RESUMO
In traditional Korean medicine, the 16-herb concoction Bojanggunbi-tang (BGT) is used to treat various gastrointestinal (GI) diseases. In this study, we investigated the regulatory mechanism underlying the influence of BGT on the interstitial cells of Cajal (ICCs), pacemaker cells in the GI tract. Within 12 h of culturing ICCs in the small intestines of mice, the pacemaker potential of ICCs was recorded through an electrophysiological method. An increase in the BGT concentration induced depolarization and decreased firing frequency. This reaction was suppressed by cholinergic receptor muscarinic 3 (CHRM3) antagonists, as well as 5-hydroxytryptamine receptor (5HTR) 3 and 4 antagonists. Nonselective cation channel inhibitors, such as thapsigargin and flufenamic acid, along with protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) inhibitors, also suppressed the BGT reaction. Guanylate cyclase and protein kinase G (PKG) antagonists inhibited BGT, but adenylate cyclase and protein kinase A antagonists had no effect. In conclusion, we demonstrated that BGT acts through CHRM3, 5HTR3, and 5HTR4 to regulate intracellular Ca2+ concentrations and the PKC, MAPK, guanylate cycle, and PKG signaling pathways.
Assuntos
Células Intersticiais de Cajal , Animais , Camundongos , Potenciais da Membrana , Células Intersticiais de Cajal/metabolismo , Transdução de Sinais , Intestino Delgado/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Colinérgicos/metabolismo , Colinérgicos/farmacologia , Camundongos Endogâmicos BALB C , Células CultivadasRESUMO
The classical Hodgkin-Huxley (HH) point-neuron model of action potential generation is four-dimensional. It consists of four ordinary differential equations describing the dynamics of the membrane potential and three gating variables associated to a transient sodium and a delayed-rectifier potassium ionic currents. Conductance-based models of HH type are higher-dimensional extensions of the classical HH model. They include a number of supplementary state variables associated with other ionic current types, and are able to describe additional phenomena such as subthreshold oscillations, mixed-mode oscillations (subthreshold oscillations interspersed with spikes), clustering and bursting. In this manuscript we discuss biophysically plausible and phenomenological reduced models that preserve the biophysical and/or dynamic description of models of HH type and the ability to produce complex phenomena, but the number of effective dimensions (state variables) is lower. We describe several representative models. We also describe systematic and heuristic methods of deriving reduced models from models of HH type.
Assuntos
Modelos Neurológicos , Neurônios , Neurônios/fisiologia , Potenciais de Ação/fisiologia , Potenciais da Membrana/fisiologia , BiofísicaRESUMO
The action of the pulsed electric field of the subnanosecond range on Jurkat, HEK 293, and U-87 MG human cell lines was studied. The cells were treated in a waveguide in 0.18 ml electrodeless Teflon cuvettes. The electric field strength in the cell culture medium was ~2 kV/cm, the pulse duration was ~1 ns, the leading edge was 150 ps, the frequency was 100 Hz, and the treatment time was 5 min. According to estimates, the change of the transmembrane potential during the pulse was ~20 mV and we assume that it was insufficient for electroporation. Jurkat and HEK 293 cells appeared to be more resistant to the treatment than U-87 MG cells. We have observed that the impulses with the above-mentioned parameters can cause a noticeable change in the mitochondrial activity of U-87 MG cells. © 2022 Bioelectromagnetics Society.
Assuntos
Eletricidade , Eletroporação , Contagem de Células , Células HEK293 , Humanos , Potenciais da MembranaRESUMO
Hydroxychloroquine (HCQ) is a derivative of the antimalaria drug chloroquine primarily prescribed for autoimmune diseases. Recent attempts to repurpose HCQ in the treatment of corona virus disease 2019 has raised concerns because of its propensity to prolong the QT-segment on the electrocardiogram, an effect associated with increased pro-arrhythmic risk. Since chirality can affect drug pharmacological properties, we have evaluated the functional effects of the R(-) and S(+) enantiomers of HCQ on six ion channels contributing to the cardiac action potential and on electrophysiological parameters of isolated Purkinje fibers. We found that R(-)HCQ and S(+)HCQ block human Kir2.1 and hERG potassium channels in the 1 µM-100 µM range with a 2-4 fold enantiomeric separation. NaV1.5 sodium currents and CaV1.2 calcium currents, as well as KV4.3 and KV7.1 potassium currents remained unaffected at up to 90 µM. In rabbit Purkinje fibers, R(-)HCQ prominently depolarized the membrane resting potential, inducing autogenic activity at 10 µM and 30 µM, while S(+)HCQ primarily increased the action potential duration, inducing occasional early afterdepolarization at these concentrations. These data suggest that both enantiomers of HCQ can alter cardiac tissue electrophysiology at concentrations above their plasmatic levels at therapeutic doses, and that chirality does not substantially influence their arrhythmogenic potential in vitro.
Assuntos
Antimaláricos/química , Antimaláricos/farmacologia , Coração/efeitos dos fármacos , Hidroxicloroquina/química , Hidroxicloroquina/farmacologia , Canais Iônicos/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Animais , Arritmias Cardíacas/induzido quimicamente , Eletrocardiografia , Técnicas Eletrofisiológicas Cardíacas , Canais de Potássio Éter-A-Go-Go , Humanos , Potenciais da Membrana/efeitos dos fármacos , Técnicas de Patch-Clamp , Ramos Subendocárdicos/efeitos dos fármacos , Coelhos , EstereoisomerismoRESUMO
Neurofeedback (NF) as a treatment for Attention Deficit Hyperactivity Disorder (ADHD) has been evaluated in several trials, but the specificity and generalizability of effects remain unclear. This four-arm randomized controlled trial evaluated the efficacy of Slow Cortical Potential (SCP; standard NF protocol) and Live Z-score (LZS; non-standard NF protocol) delivered in high-frequency format (five sessions per week during five weeks), compared to Working-memory training (WMT; active comparator) and Treatment-as-usual (TAU; passive comparator). N = 202 children/adolescents aged 9 to 17 years with ADHD participated. The primary outcome measure was multi-report (self-, teacher-, and parent-report) ADHD core symptoms on the Conners-3, assessed at baseline, posttreatment, and 6-months follow-up. Data were analyzed using a linear mixed model. Between-group differences were scarce and did not show a distinct pattern. Superiority of LZS over TAU at endpoint were observed for teacher-rated measures only, while significant differences between SCP and TAU were restricted to posttreatment measurements. Contrary to our expectations, LZS outperformed SCP at endpoint for teacher-rated hyperactivity (-5.37; 95% CI: -10.14 to -0.60; p = .028; d = -.36) and overall ADHD symptoms (-2.20; -4.18 to -0.22; p = .030; d = -.41). There was no indication that either form of NF was superior to WMT. No severe adverse events were reported during the trial, whereas transient stress-related problems were quite frequent. Overall, the results from this pragmatic trial do not provide convincing support for broad implementation of NF in child and adolescent psychiatric services. Future research should try to clarify for whom and under what circumstances NF might be a viable treatment option.
Assuntos
Transtorno do Deficit de Atenção com Hiperatividade , Neurorretroalimentação , Adolescente , Transtorno do Deficit de Atenção com Hiperatividade/terapia , Criança , Humanos , Potenciais da Membrana , Memória de Curto Prazo , Neurorretroalimentação/métodos , Resultado do TratamentoRESUMO
Latrotoxins (LaTXs) are presynaptic pore-forming neurotoxins found in the venom of Latrodectus spiders. The venom contains a toxic cocktail of seven LaTXs, with one of them targeting vertebrates (α-latrotoxin (α-LTX)), five specialized on insects (α, ß, γ, δ, ε- latroinsectotoxins (LITs), and one on crustaceans (α-latrocrustatoxin (α-LCT)). LaTXs bind to specific receptors on the surface of neuronal cells, inducing the release of neurotransmitters either by directly stimulating exocytosis or by forming Ca2+-conductive tetrameric pores in the membrane. Despite extensive studies in the past decades, a high-resolution structure of a LaTX is not yet available and the precise mechanism of LaTX action remains unclear. Here, we report cryoEM structures of the α-LCT monomer and the δ-LIT dimer. The structures reveal that LaTXs are organized in four domains. A C-terminal domain of ankyrin-like repeats shields a central membrane insertion domain of six parallel α-helices. Both domains are flexibly linked via an N-terminal α-helical domain and a small ß-sheet domain. A comparison between the structures suggests that oligomerization involves major conformational changes in LaTXs with longer C-terminal domains. Based on our data we propose a cyclic mechanism of oligomerization, taking place prior membrane insertion. Both recombinant α-LCT and δ-LIT form channels in artificial membrane bilayers, that are stabilized by Ca2+ ions and allow calcium flux at negative membrane potentials. Our comparative analysis between α-LCT and δ-LIT provides first crucial insights towards understanding the molecular mechanism of the LaTX family.
Assuntos
Viúva Negra/química , Cálcio/química , Neurotoxinas/química , Fosfatidilcolinas/química , Fosfatidiletanolaminas/química , Venenos de Aranha/química , Animais , Sítios de Ligação , Viúva Negra/patogenicidade , Cálcio/metabolismo , Clonagem Molecular , Microscopia Crioeletrônica , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Transporte de Íons , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Potenciais da Membrana/fisiologia , Modelos Moleculares , Neurotoxinas/genética , Neurotoxinas/metabolismo , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Venenos de Aranha/genética , Venenos de Aranha/metabolismoRESUMO
ATP-dependent P2X3 receptors play a crucial role in the sensitization of nerve fibers and pathological pain pathways. They are also involved in pathways triggering cough and may contribute to the pathophysiology of endometriosis and overactive bladder. However, despite the strong therapeutic rationale for targeting P2X3 receptors, preliminary antagonists have been hampered by off-target effects, including severe taste disturbances associated with blocking the P2X2/3 receptor heterotrimer. Here we present a P2X3 receptor antagonist, eliapixant (BAY 1817080), which is both highly potent and selective for P2X3 over other P2X subtypes in vitro, including P2X2/3. We show that eliapixant reduces inflammatory pain in relevant animal models. We also provide the first in vivo experimental evidence that P2X3 antagonism reduces neurogenic inflammation, a phenomenon hypothesised to contribute to several diseases, including endometriosis. To test whether eliapixant could help treat endometriosis, we confirmed P2X3 expression on nerve fibers innervating human endometriotic lesions. We then demonstrate that eliapixant reduces vaginal hyperalgesia in an animal model of endometriosis-associated dyspareunia, even beyond treatment cessation. Our findings indicate that P2X3 antagonism could alleviate pain, including non-menstrual pelvic pain, and modify the underlying disease pathophysiology in women with endometriosis. Eliapixant is currently under clinical development for the treatment of disorders associated with hypersensitive nerve fibers.
Assuntos
Fibras Nervosas/efeitos dos fármacos , Fibras Nervosas/metabolismo , Antagonistas do Receptor Purinérgico P2X/farmacologia , Receptores Purinérgicos P2X3/metabolismo , Distúrbios Somatossensoriais/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Linhagem Celular , Modelos Animais de Doenças , Feminino , Expressão Gênica , Humanos , Hiperalgesia/tratamento farmacológico , Hiperalgesia/etiologia , Hiperalgesia/metabolismo , Hiperalgesia/fisiopatologia , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Doenças Neuroinflamatórias/tratamento farmacológico , Doenças Neuroinflamatórias/etiologia , Doenças Neuroinflamatórias/metabolismo , Doenças Neuroinflamatórias/patologia , Ratos , Receptores Purinérgicos P2X3/genética , Distúrbios Somatossensoriais/tratamento farmacológico , Distúrbios Somatossensoriais/etiologiaRESUMO
INTRODUCTION: Peripheral artery disease (PAD) is a highly morbid condition in which impaired blood flow to the limbs leads to pain and tissue loss. Previously we identified 670 nm electromagnetic energy (R/NIR) to increase nitric oxide levels in cells and tissue. NO elicits relaxation of smooth muscle (SMC) by stimulating potassium efflux and membrane hyperpolarization. The actions of energy on ion channel activity have yet to be explored. Here we hypothesized R/NIR stimulates vasodilation through activation of potassium channels in SMC. METHODS: Femoral arteries or facial arteries from C57Bl/6 and Slo1-/- mice were isolated, pressurized to 60 mmHg, pre-constricted with U46619, and irradiated twice with energy R/NIR (10 mW/cm2 for 5 min) with a 10 min dark period between irradiations. Single-channel K+ currents were recorded at room temperature from cell-attached and excised inside-out membrane patches of freshly isolated mouse femoral arterial muscle cells using the patch-clamp technique. RESULTS: R/NIR stimulated vasodilation requires functional activation of the large conductance potassium channels. There is a voltage dependent outward current in SMC with light stimulation, which is due to increases in the open state probability of channel opening. R/NIR modulation of channel opening is eliminated pharmacologically (paxilline) and genetically (BKca α subunit knockout). There is no direct action of light to modulate channel activity as excised patches did not increase the open state probability of channel opening. CONCLUSION: R/NIR vasodilation requires indirect activation of the BKca channel.
Assuntos
Radiação Eletromagnética , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/efeitos da radiação , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/efeitos da radiação , Transdução de Sinais/efeitos da radiação , Vasodilatação/efeitos da radiação , Animais , Estimulação Elétrica/métodos , Terapia por Estimulação Elétrica/métodos , Artéria Femoral/metabolismo , Técnicas de Inativação de Genes , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Potenciais da Membrana/efeitos da radiação , Camundongos , Camundongos Knockout , Óxido Nítrico/metabolismo , Técnicas de Patch-Clamp , Doença Arterial Periférica/metabolismo , Doença Arterial Periférica/terapiaRESUMO
During the last decade, cardiac optogenetics has turned into an essential tool for investigating cardiac function in general and for assessing functional interactions between different myocardial cell types in particular. To advance exploitation of the unique research opportunities offered by this method, we develop a panoramic opto-electrical measurement and stimulation (POEMS) system for mouse hearts. The core of the experimental platform is composed of 294 optical fibers and 64 electrodes that form a cup which embraces the entire ventricular surface of mouse hearts and enables straightforward 'drop&go' experimentation. The flexible assignment of fibers and electrodes to recording or stimulation tasks permits a precise tailoring of experiments to the specific requirements of individual optogenetic constructs thereby avoiding spectral congestion. Validation experiments with hearts from transgenic animals expressing the optogenetic voltage reporters ASAP1 and ArcLight-Q239 demonstrate concordance of simultaneously recorded panoramic optical and electrical activation maps. The feasibility of single fiber optical stimulation is proven with hearts expressing the optogenetic voltage actuator ReaChR. Adaptation of the POEMS system to larger hearts and incorporation of additional sensors can be achieved by redesigning the system-core accordingly.
Assuntos
Coração/fisiologia , Optogenética/métodos , Animais , Técnicas Eletrofisiológicas Cardíacas , Frequência Cardíaca , Potenciais da Membrana , Camundongos , Camundongos Transgênicos , Miócitos Cardíacos/fisiologia , Optogenética/instrumentação , Imagens com Corantes Sensíveis à VoltagemRESUMO
The ability to directly observe membrane potential charging dynamics across a full microscopic field of view is vital for understanding interactions between a biological system and a given electrical stimulus. Accurate empirical knowledge of cell membrane electrodynamics will enable validation of fundamental hypotheses posited by the single shell model, which includes the degree of voltage change across a membrane and cellular sensitivity to external electric field non-uniformity and directionality. To this end, we have developed a high-speed strobe microscopy system with a time resolution of ~ 6 ns that allows us to acquire time-sequential data for temporally repeatable events (non-injurious electrostimulation). The imagery from this system allows for direct comparison of membrane voltage change to both computationally simulated external electric fields and time-dependent membrane charging models. Acquisition of a full microscope field of view enables the selection of data from multiple cell locations experiencing different electrical fields in a single image sequence for analysis. Using this system, more realistic membrane parameters can be estimated from living cells to better inform predictive models. As a proof of concept, we present evidence that within the range of membrane conductivity used in simulation literature, higher values are likely more valid.
Assuntos
Membrana Celular/ultraestrutura , Eletroporação/métodos , Fotografação/métodos , Análise de Célula Única/métodos , Animais , Células CHO , Cricetulus , Potenciais da MembranaRESUMO
Background: Vitamin D (vitD) deficiency is highly prevalent in patients with pulmonary arterial hypertension (PAH). Moreover, PAH-patients with lower levels of vitD have worse prognosis. We hypothesize that recovering optimal levels of vitD in an animal model of PAH previously depleted of vitD improves the hemodynamics, the endothelial dysfunction and the ionic remodeling. Methods: Male Wistar rats were fed a vitD-free diet for five weeks and then received a single dose of Su5416 (20 mg/Kg) and were exposed to vitD-free diet and chronic hypoxia (10% O2) for three weeks to induce PAH. Following this, vitD deficient rats with PAH were housed in room air and randomly divided into two groups: (a) continued on vitD-free diet or (b) received an oral dose of 100,000 IU/Kg of vitD plus standard diet for three weeks. Hemodynamics, pulmonary vascular remodeling, pulmonary arterial contractility, and K+ currents were analyzed. Results: Recovering optimal levels of vitD improved endothelial function, measured by an increase in the endothelium-dependent vasodilator response to acetylcholine. It also increased the activity of TASK-1 potassium channels. However, vitD supplementation did not reduce pulmonary pressure and did not ameliorate pulmonary vascular remodeling and right ventricle hypertrophy. Conclusions: Altogether, these data suggest that in animals with PAH and severe deficit of vitD, restoring vitD levels to an optimal range partially improves some pathophysiological features of PAH.
Assuntos
Endotélio Vascular/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Proteínas do Tecido Nervoso/metabolismo , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Hipertensão Arterial Pulmonar , Deficiência de Vitamina D , Vitamina D , Animais , Endotélio Vascular/patologia , Masculino , Hipertensão Arterial Pulmonar/tratamento farmacológico , Hipertensão Arterial Pulmonar/metabolismo , Hipertensão Arterial Pulmonar/patologia , Ratos , Ratos Wistar , Vitamina D/farmacocinética , Vitamina D/farmacologia , Deficiência de Vitamina D/tratamento farmacológico , Deficiência de Vitamina D/metabolismo , Deficiência de Vitamina D/patologiaRESUMO
Ion channels are attractive drug targets for many therapeutic applications. However, high-throughput screening (HTS) of drug candidates is difficult and remains very expensive. We thus assessed the suitability of the bioluminescence resonance energy transfer (BRET) technique as a new HTS method for ion-channel studies by taking advantage of our recently characterized intra- and intermolecular BRET probes targeting the transient receptor potential vanilloid type 1 (TRPV1) ion channel. These BRET probes monitor conformational changes during TRPV1 gating and subsequent coupling with calmodulin, two molecular events that are intractable using reference techniques such as automated calcium assay (ACA) and automated patch-clamp (APC). We screened the small-sized Prestwick chemical library, encompassing 1200 compounds with high structural diversity, using either intra- and intermolecular BRET probes or ACA. Secondary screening of the detected hits was done using APC. Multiparametric analysis of our results shed light on the capability of calmodulin inhibitors included in the Prestwick library to inhibit TRPV1 activation by capsaicin. BRET was the lead technique for this identification process. Finally, we present data exemplifying the use of intramolecular BRET probes to study other transient receptor potential (TRP) channels and non-TRPs ion channels. Knowing the ease of use of BRET biosensors and the low cost of the BRET technique, these assays may advantageously be included for extending ion-channel drug screening. SIGNIFICANCE STATEMENT: This study screened a chemical library against TRPV1 ion channel using bioluminescence resonance energy transfer (BRET) molecular probes and compared the results with the ones obtained using reference techniques such as automated calcium assay and automated patch-clamp. Multiparametric analysis of our results shed light on the capability of calmodulin antagonists to inhibit chemical activation of TRPV1 and indicates that BRET probes may advantageously be included in ion channel drug screening campaigns.
Assuntos
Técnicas de Transferência de Energia por Ressonância de Bioluminescência/métodos , Descoberta de Drogas/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Ensaios de Triagem em Larga Escala/métodos , Canais de Cátion TRPV/metabolismo , Bioensaio/métodos , Cálcio/química , Calmodulina/antagonistas & inibidores , Células HEK293 , Humanos , Ligantes , Potenciais da Membrana/efeitos dos fármacos , Técnicas de Patch-Clamp , Bibliotecas de Moléculas Pequenas , Canais de Cátion TRPV/agonistas , Canais de Cátion TRPV/antagonistas & inibidoresRESUMO
Parkinson's disease is characterized by both hypokinetic and hyperkinetic symptoms. While increased subthalamic burst discharges have a direct causal relationship with the hypokinetic manifestations (e.g., rigidity and bradykinesia), the origin of the hyperkinetic symptoms (e.g., resting tremor and propulsive gait) has remained obscure. Neuronal burst discharges are presumed to be autonomous or less responsive to synaptic input, thereby interrupting the information flow. We, however, demonstrate that subthalamic burst discharges are dependent on cortical glutamatergic synaptic input, which is enhanced by A-type K+ channel inhibition. Excessive top-down-triggered subthalamic burst discharges then drive highly correlative activities bottom-up in the motor cortices and skeletal muscles. This leads to hyperkinetic behaviors such as tremors, which are effectively ameliorated by inhibition of cortico-subthalamic AMPAergic synaptic transmission. We conclude that subthalamic burst discharges play an imperative role in cortico-subcortical information relay, and they critically contribute to the pathogenesis of both hypokinetic and hyperkinetic parkinsonian symptoms.
Assuntos
Globo Pálido/fisiopatologia , Hipercinese/fisiopatologia , Córtex Motor/fisiopatologia , Doença de Parkinson Secundária/fisiopatologia , Núcleo Subtalâmico/fisiopatologia , Tremor/fisiopatologia , 4-Aminopiridina/farmacologia , 6-Ciano-7-nitroquinoxalina-2,3-diona/farmacologia , Animais , Agonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Feminino , Globo Pálido/efeitos dos fármacos , Globo Pálido/metabolismo , Ácido Glutâmico/metabolismo , Ácido Glutâmico/farmacologia , Humanos , Hipercinese/metabolismo , Masculino , Potenciais da Membrana/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Córtex Motor/efeitos dos fármacos , Córtex Motor/metabolismo , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiopatologia , Optogenética/métodos , Doença de Parkinson Secundária/metabolismo , Ratos , Ratos Wistar , Núcleo Subtalâmico/efeitos dos fármacos , Núcleo Subtalâmico/metabolismo , Sinapses/efeitos dos fármacos , Sinapses/metabolismo , Sinapses/patologia , Transmissão Sináptica , Tremor/metabolismo , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/farmacologiaRESUMO
ROS are known to be accumulated in stigmas of different species and can possibly perform different functions important for plant reproduction. Here we tested the assumption that one of their functions is to control membrane potential and provoke synthesis of unique proteins in germinating pollen. We used spectrofluorometry and spectrophotometry to detect H2 O2 in stigma exudate, quantitative fluorescent microscopy of pollen tubes and flow cytometry of pollen protoplasts to reveal effects on membrane potential, and a label-free quantification approach to study pollen proteome changes after H2 O2 treatment. We found that in both growing pollen tubes and pollen protoplasts exudate causes plasmalemma hyperpolarization similar to that provoked by H2 O2 . This effect is abolished by catalase treatment and the ROS quencher, MnTMPP. Inhibitory analysis indicates probable participation of Ca2+ - and K+ -conducting channels in the observed hyperpolarization. For a deeper understanding of pollen response, we analysed proteome alterations in H2 O2 -treated pollen grains. We found 50 unique proteins and 20 differently accumulated proteins that are mainly involved in cell metabolism, energetics, protein synthesis and folding. Observed hyperpolarization and proteome alterations agree well with previously reported stimulation of pollen germination by H2 O2 and sensitivity of Ca2+ - and K+ -conducting channels to this ROS. Thus, H2 O2 is one of the active substances in tobacco stigma exudate that stimulates various physiological processes in germinating pollen.
Assuntos
Nicotiana , Tubo Polínico , Exsudatos e Transudatos , Peróxido de Hidrogênio , Potenciais da Membrana , Pólen , ProteomaRESUMO
BACKGROUND/AIMS: Tea, produced from the evergreen Camellia sinensis, has reported therapeutic properties against multiple pathologies, including hypertension. Although some studies validate the health benefits of tea, few have investigated the molecular mechanisms of action. The KCNQ5 voltage-gated potassium channel contributes to vascular smooth muscle tone and neuronal M-current regulation. METHODS: We applied electrophysiology, myography, mass spectrometry and in silico docking to determine effects and their underlying molecular mechanisms of tea and its components on KCNQ channels and arterial tone. RESULTS: A 1% green tea extract (GTE) hyperpolarized cells by augmenting KCNQ5 activity >20-fold at resting potential; similar effects of black tea were inhibited by milk. In contrast, GTE had lesser effects on KCNQ2/Q3 and inhibited KCNQ1/E1. Tea polyphenols epicatechin gallate (ECG) and epigallocatechin-3-gallate (EGCG), but not epicatechin or epigallocatechin, isoform-selectively hyperpolarized KCNQ5 activation voltage dependence. In silico docking and mutagenesis revealed that activation by ECG requires KCNQ5-R212, at the voltage sensor foot. Strikingly, ECG and EGCG but not epicatechin KCNQ-dependently relaxed rat mesenteric arteries. CONCLUSION: KCNQ5 activation contributes to vasodilation by tea; ECG and EGCG are candidates for future anti-hypertensive drug development.
Assuntos
Catequina/análogos & derivados , Canais de Potássio KCNQ/química , Canal de Potássio KCNQ1/química , Artérias Mesentéricas/efeitos dos fármacos , Extratos Vegetais/farmacologia , Chá/química , Animais , Sítios de Ligação , Catequina/química , Catequina/farmacologia , Canais de Potássio KCNQ/agonistas , Canais de Potássio KCNQ/genética , Canais de Potássio KCNQ/metabolismo , Canal de Potássio KCNQ1/antagonistas & inibidores , Canal de Potássio KCNQ1/genética , Canal de Potássio KCNQ1/metabolismo , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Artérias Mesentéricas/fisiologia , Leite/química , Simulação de Acoplamento Molecular , Miografia , Oócitos/citologia , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Técnicas de Patch-Clamp , Extratos Vegetais/química , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ratos , Ratos Wistar , Técnicas de Cultura de Tecidos , Vasodilatação/efeitos dos fármacos , Vasodilatação/fisiologia , Xenopus laevisRESUMO
Lupeol is known to be plentiful in fruits or plant barks and has an antimicrobial effect, however, its mode of action(s) has yet to be determined. To elucidate lupeol generates nitric oxide (NO), which is recognized for possessing an antimicrobial activity, intracellular NO was measured in Escherichia coli using DAF-FM. Using the properties of NO passing through plasma membrane easily, increased malondialdehyde levels have shown that lupeol causes lipid peroxidation, and the resulting membrane depolarization was confirmed by DiBAC4(3). These data indicated that lupeol-induced NO is related to the destruction of bacterial membrane. Further study was performed to examine whether NO, known as a cell proliferation inhibitor, affects bacterial cell division. As a result, DAPI staining verified that lupeol promotes cell division arrest, and followed by early apoptosis is observed in Annexin V/PI double staining. Even though these apoptotic hallmarks appeared, the endonuclease failed to perform properly with supporting data of decreased intracellular Mg2+ and Ca2+ levels without DNA fragmentation, which is confirmed using a TUNEL assay. These findings indicated that lupeol-induced NO occurs DNA fragmentation-independent bacterial apoptosis-like death (ALD). Additionally, lupeol triggers DNA filamentation and morphological changes in response to DNA repair system called SOS system. In accordance with the fact that ALD deems to SOS response, and that the RecA is considered as a caspase-like protein, increase in caspase-like protein activation occurred in E. coli wild-type, and no ΔRecA mutant. In conclusion, these results demonstrated that the antibacterial mode of action(s) of lupeol is an ALD while generating NO.
Assuntos
Antibacterianos/farmacologia , Apoptose/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Óxido Nítrico/fisiologia , Triterpenos Pentacíclicos/farmacologia , Cálcio/metabolismo , Divisão Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Fragmentação do DNA , DNA Bacteriano/efeitos dos fármacos , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Avaliação Pré-Clínica de Medicamentos , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Magnésio/metabolismo , Lipídeos de Membrana/metabolismo , Potenciais da Membrana/efeitos dos fármacos , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/biossíntese , Norfloxacino/farmacologia , Recombinases Rec A/metabolismo , Resposta SOS em GenéticaRESUMO
Calcium-activated potassium (KCa) channels contribute to multiple neuronal properties including spike frequency and afterhyperpolarizing potentials (AHPs). KCa channels are classified as KCa1.1, KCa2, or KCa3.1 based on single-channel conductance and pharmacology. Ca2+-dependent AHPs in vertebrates are categorized as fast, medium, or slow. Fast and medium AHPs are generated by KCa1.1 and KCa2 channels, respectively. The KCa subtype responsible for slow AHPs is unclear. Prolonged, Ca2+-dependent AHPs have been described in several leech neurons. Unfortunately, apamin and other KCa blockers often prove ineffective in the leech. An alternative approach is to utilize KCa modulators, which alter channel sensitivity to Ca2+. Vertebrate KCa2 channels are targeted selectively by the positive modulator CyPPA and the negative modulator NS8593. Here we show that AHPs in identified motor and mechanosensory leech neurons are enhanced by CyPPA and suppressed by NS8593. Our results indicate that KCa2 channels underlie prolonged AHPs in these neurons and suggest that KCa2 modulators may serve as effective tools to explore the role of KCa channels in leech physiology.
Assuntos
Hirudo medicinalis/efeitos dos fármacos , Hirudo medicinalis/fisiologia , 1-Naftilamina/análogos & derivados , 1-Naftilamina/farmacologia , Animais , Cálcio/metabolismo , Potenciais da Membrana , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/fisiologia , Canais de Potássio Cálcio-Ativados/metabolismo , Pirazóis/farmacologia , Pirimidinas/farmacologiaRESUMO
Antimicrobial peptides (AMPs) are promising antibacterial agents often hindered by their undesired hemolytic activity. Inspired by gramicidin S (GS), a well-known cyclodecapeptide, we synthesized a panel of antibacterial cyclopeptidomimetics using ß,γ-diamino acids (ß,γ-DiAAs). We observed that peptidomimetic CP-2 displays a bactericidal activity similar to that of GS while possessing lower side-effects. Moreover, extensive studies revealed that CP-2 likely kills bacteria through membrane disruption. Altogether, CP-2 is a promising membrane-active antibiotic with therapeutic potential.
Assuntos
Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Gramicidina/farmacologia , Peptidomiméticos/farmacologia , Antibacterianos/síntese química , Antibacterianos/química , Relação Dose-Resposta a Droga , Gramicidina/síntese química , Gramicidina/química , Potenciais da Membrana/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Conformação Molecular , Peptidomiméticos/síntese química , Peptidomiméticos/química , Relação Estrutura-AtividadeRESUMO
AIMS: Previous studies have shown the intake of omega-3 polyunsaturated fatty acids is associated with low rates of obesity and ischaemic pathologies. Omega-3 also have anti-inflammatory and plaque-stabilization effects and regulate vasodilation and constriction. However, there are few studies of the role of omega-3 in flow-induced vasodilation involving Ca2+-permeable ion channel TRPV4 in high-fat diet-induced obese (DIO) mouse. Here, we determined whether omega-3 protect against vascular dysfunction induced by a high-fat diet by enhancing TRPV4 activity and subsequently improving flow-mediated vasodilation. METHODS AND RESULTS: Flow-mediated vasodilation in second-order mesenteric arteries from mice was measured using a pressure myograph. The intracellular Ca2+ concentration in response to flow and GSK1016790A (a TRPV4 agonist) was measured by Fluo-4 fluorescence. Whole-cell current was measured by patch clamp. Cell membrane tether force was measured by atomic force microscopy. Impairment of flow-mediated vasodilation in arteries and Ca2+ influx in endothelial cells from DIO mice was restored by omega-3 treatment. The improved flow-induced vasodilation was inhibited by the TRPV4 antagonist HC067047 and in TRPV4-/- mice. Omega-3 treatment enhanced endothelial TRPV4 activity and altered cell membrane mechanic property, as indicated by enhanced GSK1016790A-induced Ca2+ influx and whole-cell current and altered membrane mean tether force in endothelial cells from DIO mice. CONCLUSION: Omega-3 improve vascular function by improving flow-induced vasodilation via enhancing TRPV4 activity in the endothelium of obese mice which may be related to improved cell membrane physical property. Activation of TRPV4 in endothelium plays an important role in the protective mechanisms of omega-3 against vascular dysfunction in obesity by improving flow-mediated vasodilation.