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1.
Nature ; 580(7802): 288-293, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32269335

RESUMO

Inactivation is the process by which ion channels terminate ion flux through their pores while the opening stimulus is still present1. In neurons, inactivation of both sodium and potassium channels is crucial for the generation of action potentials and regulation of firing frequency1,2. A cytoplasmic domain of either the channel or an accessory subunit is thought to plug the open pore to inactivate the channel via a 'ball-and-chain' mechanism3-7. Here we use cryo-electron microscopy to identify the molecular gating mechanism in calcium-activated potassium channels by obtaining structures of the MthK channel from Methanobacterium thermoautotrophicum-a purely calcium-gated and inactivating channel-in a lipid environment. In the absence of Ca2+, we obtained a single structure in a closed state, which was shown by atomistic simulations to be highly flexible in lipid bilayers at ambient temperature, with large rocking motions of the gating ring and bending of pore-lining helices. In Ca2+-bound conditions, we obtained several structures, including multiple open-inactivated conformations, further indication of a highly dynamic protein. These different channel conformations are distinguished by rocking of the gating rings with respect to the transmembrane region, indicating symmetry breakage across the channel. Furthermore, in all conformations displaying open channel pores, the N terminus of one subunit of the channel tetramer sticks into the pore and plugs it, with free energy simulations showing that this is a strong interaction. Deletion of this N terminus leads to functionally non-inactivating channels and structures of open states without a pore plug, indicating that this previously unresolved N-terminal peptide is responsible for a ball-and-chain inactivation mechanism.


Assuntos
Microscopia Crioeletrônica , Ativação do Canal Iônico , Methanobacterium/química , Canais de Potássio Cálcio-Ativados/antagonistas & inibidores , Canais de Potássio Cálcio-Ativados/ultraestrutura , Cálcio/metabolismo , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Modelos Moleculares , Canais de Potássio Cálcio-Ativados/química , Canais de Potássio Cálcio-Ativados/metabolismo , Estrutura Secundária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Termodinâmica
2.
Eur J Pharmacol ; 866: 172828, 2020 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-31790651

RESUMO

The venoarteriolar reflex is a local mechanism that induces vasoconstriction during venous congestion in various tissues, including skin. This response is thought to play a critical role in minimizing capillary damage or edema resulting from overperfusion, though factors that modulate this response remain largely unknown. Here, we hypothesized that nitric oxide synthase (NOS), cyclooxygenase (COX), and Ca2+-activated, ATP-sensitive, and voltage-gated K+ channels (KCa, KATP, and KV channels, respectively) modulate the venoarteriolar reflex in human skin. Cutaneous blood flow (laser-Doppler flowmetry) was monitored during a 3-min pre-occlusion baseline and following a 3-min venous occlusion of 45 mmHg, the latter maneuver was used to induce the venoarteriolar reflex. The venoarteriolar reflex was assessed at the following forearm skin sites: Experiment 1 (n = 11): 1) lactated Ringer solution (Control), 2) 10 mM Nω-nitro-L-arginine (NOS inhibitor), 3) 10 mM ketorolac (COX inhibitor), and 4) combined NOS + COX inhibition; Experiment 2 (n = 15): 1) lactated Ringer solution (Control), 2) 50 mM tetraethylammonium (KCa channel blocker), 3) 5 mM glybenclamide (KATP channel blocker), and 4) 10 mM 4-aminopyridine (KV channel blocker). Separate and combined NOS and COX inhibition as well as KATP channel blocker had no effect on venoarteriolar reflex. Conversely, venoarteriolar reflex was attenuated by KCa channel blockade (36-38%) and augmented by KV channel blockade (38-55%). We showed that KCa and KV channels modulate the venoarteriolar reflex with minimum roles of NOS, COX, and KATP channels in human non-glabrous forearm skin in vivo. Thus, cutaneous venoarteriolar reflex changes could reflect altered K+ channel function.


Assuntos
Arteríolas/fisiologia , Canais de Potássio Cálcio-Ativados/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Pele/irrigação sanguínea , Pele/metabolismo , Veias/fisiologia , Adulto , Humanos , Canais KATP/metabolismo , Masculino , Óxido Nítrico Sintase/metabolismo , Prostaglandina-Endoperóxido Sintases/metabolismo , Fluxo Sanguíneo Regional
3.
Clin Interv Aging ; 14: 1579-1587, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31564840

RESUMO

Background: Aging leads to structural and functional changes in the vasculature characterized by arterial endothelial dysfunction and stiffening of large elastic arteries and is a predominant risk factor for cardiovascular disease, the leading cause of morbidity and mortality in modern societies. Although exercise reduces the risk of many age-related diseases, including cardiovascular disease, the mechanisms underlying the beneficial effects of exercise on age-related endothelial function fully elucidated. Purpose: The present study explored the effects of exercise on the impaired endothelium-derived hyperpolarizing factor (EDHF)-mediated vasodilation in aged arteries and on the involvement of the transient receptor potential vanilloid 4 (TRPV4) channel and the small-conductance calcium-activated potassium (KCa2.3) channel signaling in this process. Methods: Male Sprague-Dawley rats aged 19-21 months were randomly assigned to a sedentary group or to an exercise group. Two-month-old rats were used as young controls. Results: We found that TRPV4 and KCa2.3 isolated from primary cultured rat aortic endothelial cells pulled each other down in co-immunoprecipitation assays, indicating that the two channels could physically interact. Using ex vivo functional arterial tension assays, we found that EDHF-mediated relaxation induced by acetylcholine or by the TRPV4 activator GSK1016790A was markedly decreased in aged rats compared with that in young rats and was significantly inhibited by TRPV4 or KCa2.3 blockers in both young and aged rats. However, exercise restored both the age-related and the TRPV4-mediated and KCa2.3-mediated EDHF responses. Conclusion: These results suggest an important role for the TRPV4-KCa2.3 signaling undergirding the beneficial effect of exercise to ameliorate age-related arterial dysfunction.


Assuntos
Fatores Biológicos/metabolismo , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Canais de Potássio Cálcio-Ativados/metabolismo , Canais de Cátion TRPV/metabolismo , Animais , Doenças Cardiovasculares/fisiopatologia , Endotélio Vascular/fisiopatologia , Masculino , Ratos , Ratos Sprague-Dawley , Vasodilatação/fisiologia
4.
Mol Cell Neurosci ; 101: 103410, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31644953

RESUMO

Whereas ß2-adrenoceptor (ß2-AR) has been reported to reduce GABAergic activity in the prefrontal cortex (PFC), the underlying cellular and molecular mechanisms have not been completely determined. Here, we showed that ß2-AR agonist Clenbuterol (Clen) decreased GABAergic transmission onto PFC layer V/VI pyramidal neurons via a presynaptic mechanism without altering postsynaptic GABA receptors. Clen decreased the action potential firing rate but increased the burst afterhyperpolarization (AHP) amplitude in PFC interneurons. Application of L-type Ca2+ channel or charybdotoxin-sensitive Ca2+-activated K+ channel inhibitors blocked Clen-induced decreases in action potential firing rate, spontaneous inhibitory postsynaptic current (sIPSC) frequency and Clen-induced enhancement of AHP amplitude, suggesting that the effects of Clen involves L-type Ca2+ Channels and charybdotoxin-sensitive Ca2+-activated K+ channels. Our results provide a potential cellular mechanism by which Clen controls GABAergic neuronal activity in PFC.


Assuntos
Canais de Cálcio Tipo L/metabolismo , Canais de Potássio Cálcio-Ativados/metabolismo , Córtex Pré-Frontal/metabolismo , Ácido gama-Aminobutírico/metabolismo , Potenciais de Ação , Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Animais , Charibdotoxina/farmacologia , Clembuterol/farmacologia , Potenciais Pós-Sinápticos Inibidores , Interneurônios/efeitos dos fármacos , Interneurônios/metabolismo , Interneurônios/fisiologia , Córtex Pré-Frontal/citologia , Córtex Pré-Frontal/fisiologia , Ratos , Ratos Sprague-Dawley , Receptores Adrenérgicos beta 2/metabolismo
5.
J Pharmacol Sci ; 141(1): 25-31, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31533896

RESUMO

This study was devoted to elucidating the interferon (IFN)-γ-induced signaling pathway and the interaction between protein kinase G (PKG) and protein kinase A (PKA) through large-conductance Ca(2+)-activated K(+) channels in human cardiac fibroblasts. The IK currents were recorded using a whole-cell patch clamp method. A large depolarization (+50 mV) and a high Ca2+ concentration (pCa 6.0) were used in the internal pipette solution to activate only the KCa channels. Iberiotoxin (Ibtx), which selectively inhibits BKCa channels at a concentration of 100 nmol/l, caused a significant reduction of basal IK. Adding IFN-γ in the presence of Ibtx had no effect on IK. Application of the IFN-γ caused a significant reduction in total K+ current amplitude, recorded with a 500 ms depolarizing pulse duration, to +50 mV from a holding potential of -80 mV. We tested the involvement of the sGC/cGMP/PKG signaling pathway by using specific PKG inhibitor KT 5823, potent sGC inhibitor NS 2028, and specific sGC agonist BAY 41-8543. The obtained data confirmed that only sGC participated in the IFN-γ-mediated BKCa channel modulation, which was mediated further by PKA. This study represents first evidence about the participation of the IFN-γ in the mechanisms responsible for BKCa modulation in HCFs. We also believe that this process occurs via negative crosstalk between the PKG- and PKA-associated pathways.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Fibroblastos/metabolismo , Interferon gama/farmacologia , Miocárdio/citologia , Canais de Potássio Cálcio-Ativados/metabolismo , Proteína Quinase C/fisiologia , Transdução de Sinais/fisiologia , Células Cultivadas , Humanos
6.
Nihon Yakurigaku Zasshi ; 154(3): 108-113, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-31527359

RESUMO

Similar to calcium (Ca2+) and chloride (Cl-) ion channels/transporters, potassium (K+) channels have been recognized as a crucial cancer treatment target. Recent studies have provided convincing evidences of positive correlation between elevated expression levels of Ca2+-activated K+ (KCa) channels and cancer proliferation, metastasis, and poor patient prognosis. In cancer cells, KCa1.1 and KCa3.1 KCa channels are co-localized with Ca2+-permeable Orai/TRP channels to provide a positive-feedback loop for Ca2+ entry. They are responsible for the promotion of cell growth and metastasis in the different types of cancer, and are therefore potential therapeutic targets and biomarkers for cancer. We determined the epigenetic and post-transcriptional dysregulation of KCa3.1 by class I histone deacetylase inhibitors in breast and prostate cancer cells. We further determined the transcriptional repression and protein degradation of KCa1.1 by vitamin D receptor agonists and androgen receptor antagonists, which are expected as potential therapeutic drugs for triple-negative breast cancer. The anti-inflammatory cytokine, interleukin-10 (IL-10) is an immunosuppressive factor involved in tumorigenesis, and plays a crucial role in escape from tumor immune surveillance. We determined KCa3.1 activators are a possible therapeutic option to suppress the tumor-promoting activities of IL-10. These results may provide new insights into cancer treatment focused on Ca2+-activated K+ channels.


Assuntos
Neoplasias da Mama/patologia , Inibidores de Histona Desacetilases/farmacologia , Canais de Potássio Cálcio-Ativados/metabolismo , Neoplasias da Próstata/patologia , Antagonistas de Receptores de Andrógenos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , Epigênese Genética , Feminino , Humanos , Vigilância Imunológica , Interleucina-10/metabolismo , Masculino , Proteólise , Processamento Pós-Transcricional do RNA , Receptores de Calcitriol/agonistas
7.
Nat Struct Mol Biol ; 26(9): 816-822, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31488908

RESUMO

Dynamic protein molecules are defined by their spatiotemporal characteristics and should thus be represented by models incoporating both characteritics. Structural biology enables determination of atomic structures of individual conformational states of a given protein. Obtaining the complementary temporal information of a given time resolution, which can be directly linked to the corresponding atomic structures, requires identifying at each time point the specific conformational state adopted by the protein. Here, we examine individual regulator of conductance to K+ (RCK) domains in the regulatory module of the MthK channel by monitoring in real time the orientation of an α-helix that is conformational-state-specific. The acquired dynamic information that specifies an RCK domain's multi-state conformational changes, combined with already available corresponding atomic structures, enables us to establish an experiment-based spatiotemporal representation of an RCK domain, and to deduce a quantitative mechanistic model of the channel.


Assuntos
Canais de Potássio Cálcio-Ativados/química , Canais de Potássio Cálcio-Ativados/metabolismo , Potássio/metabolismo , Conformação Proteica , Análise Espaço-Temporal
8.
Nat Struct Mol Biol ; 26(9): 802-807, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31488909

RESUMO

Conformational changes within typical protein molecules are rapid and small, making their quantitative resolution challenging. These changes generally involve rotational motions and may thus be resolved by determining changes in the orientation of a fluorescent label that assumes a unique orientation in each conformation. Here, by analyzing fluorescence intensities collected using a polarization microscope at a rate of 50 frames per second, we follow the changes of 10-16° in the orientation of a single bifunctional rhodamine molecule attached to a regulator of conductance to K+ (RCK) domain of the MthK channel, and thus, the transitions between its three conformational states, with effective standard deviation (σ) of 2-5°. Based on available crystal structures, the position of the fluorophore's center differs by 3.4-8.1 Å among the states. Thus, the present approach allows the resolution of protein conformational changes involving ångström-scale displacements.


Assuntos
Polarização de Fluorescência , Methanobacterium/enzimologia , Canais de Potássio Cálcio-Ativados/química , Canais de Potássio Cálcio-Ativados/metabolismo , Conformação Proteica , Microscopia de Polarização
9.
Nat Struct Mol Biol ; 26(9): 808-815, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31488910

RESUMO

Allosteric proteins transition among different conformational states in a ligand-dependent manner. Upon resolution of a protein's individual states, one can determine the probabilities of these states, thereby dissecting the energetic mechanisms underlying their conformational changes. Here we examine individual regulator of conductance to K+ (RCK) domains that form the regulatory module of the Ca2+-activated MthK channel. Each domain adopts multiple conformational states differing on an ångström scale. The probabilities of these different states of the domain, assessed in different Ca2+ concentrations, allowed us to fully determine a six-state model that is minimally required to account for the energetic characteristics of the Ca2+-dependent conformational changes of an RCK domain. From the energetics of this domain, we deduced, in the framework of statistical mechanics, an analytic model that quantitatively predicts the experimentally observed Ca2+ dependence of the channel's open probability.


Assuntos
Cálcio/metabolismo , Methanobacterium/enzimologia , Canais de Potássio Cálcio-Ativados/química , Canais de Potássio Cálcio-Ativados/metabolismo , Conformação Proteica , Domínios Proteicos
10.
Int J Mol Sci ; 20(15)2019 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-31370156

RESUMO

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 fármacos
11.
Am J Physiol Heart Circ Physiol ; 317(2): H357-H363, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31199187

RESUMO

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 Sinais
12.
Am J Physiol Regul Integr Comp Physiol ; 317(1): R113-R120, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31091157

RESUMO

Our objective in this study was to examine the separate and combined effects of potassium (K+) channels and nitric oxide synthase (NOS) on cutaneous vasodilation and sweating in older men during rest and exercise in the heat. In 13 habitually active men (61 ± 4 yr), cutaneous vascular conductance and local sweat rate were assessed at six dorsal forearm skin sites continuously perfused with either 1) lactated Ringer (control), 2) 10 mM NG-nitro-l-arginine methyl ester (l-NAME, NOS inhibitor), 3) 50 mM tetraethylammonium (TEA; Ca2+-activated K+ channel blocker), 4) 5 mM glybenclamide (GLY; ATP-sensitive K+ channel blocker), 5) 50 mM TEA + 10 mM l-NAME, and 6) 5 mM GLY + 10 mM l-NAME via microdialysis. Participants rested in non-heat stress (25°C) and heat stress (35°C) conditions for ∼60 min each, followed by 50 min of moderate-intensity cycling (∼55% V̇o2peak) and 30 min of recovery in the heat. During rest and exercise in the heat, l-NAME, TEA + l-NAME, and GLY + l-NAME attenuated CVC relative to control (all P ≤ 0.05), although l-NAME was not different from TEA + l-NAME or GLY + l-NAME (all P > 0.05). TEA attenuated CVC during rest, whereas GLY attenuated CVC during exercise (both P ≤ 0.05). Additionally, whereas neither l-NAME nor TEA altered sweating throughout the protocol (all P > 0.05), combined TEA + l-NAME attenuated sweating during exercise in the heat (P ≤ 0.05). We conclude that in habitually active older men blockade of KCa and KATP channels attenuates cutaneous vasodilation during rest and exercise in the heat, respectively, and these effects are NOS dependent. Furthermore, combined NOS inhibition and KCa channel blockade attenuates sweating during exercise in the heat.


Assuntos
Resposta ao Choque Térmico/fisiologia , Canais KATP/antagonistas & inibidores , Óxido Nítrico Sintase/antagonistas & inibidores , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio Cálcio-Ativados/antagonistas & inibidores , Vasodilatação/efeitos dos fármacos , Idoso , Inibidores Enzimáticos/farmacologia , Glibureto/administração & dosagem , Glibureto/farmacologia , Humanos , Hipoglicemiantes/administração & dosagem , Hipoglicemiantes/farmacologia , Canais KATP/metabolismo , Masculino , Pessoa de Meia-Idade , NG-Nitroarginina Metil Éster/administração & dosagem , NG-Nitroarginina Metil Éster/farmacologia , Canais de Potássio Cálcio-Ativados/metabolismo , Sudorese/efeitos dos fármacos , Tetraetilamônio/administração & dosagem , Tetraetilamônio/farmacologia
13.
J Pharmacol Sci ; 140(1): 48-53, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31088764

RESUMO

Nobiletin is a one of the polymethoxyflavones contained in the peel of citrus fruits, such as Citrus depressa. In this study, the effect of nobiletin-induced relaxation on phenylephrine (PE)-induced contraction of endothelium-denuded rat aorta was investigated. Nobiletin inhibited PE- or KCl-induced contractions in a concentration-dependent manner in endothelium-intact and -denuded aortas. However, this relaxation was stronger in PE-induced contractions than in KCl-induced contractions; moreover, the nobiletin-induced relaxation was significantly increased on PE-induced contraction in endothelium-intact aorta. ODQ significantly inhibited the nobiletin-induced relaxation in endothelium-denuded aorta; however, SQ22536 did not affect the relaxation. In addition, IBMX synergistically enhanced the nobiletin-induced relaxation. Nobiletin increased cGMP levels in aorta. Also, IBMX significantly increased cGMP content in aorta, and ODQ significantly reduced cGMP levels. Nobiletin-induced relaxation was significantly inhibited by the Ca2+-activated K+ (BK) channel inhibitor iberiotoxin (IbTX) and the ATP-sensitive K+ (KATP) channel inhibitor glybenclamide. Sodium nitroprusside-induced relaxation was suppressed by IbTX, but not by glybenclamide. These results suggest that nobiletin inhibits PE-induced contractions of endothelium-denuded rat aorta by increasing cGMP levels via GC activation. Moreover, the present findings indicate the possibility that nobiletin opened BK channels by a cGMP-related signal, but KATP channels were opened by a cGMP-nonrelated signal in rat aorta.


Assuntos
Aorta/efeitos dos fármacos , Flavonas/farmacologia , Vasodilatação/efeitos dos fármacos , 1-Metil-3-Isobutilxantina/farmacologia , Animais , Aorta/metabolismo , Citrus/química , GMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Endotélio Vascular , Flavonas/isolamento & purificação , Técnicas In Vitro , Masculino , Fenilefrina/antagonistas & inibidores , Canais de Potássio Cálcio-Ativados/metabolismo , Ratos Wistar , Vasoconstrição/efeitos dos fármacos
14.
J Med Food ; 22(4): 393-407, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30990753

RESUMO

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 Wistar
15.
Dokl Biol Sci ; 484(1): 10-12, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31016496

RESUMO

Endothelium-dependent relaxation mechanisms have been studied in phenylephrine-precontracted capsules of bovine mesenteric lymph nodes studied in vitro. Tetraethylammonium chloride and TRAM-34 in a solution with L-NAME and Indomethacin, which suppress the production NO and prostacyclin of endothelium, increased the tone of the lymph nodes. We believe that in bovine mesenteric lymph nodes, the dilation mechanism is mediated by hyperpolarization of the endothelium, which is associated with activation of large- and intermedium conductance Ca2+-activated potassium channels.


Assuntos
Endotélio/fisiologia , Linfonodos/fisiologia , Potenciais da Membrana , Animais , Bovinos , Endotélio/efeitos dos fármacos , Endotélio/metabolismo , Epoprostenol/metabolismo , Indometacina/farmacologia , Linfonodos/efeitos dos fármacos , Linfonodos/metabolismo , Masculino , Mesentério , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/metabolismo , Canais de Potássio Cálcio-Ativados/metabolismo , Pirazóis/farmacologia
16.
Int J Mol Sci ; 20(6)2019 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-30893836

RESUMO

Effective delivery of oxygen and essential nutrients to vital organs and tissues throughout the body requires adequate blood flow supplied through resistance vessels. The intimate relationship between intracellular calcium ([Ca2+]i) and regulation of membrane potential (Vm) is indispensable for maintaining blood flow regulation. In particular, Ca2+-activated K⁺ (KCa) channels were ascertained as transducers of elevated [Ca2+]i signals into hyperpolarization of Vm as a pathway for decreasing vascular resistance, thereby enhancing blood flow. Recent evidence also supports the reverse role for KCa channels, in which they facilitate Ca2+ influx into the cell interior through open non-selective cation (e.g., transient receptor potential; TRP) channels in accord with robust electrical (hyperpolarization) and concentration (~20,000-fold) transmembrane gradients for Ca2+. Such an arrangement supports a feed-forward activation of Vm hyperpolarization while potentially boosting production of nitric oxide. Furthermore, in vascular types expressing TRP channels but deficient in functional KCa channels (e.g., collecting lymphatic endothelium), there are profound alterations such as downstream depolarizing ionic fluxes and the absence of dynamic hyperpolarizing events. Altogether, this review is a refined set of evidence-based perspectives focused on the role of the endothelial KCa and TRP channels throughout multiple experimental animal models and vascular types. We discuss the diverse interactions among KCa and TRP channels to integrate Ca2+, oxidative, and electrical signaling in the context of cardiovascular physiology and pathology. Building from a foundation of cellular biophysical data throughout a wide and diverse compilation of significant discoveries, a translational narrative is provided for readers toward the treatment and prevention of chronic, age-related cardiovascular disease.


Assuntos
Envelhecimento/metabolismo , Cálcio/metabolismo , Fenômenos Fisiológicos Cardiovasculares , Doença Crônica , Canais de Potássio Cálcio-Ativados/metabolismo , Canais de Cátion TRPV/metabolismo , Animais , Humanos
17.
Mol Pharmacol ; 95(4): 376-385, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30739093

RESUMO

The neurohypophyseal hormone oxytocin (OT) regulates biologic functions in both peripheral tissues and the central nervous system. In the central nervous system, OT influences social processes, including peer relationships, maternal-infant bonding, and affiliative social relationships. In mammals, the nonapeptide OT structure is highly conserved with leucine in the eighth position (Leu8-OT). In marmosets (Callithrix), a nonsynonymous nucleotide substitution in the OXT gene codes for proline in the eighth residue position (Pro8-OT). OT binds to its cognate G protein-coupled receptor (OTR) and exerts diverse effects, including stimulation (Gs) or inhibition (Gi/o) of adenylyl cyclase, stimulation of potassium channel currents (Gi), and activation of phospholipase C (Gq). Chinese hamster ovary cells expressing marmoset or human oxytocin receptors (mOTRs or hOTRs, respectively) were used to characterize OT signaling. At the mOTR, Pro8-OT was more efficacious than Leu8-OT in measures of Gq activation, with both peptides displaying subnanomolar potencies. At the hOTR, neither the potency nor efficacy of Pro8-OT and Leu8-OT differed with respect to Gq signaling. In both mOTR- and hOTR-expressing cells, Leu8-OT was more potent and modestly more efficacious than Pro8-OT in inducing hyperpolarization. In mOTR cells, Leu8-OT-induced hyperpolarization was modestly inhibited by pretreatment with pertussis toxin (PTX), consistent with a minor role for Gi/o activation; however, the Pro8-OT response in mOTR and hOTR cells was PTX insensitive. These findings are consistent with membrane hyperpolarization being largely mediated by a Gq signaling mechanism leading to Ca2+-dependent activation of K+ channels. Evaluation of the influence of apamin, charybdotoxin, paxilline, and TRAM-34 demonstrated involvement of both intermediate and large conductance Ca2+-activated K+ channels.


Assuntos
Cálcio/metabolismo , Leucina/metabolismo , Ocitocina/metabolismo , Canais de Potássio Cálcio-Ativados/metabolismo , Potássio/metabolismo , Prolina/metabolismo , Receptores de Ocitocina/metabolismo , Animais , Células CHO , Cricetulus , Humanos , Potenciais da Membrana/fisiologia , Transdução de Sinais/fisiologia , Fosfolipases Tipo C/metabolismo
18.
Chem Biol Interact ; 300: 35-39, 2019 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-30629953

RESUMO

Azoxystrobin, a broad-spectrum fungicide, has been increasingly used in the agricultural industry. In Japan in 2018, azoxystrobin at five times the normal limit was detected in a shipment of Australian barley that had been used in food products. Therefore, the effects of azoxystrobin need to be carefully examined to predict potential adverse reactions in humans. In this study, the effects of azoxystrobin on the membrane potential and intracellular Ca2+ levels of thymocytes have been photochemically examined using flow cytometry. Azoxystrobin hyperpolarized plasma membrane potential. This hyperpolarization appeared to be due to the activation of Ca2+-dependent K+ channels, as both the removal of extracellular Ca2+ and addition of charybdotoxin attenuated the observed hyperpolarization. In the presence of quinine, an anti-malarial drug that blocks Ca2+-dependent K+ channels, azoxystrobin depolarized the membranes instead. Azoxystrobin increased intracellular Ca2+ levels in a concentration-dependent manner through the influx of extracellular Ca2+ and intracellular release of Ca2+, as confirmed by reduction in azoxystrobin-induced response in the absence of extracellular Ca2+. It appears likely that azoxystrobin at micromolar concentrations modifies membrane ion permeability in thymocytes. Since changes in membrane potential and intracellular Ca2+ levels occur during typical physiological lymphocyte responses, azoxystrobin may disturb lymphocyte function.


Assuntos
Fungicidas Industriais/farmacologia , Pirimidinas/farmacologia , Estrobilurinas/farmacologia , Animais , Cálcio/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Masculino , Potenciais da Membrana/efeitos dos fármacos , Canais de Potássio Cálcio-Ativados/antagonistas & inibidores , Canais de Potássio Cálcio-Ativados/metabolismo , Quinina/farmacologia , Ratos , Ratos Wistar , Timócitos/citologia , Timócitos/efeitos dos fármacos , Timócitos/metabolismo
19.
Am J Physiol Endocrinol Metab ; 316(4): E646-E659, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30694690

RESUMO

Pancreatic α-cells exhibit oscillations in cytosolic Ca2+ (Ca2+c), which control pulsatile glucagon (GCG) secretion. However, the mechanisms that modulate α-cell Ca2+c oscillations have not been elucidated. As ß-cell Ca2+c oscillations are regulated in part by Ca2+-activated K+ (Kslow) currents, this work investigated the role of Kslow in α-cell Ca2+ handling and GCG secretion. α-Cells displayed Kslow currents that were dependent on Ca2+ influx through L- and P/Q-type voltage-dependent Ca2+ channels (VDCCs) as well as Ca2+ released from endoplasmic reticulum stores. α-Cell Kslow was decreased by small-conductance Ca2+-activated K+ (SK) channel inhibitors apamin and UCL 1684, large-conductance Ca2+-activated K+ (BK) channel inhibitor iberiotoxin (IbTx), and intermediate-conductance Ca2+-activated K+ (IK) channel inhibitor TRAM 34. Moreover, partial inhibition of α-cell Kslow with apamin depolarized membrane potential ( Vm) (3.8 ± 0.7 mV) and reduced action potential (AP) amplitude (10.4 ± 1.9 mV). Although apamin transiently increased Ca2+ influx into α-cells at low glucose (42.9 ± 10.6%), sustained SK (38.5 ± 10.4%) or BK channel inhibition (31.0 ± 11.7%) decreased α-cell Ca2+ influx. Total α-cell Ca2+c was similarly reduced (28.3 ± 11.1%) following prolonged treatment with high glucose, but it was not decreased further by SK or BK channel inhibition. Consistent with reduced α-cell Ca2+c following prolonged Kslow inhibition, apamin decreased GCG secretion from mouse (20.4 ± 4.2%) and human (27.7 ± 13.1%) islets at low glucose. These data demonstrate that Kslow activation provides a hyperpolarizing influence on α-cell Vm that sustains Ca2+ entry during hypoglycemic conditions, presumably by preventing voltage-dependent inactivation of P/Q-type VDCCs. Thus, when α-cell Ca2+c is elevated during secretagogue stimulation, Kslow activation helps to preserve GCG secretion.


Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Células Secretoras de Glucagon/metabolismo , Glucagon/metabolismo , Glucose/metabolismo , Canais de Potássio Cálcio-Ativados/metabolismo , Alcanos/farmacologia , Animais , Apamina/farmacologia , Canais de Cálcio Tipo L/metabolismo , Canais de Cálcio Tipo P/metabolismo , Canais de Cálcio Tipo Q/metabolismo , Retículo Endoplasmático/metabolismo , Camundongos , Camundongos Transgênicos , Técnicas de Patch-Clamp , Peptídeos/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio Cálcio-Ativados/antagonistas & inibidores , Pirazóis/farmacologia , Compostos de Quinolínio/farmacologia
20.
Neuropharmacology ; 144: 133-142, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30366003

RESUMO

Sudden and complete unilateral loss of peripheral vestibular inputs evokes characteristic vestibular syndrome comprised of posturo-locomotor, oculomotor, vegetative and cognitive symptoms. Subsequently to the vestibular insult, a neurophysiological process called central vestibular compensation promotes the progressive restoration of the posture and balance. The modulation of the excitability of vestibular secondary neurons has been demonstrated to be a key process of this mechanism. However, the molecular mechanisms that support this modulatory process have thus far not been fully identified. The present study used a combination of a radio-labeled apamin binding experiment and a functional assessment of the vestibular function to demonstrate that unilateral vestibular neurectomy (UVN) induces both ipsi- and contralateral up-regulation of the apamin-sensitive calcium-activated small conductance K+ (SK) channels, within the first days following the insult. We also demonstrate that apamin administration during the acute phase of the vestibular syndrome significantly reduces both the posturo-locomotor and vestibulo-ocular deficits induced by the UVN. This is illustrated by the reduction of both the spontaneous nystagmus and the static and dynamic balance unsteadiness. These data suggest that the regulation of SK channel expression may be part of the vestibular compensation process. It is also indicated that the pharmacological modulation of SK channels may be a potential way to alleviate the vestibular syndrome.


Assuntos
Apamina/farmacologia , Atividade Motora/efeitos dos fármacos , Neurotransmissores/farmacologia , Nistagmo Patológico/tratamento farmacológico , Equilíbrio Postural/efeitos dos fármacos , Doenças Vestibulares/tratamento farmacológico , Animais , Gatos , Modelos Animais de Doenças , Movimentos Oculares/efeitos dos fármacos , Movimentos Oculares/fisiologia , Lateralidade Funcional , Masculino , Atividade Motora/fisiologia , Nistagmo Patológico/metabolismo , Equilíbrio Postural/fisiologia , Postura/fisiologia , Canais de Potássio Cálcio-Ativados/metabolismo , Recuperação de Função Fisiológica/efeitos dos fármacos , Recuperação de Função Fisiológica/fisiologia , Doenças Vestibulares/metabolismo , Nervo Vestibular/lesões
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