RESUMO
CONTEXT: The Tongmai Yangxin pill (TMYX) has potential clinical effects on no-reflow (NR); however, the effective substances and mechanisms remain unclear. OBJECTIVE: This study evaluates the cardioprotective effects and molecular mechanisms of TMYX against NR. MATERIALS AND METHODS: We used a myocardial NR rat model to confirm the effect and mechanism of action of TMYX in alleviating NR. Sprague-Dawley (SD) rats were divided into Control (Con), sham, NR, TMYX (4.0 g/kg), and sodium nitroprusside (SNP, 5.0 mg/kg), and received their treatments once a day for one week. In vitro studies in isolated coronary microvasculature of NR rats and in silico network pharmacology analyses were performed to reveal the underlying mechanisms of TMYX and determine the main components, targets, and pathways of TMYX, respectively. RESULTS: TMYX (4.0 g/kg) showed therapeutic effects on NR by improving the cardiac structure and function, reducing NR, ischemic areas, and cardiomyocyte injury, and decreasing the expression of cardiac troponin I (cTnI). Moreover, the mechanism of TMYX predicted by network pharmacology is related to the HIF-1, NF-κB, and TNF signaling pathways. In vivo, TMYX decreased the expression of MPO, NF-κB, and TNF-α and increased the expression of GPER, p-ERK, and HIF-1α. In vitro, TMYX enhanced the diastolic function of coronary microvascular cells; however, this effect was inhibited by G-15, H-89, L-NAME, ODQ and four K+ channel inhibitors. CONCLUSIONS: TMYX exerts its pharmacological effects in the treatment of NR via multiple targets. However, the contribution of each pathway was not detected, and the mechanisms should be further investigated.
Assuntos
NF-kappa B , Canais de Potássio , Animais , Ratos , Isquemia , Miócitos Cardíacos , NF-kappa B/metabolismo , Canais de Potássio/metabolismo , Ratos Sprague-Dawley , Medicamentos de Ervas Chinesas/farmacologiaRESUMO
Pks13 was identified as a key enzyme involved in the final step of mycolic acid biosynthesis. We previously identified antitubercular coumestans that targeted Pks13-TE, and these compounds exhibited high potency both in vitro and in vivo. However, lead compound 8 presented potential safety concerns because it inhibits the hERG potassium channel in electrophysiology patch-clamp assays (IC50 = 0.52 µM). By comparing the Pks13-TE-compound 8 complex and the ligand-binding pocket of the hERG ion channel, fluoro-substituted and oxazine-containing coumestans were designed and synthesized. Fluoro-substituted compound 23 and oxazine-containing coumestan 32 showed excellent antitubercular activity against both drug-susceptible and drug-resistant Mtb strains (MIC = 0.0039-0.0078 µg/mL) and exhibited limited hERG inhibition (IC50 ≥ 25 µM). Moreover, 32 exhibited improved metabolic stability relative to parent compound 8 while showing favorable bioavailability in mouse models via serum inhibition titration assays.
Assuntos
Infecções por Mycobacterium , Mycobacterium tuberculosis , Animais , Antituberculosos/química , Cumarínicos , Ligantes , Camundongos , Testes de Sensibilidade Microbiana , Ácidos Micólicos/metabolismo , Oxazinas/metabolismo , Policetídeo Sintases , Canais de Potássio/metabolismoRESUMO
It has been described that using noninvasive exposure to 40-Hz white light LED reduces amyloid-beta, a peptide thought to initiate neurotoxic events in Alzheimer's disease (AD). However, the mechanisms remain to be identified. Since AD impairs mitochondrial potassium channels and respiratory chain activity, the objectives of the current study were to determine the effect of 40-Hz white light LED on structure-function of mitoKATP channel and brain mitochondrial respiratory chain activity, production of reactive oxygen species (ROS), and ΔΨm in AD. Single mitoKATP channel was considered using a channel incorporated into the bilayer lipid membrane and expression of mitoKATP-Kir6.1 subunit as a pore-forming subunit of the channel was determined using a western blot analysis in Aß1-42 toxicity and light-treated rats. Our results indicated a severe decrease in mito-KATP channel permeation and Kir6.1 subunit expression coming from the Aß1-42-induced neurotoxicity. Furthermore, we found that Aß1-42-induced neurotoxicity decreased activities of complexes I and IV and increased ROS production and ΔΨm. Surprisingly, light therapy increased channel permeation and mitoKATP-Kir6.1 subunit expression. Noninvasive 40-Hz white light LED treatment also increased activities of complexes I and IV and decreased ROS production and ΔΨm up to ~ 70%. Here, we report that brain mito-KATP channel and respiratory chain are, at least in part, novel targets of 40-Hz white light LED therapy in AD.
Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides , Trifosfato de Adenosina/metabolismo , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/toxicidade , Animais , Encéfalo/metabolismo , Transporte de Elétrons , Canais KATP/metabolismo , Canais de Potássio/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismoRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Previous studies have shown that the active fraction of Rhodiola tangutica (Maxim.) S.H. Fu (ACRT) dilates pulmonary arteries and thwarts pulmonary artery remodelling. The dilatation effect of ACRT on pulmonary artery vascular rings could be reduced by potassium (K+) channel blockers. However the exact mechanisms of ACRT on ion channels are still unclear. AIM OF THE STUDY: This study aimed to investigate whether the effect of ACRT on K+ channels inhibits cell proliferation after pulmonary artery smooth muscle cells (PASMCs) are exposed to hypoxia. MATERIALS AND METHODS: The whole-cell patch-clamp method was used to clarify the effect of ACRT on the K+ current (IK) of rat PASMCs exposed to hypoxia. The mRNA and protein expression levels were detected using real-time quantitative polymerase chain reaction (RT-qPCR) and western blotting, respectively. The intracellular calcium (Ca2+) concentration ([Ca2+]i) values in rat PASMCs were detected by laser scanning confocal microscopy. The cell cycle and cell proliferation were assessed using flow cytometry analysis and CCK-8 and EdU assays. RESULTS: ACRT pretreatment alleviated the inhibition of IK induced by hypoxia in rat PASMCs. Compared with hypoxia, ACRT upregulated voltage-dependent K+ channel (Kv) 1.5 and big-conductance calcium-activated K+ channel (BKCa) mRNA and protein expression and downregulated voltage-dependent Ca2+ channel (Cav) 1.2 mRNA and protein expression. ACRT decreased [Ca2+]i, inhibited the promotion of cyclin D1 and proliferating cell nuclear antigen (PCNA) expression, and prevented the proliferation of rat PASMCs exposed to hypoxia. CONCLUSION: In conclusion, the present study demonstrated that ACRT plays a key role in restoring ion channel function and then inhibiting the proliferation of PASMCs under hypoxia, ACRT has preventive and therapeutic potential in hypoxic pulmonary hypertension.
Assuntos
Músculo Liso Vascular/efeitos dos fármacos , Extratos Vegetais/farmacologia , Artéria Pulmonar/efeitos dos fármacos , Rhodiola/química , Animais , Cálcio/metabolismo , Hipóxia Celular , Proliferação de Células/efeitos dos fármacos , Hipertensão Pulmonar/tratamento farmacológico , Masculino , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Técnicas de Patch-Clamp , Canais de Potássio/efeitos dos fármacos , Canais de Potássio/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Artéria Pulmonar/citologia , Ratos , Ratos Sprague-DawleyRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Leonurus sibiricus L. (Lamiaceae) is a medicinal plant known in Brazil as "rubim" or "erva de macaé". It is used for various purposes, including stomach disorders. AIM OF THE STUDY: To evaluate the effect of the ethanol extract of the aerial parts of L. sibiricus (EELs) in models of gastric damage in mice. MATERIAL AND METHODS: The effect of EELs (50, 100 and 300 mg/kg, p.o., 1 h before induction) was tested on acidified ethanol (ACEt)-induced gastric ulcers. Additionally, we tested the effect of EELs (by intraduodenal administration) in the pylorus ligation (PL) model. RESULTS: Pretreatment with EELs, at 300 mg/kg, but not 50 and 100 mg/kg, reduced the relative area of gastric ulcers induced by ACEt (p < 0.01) and lipoperoxidation (p < 0.001), and increased the sulfhydryl content (p < 0.01) in the stomach in comparison with the vehicle group. Pretreatment with N-ethylmaleimide (a blocker of non-protein sulfhydryl groups, 10 mg/kg, i.p.) or glibenclamide (a KATP channel blocker, 10 mg/kg, i.p.) inhibited the gastroprotective response caused by EELs (300 mg/kg; p < 0.001), but there were no alterations due to pretreatments with inhibitors of the synthesis of prostaglandins (indomethacin, 10 mg/kg), nitric oxide (L-NAME, 70 mg/kg) or hydrogen sulfide (DL-propargylglycine, 10 mg/kg). Treatment with EELs (300 mg/kg) reduced mucus production (p < 0.001) and the volume of gastric secretion (p < 0.001) after PL without affecting gastric acidity or pH. CONCLUSIONS: These results provide evidence that EELs exerts gastroprotective action in mice, with the participation of oxidative stress and mediation of NP-SH, KATP channels and mucus production.
Assuntos
Leonurus/química , Fitoterapia , Extratos Vegetais/farmacologia , Úlcera Gástrica/prevenção & controle , Animais , Inibidores Enzimáticos/farmacologia , Etanol/toxicidade , Etilmaleimida/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Glibureto/farmacologia , Hipoglicemiantes/farmacologia , Masculino , Camundongos , Óxido Nítrico/metabolismo , Extratos Vegetais/química , Canais de Potássio/genética , Canais de Potássio/metabolismo , Prostaglandinas/genética , Prostaglandinas/metabolismo , Distribuição Aleatória , Compostos de Sulfidrila/metabolismoRESUMO
Little is known about the effect of lead on the activity of the vacuolar K+ channels. Here, the patch-clamp technique was used to compare the impact of lead (PbCl2) on the slow-activating (SV) and fast-activating (FV) vacuolar channels. It was revealed that, under symmetrical 100-mM K+, the macroscopic currents of the SV channels exhibited a typical slow activation and a strong outward rectification of the steady-state currents, while the macroscopic currents of the FV channels displayed instantaneous currents, which, at the positive potentials, were about three-fold greater compared to the one at the negative potentials. When PbCl2 was added to the bath solution at a final concentration of 100 µM, it decreased the macroscopic outward currents of both channels but did not change the inward currents. The single-channel recordings demonstrated that cytosolic lead causes this macroscopic effect by a decrease of the single-channel conductance and decreases the channel open probability. We propose that cytosolic lead reduces the current flowing through the SV and FV channels, which causes a decrease of the K+ fluxes from the cytosol to the vacuole. This finding may, at least in part, explain the mechanism by which cytosolic Pb2+ reduces the growth of plant cells.
Assuntos
Beta vulgaris/crescimento & desenvolvimento , Chumbo/farmacologia , Canais de Potássio/metabolismo , Vacúolos/metabolismo , Beta vulgaris/efeitos dos fármacos , Beta vulgaris/metabolismo , Citosol/efeitos dos fármacos , Citosol/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Técnicas de Patch-Clamp , Proteínas de Plantas/efeitos dos fármacos , Proteínas de Plantas/metabolismo , Canais de Potássio/efeitos dos fármacos , Vacúolos/efeitos dos fármacosRESUMO
Voltage-gated potassium (Kv) channels are a family of membrane proteins that facilitate K+ ion diffusion across the plasma membrane, regulating both resting and action potentials. Kv channels comprise four pore-forming α subunits, each with a voltage sensing domain, and they are regulated by interaction with ß subunits such as those belonging to the KCNE family. Here we conducted a comprehensive biophysical characterization of stoichiometry and protein diffusion across the plasma membrane of the epithelial KCNQ1-KCNE2 complex, combining total internal reflection fluorescence (TIRF) microscopy and a series of complementary Fluorescence Fluctuation Spectroscopy (FFS) techniques. Using this approach, we found that KCNQ1-KCNE2 has a predominant 4:4 stoichiometry, while non-bound KCNE2 subunits are mostly present as dimers in the plasma membrane. At the same time, we identified unique spatio-temporal diffusion modalities and nano-environment organization for each channel subunit. These findings improve our understanding of KCNQ1-KCNE2 channel function and suggest strategies for elucidating the subunit stoichiometry and forces directing localization and diffusion of ion channel complexes in general.
Assuntos
Canais de Potássio/química , Domínios e Motivos de Interação entre Proteínas , Análise Espectral , Potenciais de Ação , Animais , Células CHO , Cricetulus , Humanos , Ativação do Canal Iônico , Modelos Moleculares , Conformação Molecular , Técnicas de Patch-Clamp , Canais de Potássio/metabolismo , Ligação Proteica , Análise Espectral/métodos , Relação Estrutura-AtividadeRESUMO
Quercetin is a natural flavonoid which has been reported to be analgesic in different animal models of pain. However, the mechanism underlying the pain-relieving effects is still unclear. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels play critical roles in controlling pacemaker activity in cardiac and nervous systems, making the channel a new target for therapeutic exploration. In this study, we explored a series of flavonoids for their modulation on HCN channels. Among all tested flavonoids, quercetin was the most potent inhibitor for HCN channels with an IC50 value of 27.32 ± 1.19 µM for HCN2. Furthermore, quercetin prominently left shifted the voltage-dependent activation curves of HCN channels and decelerated deactivation process. The results presented herein firstly characterize quercetin as a novel and potent inhibitor for HCN channels, which represents a novel structure for future drug design of HCN channel inhibitors.
Assuntos
Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/antagonistas & inibidores , Quercetina/farmacologia , Animais , Células COS , Chlorocebus aethiops , Avaliação Pré-Clínica de Medicamentos , Fenômenos Eletrofisiológicos , Flavonoides/química , Flavonoides/farmacologia , Flavonóis/química , Flavonóis/farmacologia , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Proteínas Musculares/antagonistas & inibidores , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Técnicas de Patch-Clamp , Canais de Potássio/genética , Canais de Potássio/metabolismo , Quercetina/química , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relação Estrutura-AtividadeRESUMO
Oxytocin, a hypothalamic neuropeptide essential for breastfeeding, is mainly produced in oxytocin neurons in the supraoptic nucleus (SON) and paraventricular nucleus. However, mechanisms underlying oxytocin secretion, specifically the involvement of hyperpolarization-activated cyclic nucleotide-gated channel 3 (HCN3) in oxytocin neuronal activity, remain unclear. Using a rat model of intermittent and continuous pup deprivation (PD) at the middle stage of lactation, we analyzed the contribution of HCN3 in oxytocin receptor (OTR)-associated signaling cascade to oxytocin neuronal activity in the SON. PD caused maternal depression, anxiety, milk shortage, involution of the mammary glands, and delays in uterine recovery, particularly in continuous PD. PD increased hypothalamic but not plasma oxytocin levels in enzyme-linked immunosorbent assay. In the SON, PD increased c-Fos expression but reduced expressions of cyclooxygenase-2 and HCN3 in Western blots and/or immunohistochemistry. Moreover, PD significantly increased the molecular association of OTR with HCN3 in coimmunoprecipitation. In brain slices, inhibition of HCN3 activity with DK-AH269 blocked prostaglandin E2-evoked increase in the firing activity and burst discharge in oxytocin neurons in patch-clamp recordings. In addition, oxytocin-evoked increase in the molecular association between OTR and HCN3 in brain slices of the SON was blocked by pretreatment with indomethacin, an inhibitor of cyclooxygenase-2. These results indicate that normal activity of oxytocin neurons is under the regulation of an oxytocin receptor-cyclooxygenase-2-HCN3 pathway and that PD disrupts maternal behavior through increasing intranuclear oxytocin secretion in the SON but likely reducing bolus oxytocin release into the blood through inhibition of HCN3 activity.
Assuntos
Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Lactação/metabolismo , Privação Materna , Neurônios/metabolismo , Ocitocina/metabolismo , Canais de Potássio/metabolismo , Animais , Animais Recém-Nascidos , Benzazepinas/farmacologia , Feminino , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/antagonistas & inibidores , Hipotálamo/efeitos dos fármacos , Hipotálamo/metabolismo , Lactação/efeitos dos fármacos , Lactação/psicologia , Masculino , Neurônios/efeitos dos fármacos , Ratos , Ratos Sprague-DawleyRESUMO
The GABA analog phenibut (ß-Phenyl-GABA) is a GABAB receptor agonist that has been licensed for various uses in Russia. Phenibut is also available as a dietary supplement from online vendors worldwide, and previous studies have indicated that phenibut overdose results in intoxication, withdrawal symptoms, and addiction. F-phenibut (ß-(4-Fluorophenyl)-GABA), a derivative of phenibut, has not been approved for clinical use. However, it is also available as a nootropic supplement from online suppliers. F-phenibut binds to GABAB with a higher affinity than phenibut; therefore, F-phenibut may lead to more serious intoxication than phenibut. However, the mechanisms by which F-phenibut acts on GABAB receptors and influences neuronal function remain unknown. In the present study, we compared the potency of F-phenibut, phenibut, and the GABAB agonist (±)-baclofen (baclofen) using in vitro patch-clamp recordings obtained from mouse cerebellar Purkinje cells slice preparations Our findings indicate that F-phenibut acted as a potent GABAB agonist. EC50 of outward current density evoked by the three GABAB agonists decreased in the following order: phenibut (1362 µM) > F-phenibut (23.3 µM) > baclofen (6.0 µM). The outward current induced by GABAB agonists was an outward-rectifying K+ current, in contrast to the previous finding that GABAB agonists activates an inward-rectifying K+ current. The K+ current recorded in the present study was insensitive to extracellular Ba2+, intra- or extracellular Cs+, and intra- or extracellular tetraethylammonium-Cl. Moreover, F-phenibut suppressed action potential generation in Purkinje cells. Thus, abuse of F-phenibut may lead to severe damage by inhibiting the excitability of GABAB-expressing neurons.
Assuntos
Agonistas dos Receptores de GABA-B/farmacologia , Canais de Potássio/metabolismo , Potássio/metabolismo , Células de Purkinje/efeitos dos fármacos , Receptores de GABA-B/efeitos dos fármacos , Ácido gama-Aminobutírico/farmacologia , Potenciais de Ação , Animais , Baclofeno/farmacologia , Relação Dose-Resposta a Droga , Feminino , Agonistas dos Receptores de GABA-B/toxicidade , Técnicas In Vitro , Masculino , Camundongos Endogâmicos ICR , Células de Purkinje/metabolismo , Receptores de GABA-B/metabolismo , Ácido gama-Aminobutírico/análogos & derivados , Ácido gama-Aminobutírico/toxicidadeRESUMO
KCNK3 is a two-pore-domain (K2P) potassium channel involved in maintaining ion homeostasis, mediating thermogenesis, controlling breath and modulating electrical membrane potential. Although the functions of this channel have been widely described in mammals, its roles in fishes are still rarely known. Here, we identified two kcnk3 genes from the euryhaline rabbitfish (Siganus canaliculatus), and their roles related to fatty acids metabolism and osmoregulation were investigated. The open reading frames of kcnk3a and kcnk3b were 1203 and 1176 bp in length, encoding 400 and 391 amino acids respectively. Multiple sequences alignment and phylogenetic analysis revealed that the two isotypes of kcnk3 were extensively presented in fishes. Quantitative real-time PCRs indicated that both genes were widely distributed in examined tissues but showed different patterns. kcnk3a primary distributed in adipose, eye, heart, and spleen tissues, while kcnk3b was mainly detectable in heart, kidney, muscle and spleen tissues. In vivo experiments showed that fish fed diets with fish oil as dietary lipid (rich in long chain polyunsaturated fatty acids, LC-PUFA) induced higher mRNA expression levels of kcnk3 genes in comparison with fish fed with plant oil diet at two different salinity environments (32 and 15). Meanwhile, the expression levels of kcnk3 genes were higher in seawater (32) than that in brackish water (15) when fishes were fed with both types of feeds. In vitro experiments with rabbitfish hepatocytes showed that LC-PUFA significantly improved hepatic kcnk3a expression level compared with treatment of linolenic acid. These results suggest that two kcnk3 genes are widely existed and they might be functionally related to fatty acids metabolism and osmoregulation in the rabbitfish.
Assuntos
Ácidos Graxos/metabolismo , Peixes/genética , Osmorregulação , Canais de Potássio/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , DNA Complementar/genética , Peixes/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Hepatócitos/metabolismo , Metabolismo dos Lipídeos/genética , Fígado/metabolismo , Filogenia , Canais de Potássio/química , Canais de Potássio/metabolismo , Salinidade , Distribuição TecidualRESUMO
Previous studies indicate that sex-related differences exist in the regulation of cutaneous vasodilation, however, the mechanisms remain unresolved. We assessed if sex-differences in young adults exist for cholinergic, nicotinic, and ß-adrenergic cutaneous vasodilation with a focus on nitric oxide synthase (NOS), cyclooxygenase (COX), and K+ channel mechanisms. In twelve young men and thirteen young women, four intradermal forearm skin sites were perfused with the following: 1) lactated Ringer's solution (control), 2) 10 mM Nω-nitro-l-arginine, a non-selective NOS inhibitor, 3) 10 mM ketorolac, a non-selective COX inhibitor, or 4) 50 mM BaCl2, a nonspecific K+ channel blocker. At all four sites, cutaneous vasodilation was induced by 1) 10 mM nicotine, a nicotinic receptor agonist, 2) 100 µM isoproterenol, a nonselective ß-adrenergic receptor agonist, and 3) 2 mM and 2000 mM acetylcholine, an acetylcholine receptor agonist. Nicotine and isoproterenol were administered for 3 min, whereas each acetylcholine dose was administered for 25 min. Regardless of treatment site, cutaneous vasodilation in response to nicotine and a high dose of acetylcholine (2000 mM) were lower in women than men. By contrast, isoproterenol induced cutaneous vasodilation was greater in women vs. men. Irrespective of sex, NOS inhibition or K+ channel blockade attenuated isoproterenol-mediated cutaneous vasodilation, whereas K+ channel blockade decreased nicotine-induced cutaneous vasodilation. Taken together, our findings indicate that while the mechanisms underlying cutaneous vasodilation are comparable between young men and women, sex-related differences in the magnitude of cutaneous vasodilation do exist and this response differs as a function of the receptor agonist.
Assuntos
Vasos Sanguíneos/enzimologia , Óxido Nítrico Sintase/metabolismo , Canais de Potássio/metabolismo , Prostaglandina-Endoperóxido Sintases/metabolismo , Receptores Adrenérgicos beta/metabolismo , Receptores Colinérgicos/metabolismo , Pele/irrigação sanguínea , Vasodilatação , Agonistas Adrenérgicos beta/farmacologia , Adulto , Vasos Sanguíneos/efeitos dos fármacos , Agonistas Colinérgicos/farmacologia , Inibidores de Ciclo-Oxigenase/farmacologia , Feminino , Antebraço , Humanos , Masculino , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/antagonistas & inibidores , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio/efeitos dos fármacos , Receptores Nicotínicos/metabolismo , Fatores Sexuais , Transdução de Sinais , Vasodilatação/efeitos dos fármacos , Adulto JovemRESUMO
BACKGROUND: Cytoprotection afforded by mitochondrial ATP-sensitive K+-channel (mKATP-channel) opener diazoxide (DZ) largely depends on the activation of potassium cycle with eventual modulation of mitochondrial functions and ROS production. However, generally these effects were studied in the presence of MgâATP known to block K+ transport. Thus, the purpose of our work was the estimation of DZ effects on K+ transport, K+ cycle and ROS production in rat liver mitochondria in the absence of MgâATP. RESULTS: Without Mg·ATP, full activation of native mKATP-channel, accompanied by the increase in ATP-insensitive K+ uptake, activation of K+-cycle and respiratory uncoupling, was reached at ≤0.5 µM of DZ,. Higher diazoxide concentrations augmented ATP-insensitive K+ uptake, but not mKATP-channel activity. mKATP-channel was blocked by Mg·ATP, reactivated by DZ, and repeatedly blocked by mKATP-channel blockers glibenclamide and 5-hydroxydecanoate, whereas ATP-insensitive potassium transport was blocked by Mg2+ and was not restored by DZ. High sensitivity of potassium transport to DZ in native mitochondria resulted in suppression of mitochondrial ROS production caused by the activation of K+-cycle on sub-micromolar scale. Based on the oxygen consumption study, the share of mKATP-channel in respiratory uncoupling by DZ was found. CONCLUSIONS: The study of mKATP-channel activation by diazoxide in the absence of MgATP discloses novel, not described earlier, aspects of mKATP-channel interaction with this drug. High sensitivity of mKATP-channel to DZ results in the modulation of mitochondrial functions and ROS production by DZ on sub-micromolar concentration scale. Our experiments led us to the hypothesis that under the conditions marked by ATP deficiency affinity of mKATP-channel to DZ can increase, which might contribute to the high effectiveness of this drug in cardio- and neuroprotection.
Assuntos
Trifosfato de Adenosina/metabolismo , Diazóxido/farmacologia , Mitocôndrias Hepáticas/efeitos dos fármacos , Canais de Potássio/metabolismo , Potássio/metabolismo , Animais , Ácidos Decanoicos/farmacologia , Metabolismo Energético/efeitos dos fármacos , Feminino , Glibureto/farmacologia , Hidroxiácidos/farmacologia , Transporte de Íons/efeitos dos fármacos , Transporte de Íons/genética , Canais KATP/metabolismo , Magnésio/metabolismo , Mitocôndrias Hepáticas/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , Bloqueadores dos Canais de Potássio/metabolismo , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio/genética , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismoRESUMO
Traditional Chinese Medicines (TCMs)-containing aconitine are popular and indispensable home remedies in Asia for thousands of years due to its excellent pharmaceutical effects. Accumulating evidence has identified that repeated-dose of aconitine could cause polymorphic ventricular arrhythmias. However, underlying molecular mechanisms are still not fully understood. Hence, the present study firstly investigated the potential role of Notch1 signaling in aconitine-induced cardiotoxicity, aiming to elaborate possible molecular mechanisms involved in aconitine triggered ventricular arrhythmias. Our results showed that aconitine increased Notch1 signaling and downstream KDM5A expression in human and rat cardiomyocytes at non-detectable cytotoxic doses. Furthermore, aconitine promoted the formation of a new regulatory complex containing NICD and KDM5A in a CK2αHI regime, which then targeted to HCN4 promoter and induced re-expression of HCN4 in mature cardiomyocytes. Ultimately, HCN4-mediated If current contributed to aconitine-caused alterations in beating rate of rat cardiomyocytes. All changes aforementioned were significantly ameliorated by Notch1 inhibitor, suggesting that Notch1-mediated epigenetic regulation of HCN4 contributes to aconitine-induced ventricular myocardial dysrhythmia. Thus, our findings provide a novel toxic mechanism and position Notch1/NICD/KDM5A/HCN4 toxicity pathway as a potential target for the treatments of repeated-dose of medicine containing aconitine induced ventricular arrhythmias.
Assuntos
Aconitina/farmacologia , Arritmias Cardíacas/induzido quimicamente , Ventrículos do Coração/efeitos dos fármacos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Canais de Potássio/metabolismo , Receptor Notch1/metabolismo , Animais , Animais Recém-Nascidos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Histonas , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Canais de Potássio/genética , Ratos , Receptor Notch1/genética , Superóxidos/metabolismoRESUMO
Estrogenic deficiency is considered a risk of coronary disease in women. The phytoestrogen genistein could be a safe preventive strategy. The first aim of this work was to validate a model of cardiac stunning in which natural estrogenic deficiency rats, ie, adult young male (YM) and aged female (AgF), are compared with young female rats (YF). The second aim was to study whether the in vivo administration of genistein prevents the stunning in estrogenic deficiency rats. The third aim was to evaluate whether in our estrogenic deficiency model exists a synergy between genistein and estradiol. The fourth aim was to characterize the underlying mechanisms of genistein. Stunning was induced by ischemia/reperfusion (I/R) in isolated hearts inside a calorimeter. The left ventricular pressure (P) and total heat rate (Ht) were simultaneously measured, while diastolic contracture and muscle economy (P/Ht) were calculated. During R, P/Ht and P recovered less in AgF and YM than in YF rat hearts. Genistein through i.p. (GST-ip) improved P and P/Ht in AgF and YM, but not in YF. In YM, the cardioprotections of GST-ip and estradiol were synergistic. After ischemia, GST-ip increased SR Ca leak causing diastolic contracture. The GST-ip cardioprotection neither was affected by blockade of PI3K-Akt, NO synthases, or phosphatases, but it was sensitive to blockade of protein-kinase C and mKATP channels. Results suggest that (1) estrogenic deficiency worsens cardiac stunning, (2) GST-ip was more cardioprotective in estrogenic deficiency and synergistic with estradiol, and (3) cardioprotection of GST-ip depends on the protein-kinase C and mKATP channel pathway activation.
Assuntos
Metabolismo Energético/efeitos dos fármacos , Genisteína/farmacologia , Mitocôndrias Cardíacas/efeitos dos fármacos , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miocárdio Atordoado/prevenção & controle , Miócitos Cardíacos/efeitos dos fármacos , Fitoestrógenos/farmacologia , Canais de Potássio/metabolismo , Fatores Etários , Animais , Sinalização do Cálcio , Modelos Animais de Doenças , Estradiol/farmacologia , Feminino , Preparação de Coração Isolado , Masculino , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/patologia , Traumatismo por Reperfusão Miocárdica/enzimologia , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Miocárdio Atordoado/enzimologia , Miocárdio Atordoado/patologia , Miocárdio Atordoado/fisiopatologia , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/patologia , Proteína Quinase C/metabolismo , Ratos Sprague-Dawley , Fatores Sexuais , Função Ventricular Esquerda/efeitos dos fármacos , Pressão Ventricular/efeitos dos fármacosRESUMO
Adenosine monophosphate-activated protein kinase (AMPK), expressed/present ubiquitously in the body, contributes to metabolic regulation. In the vasculature, activation of AMPK is associated with several beneficial biological effects including enhancement of vasodilatation, reduction of oxidative stress and inhibition of inflammatory reactions. The vascular protective effects of certain anti-diabetic (metformin and sitagliptin) or lipid-lowering (simvastatin and fenofibrate) therapeutic agents, of active components of Chinese medicinal herbs (resveratrol and berberine) and of pharmacological agents (AICAR, A769662 and PT1) have been attributed to the activation of AMPK (in endothelial cells, vascular smooth muscle cells and/or perivascular adipocytes), independently of changes in the metabolic profile (eg glucose tolerance and/or plasma lipoprotein levels), leading to improved endothelium-derived nitric oxide-mediated vasodilatation and attenuated endothelium-derived cyclooxygenase-dependent vasoconstriction. By contrast, endothelial AMPK activation with pharmacological agents or by genetic modification is associated with reduced endothelium-dependent relaxations in small blood vessels and elevated systolic blood pressure. Indeed, AMPK activators inhibit endothelium-dependent hyperpolarization (EDH)-type relaxations in superior mesenteric arteries, partly by inhibiting endothelial calcium-activated potassium channel signalling. Therefore, AMPK activation is not necessarily beneficial in terms of endothelial function. The contribution of endothelial AMPK in the regulation of vascular tone, in particular in the microvasculature where EDH plays a more important role, remains to be characterized.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Endotélio Vascular/enzimologia , Músculo Liso Vascular/enzimologia , Óxido Nítrico/metabolismo , Vasoconstrição/fisiologia , Vasodilatação/fisiologia , Proteínas Quinases Ativadas por AMP/genética , Animais , Diabetes Mellitus/enzimologia , Diabetes Mellitus/fisiopatologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiopatologia , Humanos , Hipoglicemiantes/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/fisiopatologia , Canais de Potássio/metabolismo , Transdução de Sinais , Vasoconstrição/efeitos dos fármacos , Vasodilatação/efeitos dos fármacosRESUMO
Grapevine (Vitis vinifera L.), one of the most important fruit crops, is a model plant for studying the physiology of fleshy fruits. Here, we report on the characterization of a new grapevine Shaker-type K+ channel, VvK5.1. Phylogenetic analysis revealed that VvK5.1 belongs to the SKOR-like subfamily. Our functional characterization of VvK5.1 in Xenopus oocytes confirms that it is an outwardly rectifying K+ channel that displays strict K+ selectivity. Gene expression level analyses by real-time quantitative PCR showed that VvK5.1 expression was detected in berries, roots, and flowers. In contrast to its Arabidopsis thaliana counterpart that is involved in K+ secretion in the root pericycle, allowing root to shoot K+ translocation, VvK5.1 expression territory is greatly enlarged. Using in situ hybridization we showed that VvK5.1 is expressed in the phloem and perivascular cells of berries and in flower pistil. In the root, in addition to being expressed in the root pericycle like AtSKOR, a strong expression of VvK5.1 is detected in small cells facing the xylem that are involved in lateral root formation. This fine and selective expression pattern of VvK5.1 at the early stage of lateral root primordia supports a role for outward channels to switch on cell division initiation.
Assuntos
Proteínas de Plantas/metabolismo , Canais de Potássio/metabolismo , Vitis/metabolismo , Animais , DNA Complementar/genética , Flores/genética , Regulação da Expressão Gênica de Plantas , Oócitos/metabolismo , Floema/genética , Filogenia , Raízes de Plantas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Xenopus laevisRESUMO
The neurotoxins of venomous scorpion act on ion channels. Whether these neurotoxins are retained in processed Buthus martensii Karsch scorpions used in traditional Chinese medicine materials is unknown. Comprehensive mass spectrometry-based proteomic characterization of functionally active toxins in the processed medicinal scorpion material revealed 22 full-length and 44 truncated thermostable potassium channel-modulatory toxins that preserved six conserved cysteine residues capable of forming the three disulfide bonds necessary for toxicity. Additionally, a broad spectrum of degraded toxin fragments was found, indicating their relative thermal instability which enabled toxicity reduction. Furthermore, the suppression of interleukin-2 (IL-2) production in Jurkat cells and the reduced delayed-type hypersensitivity (DTH) response demonstrated that the extracts have immunoregulatory activity both in vitro and in vivo. Our work describes the first "map" of functionally active scorpion toxins in processed scorpion medicinal material, which is helpful to unveil the pharmaceutical basis of the processed scorpion medicinal material in traditional Chinese medicine. BIOLOGICAL SIGNIFICANCE: Scorpions have been used as medicinal materials in China for more than one thousand years. This is an example of the well-known "Combat poison with poison" strategy common to traditional Chinese medicine. In the past 30â¯years, extensive investigations of Chinese scorpions have indicated that the neurotoxins in the scorpion venom are the main toxic components and they target various ion channels in cell membranes. However, whether these neurotoxins are retained in processed Buthus martensii Karsch scorpions used for traditional Chinese medicine remains unknown. Our study described the thermal stability and instability of potassium channel-modulatory neurotoxins in processed scorpions and helps to understand the pharmaceutical basis underling the strategy of "combat poison with poison to cure diseases".
Assuntos
Medicina Tradicional Chinesa , Neurotoxinas/análise , Bloqueadores dos Canais de Potássio/análise , Proteoma/análise , Venenos de Escorpião/análise , Animais , Estabilidade de Medicamentos , Feminino , Células HEK293 , Humanos , Células Jurkat , Neurotoxinas/metabolismo , Peptídeos/análise , Peptídeos/metabolismo , Preparações Farmacêuticas/análise , Preparações Farmacêuticas/química , Preparações Farmacêuticas/metabolismo , Bloqueadores dos Canais de Potássio/isolamento & purificação , Bloqueadores dos Canais de Potássio/metabolismo , Canais de Potássio/metabolismo , Estabilidade Proteica , Proteoma/metabolismo , Proteômica/métodos , Ratos , Ratos Endogâmicos Lew , Venenos de Escorpião/química , Venenos de Escorpião/metabolismo , Escorpiões/química , Escorpiões/metabolismo , TemperaturaRESUMO
Pyruvic acid is important organic chemical intermediates that plays a role in cardiomyocyte pathophysiology and therapy. This study sought to explore the inotropic effects of pyruvic acid on the function of the isolated rat hearts and investigate its underlying mechanism. Pyruvic acid produced a greater negative inotropic effect compared to HCl and sodium pyruvate in a concentration-dependent pattern in the hearts. The role of low dose of pyruvic acid on heart function was regulated by pyruvic acid molecules and high dose pyruvic acid may be influenced by pyruvic acid molecules and pH. Kv channels may be involved in the pyruvic acid-induced negative inotropic effect. Finally, pyruvic acid markedly increased the level of LDH and CK and reduced the level of Ca2+Mg2+-ATPase and Na+K+-ATPase. These results suggest that pyruvic acid may modulate cardiac function at physiological or low doses but can cause damage to cardiomyocytes at high doses.
Assuntos
Coração/efeitos dos fármacos , Contração Miocárdica/efeitos dos fármacos , Miocárdio/metabolismo , Ácido Pirúvico/farmacologia , Ácido Pirúvico/toxicidade , Animais , Creatina Quinase/metabolismo , Relação Dose-Resposta a Droga , Coração/fisiopatologia , Técnicas In Vitro , L-Lactato Desidrogenase/metabolismo , Masculino , Perfusão , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio/metabolismo , Ratos WistarRESUMO
BACKGROUND AND AIM: Simaba ferruginea A.St.-Hil. Popularly known as "calunga," is a typical Brazilian cerrado plant whose rhizomes are popular for treating diarrhea. AIMS: The aim of this study was to evaluate the spasmolytic activity and the antidiarrheal effect of the ethanolic extract obtained from S. ferruginea (Sf-EtOH). METHODS: Ileal segments (1-2 cm) from male Wistar rats were mounted in isolated organ baths and connected to a force transducer, and then to an amplifier which was connected to a computer (AVS Projetos/São Paulo-SP). After stabilization for 60 min, under tension (1 gf), two submaximal contractions were induced with KCl 40 mM or carbachol 10-6 M on ileal segments. During the third tonic and sustained contraction, Sf-EtOH was added in cumulative concentrations to the organ bath. Incubations with L-NAME (10-4 M), ODQ (10-5 M), TEA+ (5 or 1 mM), glibenclamide (10-5 M), or apamine (100 nM) were prepared (n = 5), separately and used to verify the involvement of the nitric oxide synthase, guanylate cyclase, and potassium channels in the relaxing effect. The results were expressed as mean ± standard error of the mean and were statistically evaluated using one-way ANOVA followed by Bonferroni test, when necessary *p < 0.05. RESULTS: Sf-EtOH promotes relaxation on rat isolated ileum pre-contracted with CCh and KCl in a concentration-dependent manner. Sf-EtOH also inhibited ileum contractions against cumulative concentrations of carbachol (CCh), KCl, and CaCl2, shifting the curves to the right in a non-parallel manner with an Emax reduction. In the presence of potassium channel blockers, Sf-EtOH shifted the curves to the right with a reduction of Emax, suggesting the involvement of BKCa, KATP, and SKCa in its spasmolytic effect. In the presence of L-NAME or ODQ, the relaxation curves were shifted to the right, suggesting the involvement of this pathway in Sf-EtOH spasmolytic effect. CONCLUSIONS: Sf-EtOH acts in a concentration-dependent manner, involving the positive modulation of K+ channels and NO pathway.