos dos fármacos , Glicina/análogos & derivados , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/agonistas , Receptores de Canabinoides/efeitos dos fármacos , Resorcinóis/farmacologia , Animais , Aorta Torácica/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Antagonistas de Receptores de Canabinoides/farmacologia , Linhagem Celular , Relação Dose-Resposta a Droga , Células Endoteliais/metabolismo , Feminino , Glicina/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Masculino , Potenciais da Membrana , Camundongos Endogâmicos C57BL , Receptores de Canabinoides/metabolismo , Transdução de Sinais/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos
11.
Neuropsychopharmacology ; 43(3): 492-502, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-28722023

RESUMO

In fragile X syndrome (FXS), sensory hypersensitivity and impaired habituation is thought to result in attention overload and various behavioral abnormalities in reaction to the excessive and remanent salience of environment features that would normally be ignored. This phenomenon, termed sensory defensiveness, has been proposed as the potential cause of hyperactivity, hyperarousal, and negative reactions to changes in routine that are often deleterious for FXS patients. However, the lack of tools for manipulating sensory hypersensitivity has not allowed the experimental testing required to evaluate the relevance of this hypothesis. Recent work has shown that BMS-204352, a BKCa channel agonist, was efficient to reverse cortical hyperexcitability and related sensory hypersensitivity in the Fmr1-KO mouse model of FXS. In the present study, we report that exposing Fmr1-KO mice to novel or unfamiliar environments resulted in multiple behavioral perturbations, such as hyperactivity, impaired nest building and excessive grooming of the back. Reversing sensory hypersensitivity with the BKCa channel agonist BMS-204352 prevented these behavioral abnormalities in Fmr1-KO mice. These results are in support of the sensory defensiveness hypothesis, and confirm BKCa as a potentially relevant molecular target for the development of drug medication against FXS/ASD.


Assuntos
Síndrome do Cromossomo X Frágil/fisiopatologia , Asseio Animal/fisiologia , Atividade Motora/fisiologia , Comportamento de Nidação/fisiologia , Animais , Ansiolíticos/farmacologia , Diazepam/farmacologia , Modelos Animais de Doenças , Meio Ambiente , Proteína do X Frágil de Retardo Mental/genética , Proteína do X Frágil de Retardo Mental/metabolismo , Asseio Animal/efeitos dos fármacos , Indóis/farmacologia , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/agonistas , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Masculino , Camundongos Knockout , Atividade Motora/efeitos dos fármacos , Comportamento de Nidação/efeitos dos fármacos , Neurotransmissores/farmacologia , Psicotrópicos/farmacologia , Recognição (Psicologia) , Comportamento Estereotipado/efeitos dos fármacos , Comportamento Estereotipado/fisiologia
12.
Biochim Biophys Acta Biomembr ; 1860(4): 943-952, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28966112

RESUMO

BK channels are dually regulated by voltage and Ca2+, providing a cellular mechanism to couple electrical and chemical signalling. Intracellular Ca2+ concentration is sensed by a large cytoplasmic region in the channel known as "gating ring", which is formed by four tandems of regulator of conductance for K+ (RCK1 and RCK2) domains. The recent crystal structure of the full-length BK channel from Aplysia californica has provided new information about the residues involved in Ca2+ coordination at the high-affinity binding sites located in the RCK1 and RCK2 domains, as well as their cooperativity. Some of these residues have not been previously studied in the human BK channel. In this work we have investigated, through site directed mutagenesis and electrophysiology, the effects of these residues on channel activation by voltage and Ca2+. Our results demonstrate that the side chains of two non-conserved residues proposed to coordinate Ca2+ in the A. californica structure (G523 and E591) have no apparent functional role in the human BK Ca2+ sensing mechanism. Consistent with the crystal structure, our data indicate that in the human channel the conserved residue R514 participates in Ca2+ coordination in the RCK1 binding site. Additionally, this study provides functional evidence indicating that R514 also interacts with residues E902 and Y904 connected to the Ca2+ binding site in RCK2. Interestingly, it has been proposed that this interaction may constitute a structural correlate underlying the cooperative interactions between the two high-affinity Ca2+ binding sites regulating the Ca2+ dependent gating of the BK channel. This article is part of a Special Issue entitled: Beyond the Structure-Function Horizon of Membrane Proteins edited by Ute Hellmich, Rupak Doshi and Benjamin McIlwain.


Assuntos
Cálcio/metabolismo , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Domínios Proteicos , Sequência de Aminoácidos , Animais , Aplysia/genética , Aplysia/metabolismo , Sítios de Ligação/genética , Cristalografia por Raios X , Células HEK293 , Humanos , Ativação do Canal Iônico/genética , Ativação do Canal Iônico/fisiologia , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Potenciais da Membrana/genética , Potenciais da Membrana/fisiologia , Modelos Moleculares , Mutação , Técnicas de Patch-Clamp , Homologia de Sequência de Aminoácidos
13.
Pharmacol Rep ; 69(6): 1131-1139, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29128791

RESUMO

BACKGROUND: Some cannabinoids, a family of compounds derived from Cannabis sativa (marijuana), have previously shown vasodilator effects in several studies, a feature that makes them suitable for the generation of a potential treatment for hypertension. The mechanism underlying this vasodilator effect in arteries is still controversial. In this report, we explored how the synthetic cannabinoids ACPA (CB1-selective agonist) and JWH-133 (CB2-selective agonist) regulate the vascular tone of rat superior mesenteric arteries. METHODS: To screen the expression of CB1 (Cannabinoid receptor 1) and CB2 (Cannabinoid receptor 2) receptors in arterial rings or isolated smooth muscle cells obtained from the artery, immunocytochemistry, immunohistochemistry, and confocal microscopy were performed. In addition, the effects on vascular tone induced by the two cannabinoids were tested in isometric tension experiments in rings obtained from superior mesenteric arteries. The participation of voltage and calcium-activated potassium channel of big conductance (BKCa) and the role of nitric oxide (NO) release on the vascular effects induced by ACPA and JWH-133 were tested. RESULTS: CB1 and CB2 receptors were highly expressed in the rat superior mesenteric artery, in both smooth muscle and endothelium. The vasodilation effect shown by ACPA was endothelium-dependent through a mechanism involving CB1 receptors, BKCa channel activation, and NO release; meanwhile, the vasodilator effect of JWH-133 was induced by the activation of CB2 receptors located in smooth muscle and by a CB2 receptor-independent mechanism inducing NO release. CONCLUSIONS: CB1 and CB2 receptor activation in superior mesenteric artery causes vasorelaxation by mechanisms involving BKCa channels and NO release.


Assuntos
Ácidos Araquidônicos/farmacologia , Canabinoides/farmacologia , Receptor CB1 de Canabinoide/agonistas , Receptor CB2 de Canabinoide/agonistas , Animais , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/efeitos dos fármacos , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Masculino , Artéria Mesentérica Superior/efeitos dos fármacos , Artéria Mesentérica Superior/metabolismo , Microscopia Confocal , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Óxido Nítrico/metabolismo , Ratos , Ratos Wistar , Receptor CB1 de Canabinoide/metabolismo , Receptor CB2 de Canabinoide/metabolismo , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia
15.
J Gen Physiol ; 149(11): 985-1007, 2017 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-29025867

RESUMO

For those interested in the machinery of ion channel gating, the Ca2+ and voltage-activated BK K+ channel provides a compelling topic for investigation, by virtue of its dual allosteric regulation by both voltage and intracellular Ca2+ and because its large-single channel conductance facilitates detailed kinetic analysis. Over the years, biophysical analyses have illuminated details of the allosteric regulation of BK channels and revealed insights into the mechanism of BK gating, e.g., inner cavity size and accessibility and voltage sensor-pore coupling. Now the publication of two structures of an Aplysia californica BK channel-one liganded and one metal free-promises to reinvigorate functional studies and interpretation of biophysical results. The new structures confirm some of the previous functional inferences but also suggest new perspectives regarding cooperativity between Ca2+-binding sites and the relationship between voltage- and Ca2+-dependent gating. Here we consider the extent to which the two structures explain previous functional data on pore-domain properties, voltage-sensor motions, and divalent cation binding and activation of the channel.


Assuntos
Ativação do Canal Iônico , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Animais , Aplysia/fisiologia , Humanos , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética
16.
J Vasc Res ; 54(6): 329-343, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29040972

RESUMO

AIM: The objective of this study was to examine the effects of n-3 polyunsaturated fatty acids (n-3 PUFAs) on coronary arterial large conductance Ca2+-activated K+ (BK) channel function in coronary smooth muscle cells (SMCs) of streptozotocin-induced diabetic rats. METHODS: The effects of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) on coronary BK channel open probabilities were determined using the patch clamp technique. The mRNA and protein expressions of BK channel subunits were measured using qRT-PCR and Western blots. The coronary artery tension and coronary SMC Ca2+ concentrations were measured using a myograph system and fluorescence Ca2+ indicator. RESULTS: Compared to nondiabetic control rats, the BK channel function was impaired with a reduced response to EPA and DHA in freshly isolated SMCs of diabetic rats. Oral administration of n-3 PUFAs had no effects on protein expressions of BK channel subunits in nondiabetic rats, but significantly enhanced those of BK-ß1 in diabetic rats without altering BK-α protein levels. Moreover, coronary ring tension induced by iberiotoxin (a specific BK channel blocker) was increased and cytosolic Ca2+ concentrations in coronary SMCs were decreased in diabetic rats, but no changes were found in nondiabetic rats. CONCLUSIONS: n-3 PUFAs protect the coronary BK channel function and coronary vasoreactivity in diabetic rats as a result of not only increasing BK-ß1 protein expressions, but also decreasing coronary artery tension and coronary smooth muscle cytosolic Ca2+ concentrations.


Assuntos
Doença da Artéria Coronariana/prevenção & controle , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Tipo 1/tratamento farmacológico , Angiopatias Diabéticas/prevenção & controle , Ácidos Docosa-Hexaenoicos/farmacologia , Ácido Eicosapentaenoico/farmacologia , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Vasoconstrição/efeitos dos fármacos , Animais , Sinalização do Cálcio/efeitos dos fármacos , Doença da Artéria Coronariana/genética , Doença da Artéria Coronariana/metabolismo , Doença da Artéria Coronariana/fisiopatologia , Vasos Coronários/efeitos dos fármacos , Vasos Coronários/metabolismo , Vasos Coronários/fisiopatologia , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/fisiopatologia , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/fisiopatologia , Angiopatias Diabéticas/genética , Angiopatias Diabéticas/metabolismo , Angiopatias Diabéticas/fisiopatologia , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/efeitos dos fármacos , 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 , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Potenciais da Membrana , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiopatologia , Miócitos de Músculo Liso/metabolismo , Bloqueadores dos Canais de Potássio/farmacologia , Ratos Sprague-Dawley , Fatores de Tempo
17.
Epigenomics ; 9(11): 1403-1422, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28990796

RESUMO

AIM: Homocysteine (Hcy) is a sensitive marker of one-carbon metabolism. Higher Hcy levels have been associated with global DNA hypomethylation. We investigated the association between plasma Hcy and epigenome-wide DNA methylation in leukocytes. METHODS: Methylation was measured using Illumina 450 k arrays in 2035 individuals from six cohorts. Hcy-associated differentially methylated positions and regions were identified using meta-analysis. RESULTS: Three differentially methylated positions cg21607669 (SLC27A1), cg26382848 (AJUBA) and cg10701000 (KCNMA1) at chromosome 19, 14 and 10, respectively, were significantly associated with Hcy. In addition, we identified 68 Hcy-associated differentially methylated regions, the most significant of which was a 1.8-kb spanning domain (TNXB/ATF6B) at chromosome 6. CONCLUSION: We identified novel epigenetic loci associated with Hcy levels, of which specific role needs to be further validated.


Assuntos
Metilação de DNA , Epigênese Genética , Homocisteína/sangue , Leucócitos/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Proteínas de Transporte de Ácido Graxo/genética , Proteínas de Transporte de Ácido Graxo/metabolismo , Feminino , Estudo de Associação Genômica Ampla , Humanos , Proteínas com Domínio LIM/genética , Proteínas com Domínio LIM/metabolismo , 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 , Tenascina/genética , Tenascina/metabolismo
18.
Cell Tissue Res ; 370(3): 365-377, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28803422

RESUMO

Vessel disease is a kind of severe complication in diabetic patients. However, few pharmacologic agents can directly recover diabetic vascular function. Salidroside (SAL), a major ingredient from Rhodiola rosea, has been found to have an obvious hypoglycemic effect and a beneficial protection on vascular function in diabetes. However, whether SAL is a suitable treatment for diabetes has not so far been evaluated and the underlying mechanisms remain unknown. The present work aims to (1) investigate the potential effects of SAL on cerebrovascular relaxation in streptozotocin-induced diabetic rats or when exposed to acute hyperglycemia condition and (2) examine whether function of the BKCa channel is involved in SAL treatment for diabetic vascular relaxation. Our results indicate that chronic administration of 100 mg/kg/day SAL not only improves cerebrovascular relaxation but also increases BKCa ß1-subunit expressions at both protein and mRNA levels and enhances BKCa whole-cell and single-channel activities in cerebral VSMCs of diabetic rats. Correspondingly, acute application of 100 µM SAL induces cerebrovascular relaxation by activation of the BKCa channel. Furthermore, SAL activated the BKCa channel mainly through acting on the ß1-subunit in HEK293 cells transfected with hSloα+ß1 constructs. We concluded that SAL improved vasodilation in diabetic rats through restoring the function of the BKCa-ß1 subunit in cerebrovascular smooth muscle cells, which may be the underlying mechanism responsible for the vascular protection of SAL in diabetes.


Assuntos
Glucosídeos/farmacologia , Hipoglicemiantes/farmacologia , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Relaxamento Muscular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Fenóis/farmacologia , Vasodilatação/efeitos dos fármacos , Animais , Linhagem Celular , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/patologia , Células HEK293 , Humanos , Masculino , Miócitos de Músculo Liso/metabolismo , Ratos , Ratos Wistar , Estreptozocina
19.
Atherosclerosis ; 263: 227-236, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28672269

RESUMO

BACKGROUND AND AIMS: The impact of obesity on vasomotor regulation of coronary arteries and its underlying mechanisms are not completely understood and, in particular, the role of BKCa channels in the NO-mediated coronary vasodilation in obesity remains to be elucidated. METHODS: The effects of selective blockade of BKCa channel was tested on nitric oxide (NO)-mediated vasodilator responses of coronary arteries from lean and obese Zucker rats (LZR and OZR, respectively) by means of simultaneous measurements of intracellular Ca2+ concentration ([Ca2+]i) by Fura-2 fluorescence and tension in endothelium-denuded coronary arteries mounted in microvascular myographs. BKCa channel subunits expression was measured by Western blot. RESULTS: The selective BKCa channel blocker iberitoxin largely reduced the relaxations and decreases in [Ca2+]i induced by a NO donor in coronary arteries from OZR. Iberitoxin increased to a great extent both basal [Ca2+]i and tone in OZR. The agonist of the voltage-gated L-type calcium channels Bay K8644 induced an increase in [Ca2+]i and tone that was significantly smaller in arteries from OZR, which was restored to control levels in LZR after BKCa channel inhibition. Caffeine- and ryanodine-induced intracellular Ca2+ mobilization and BKCa channel ß1 subunit expression were increased in arteries from OZR. CONCLUSIONS: The present study suggests that an enhanced activity of VSM BKCa channels, associated with up-regulation of channel ß1 subunit and with a higher intracellular Ca2+ mobilization, contributes to the preserved NO-mediated vasodilatation and basal tone of coronary arteries in obesity.


Assuntos
Vasos Coronários/metabolismo , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Óxido Nítrico/metabolismo , Obesidade/metabolismo , Vasodilatação , Animais , Cálcio/metabolismo , Agonistas dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/efeitos dos fármacos , Canais de Cálcio Tipo L/metabolismo , Sinalização do Cálcio , Vasos Coronários/efeitos dos fármacos , Vasos Coronários/fisiopatologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/antagonistas & inibidores , Masculino , Doadores de Óxido Nítrico/metabolismo , Doadores de Óxido Nítrico/farmacologia , Obesidade/fisiopatologia , Bloqueadores dos Canais de Potássio/farmacologia , Ratos Zucker , Fatores de Tempo , Vasodilatação/efeitos dos fármacos
20.
J Mol Endocrinol ; 59(3): 219-233, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28676563

RESUMO

Melatonin, synthesized primarily by the pineal gland, is a neuroendocrine hormone with high membrane permeability. The vascular effects of melatonin, including vasoconstriction and vasodilation, have been demonstrated in numerous studies. However, the mechanisms underlying these effects are not fully understood. Large-conductance Ca2+-activated K+ (BKCa) channels are expressed broadly on smooth muscle cells and play an important role in vascular tone regulation. This study explored the mechanisms of myocyte BKCa channels and endothelial factors underlying the action of melatonin on the mesenteric arteries (MAs). Vascular contractility and patch-clamp studies were performed on myocytes of MAs from Wistar rats. Melatonin induced significant vasodilation on MAs. In the presence of Nω-nitro-l-arginine methyl ester (l-NAME), a potent endothelial oxide synthase (eNOS) inhibitor, melatonin elicited concentration-dependent relaxation, with lowered pIC50 The effect of melatonin was significantly attenuated in the presence of BKCa channel blocker iberiotoxin or MT1/MT2 receptor antagonist luzindole in both (+) l-NAME and (-) l-NAME groups. In the (+) l-NAME group, iberiotoxin caused a parallel rightward shift of the melatonin concentration-relaxation curve, with pIC50 lower than that of luzindole. Both inside-out and cell-attached patch-clamp recordings showed that melatonin significantly increased the open probability, mean open time and voltage sensitivity of BKCa channels. In a cell-attached patch-clamp configuration, the melatonin-induced enhancement of BKCa channel activity was significantly suppressed by luzindole. These findings indicate that in addition to the activation of eNOS, melatonin-induced vasorelaxation of MAs is partially attributable to its direct (passing through the cell membrane) and indirect (via MT1/MT2 receptors) activation of the BKCa channels on mesenteric arterial myocytes.


Assuntos
Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/agonistas , Melatonina/metabolismo , Vasodilatação , Animais , Expressão Gênica , Ativação do Canal Iônico/efeitos dos fármacos , 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 , Melatonina/farmacologia , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Ratos , Receptor MT1 de Melatonina/genética , Receptor MT1 de Melatonina/metabolismo , Receptor MT2 de Melatonina/genética , Receptor MT2 de Melatonina/metabolismo , Vasodilatação/efeitos dos fármacos , Vasodilatação/genética
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