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1.
J Cell Sci ; 135(23)2022 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-36373794

RESUMO

Mammalian (or mechanistic) target of rapamycin complex 2 (mTORC2) is a kinase complex that targets predominantly Akt family proteins, SGK1 and protein kinase C (PKC), and has well-characterized roles in mediating hormone and growth factor effects on a wide array of cellular processes. Recent evidence suggests that mTORC2 is also directly stimulated in renal tubule cells by increased extracellular K+ concentration, leading to activation of the Na+ channel, ENaC, and increasing the electrical driving force for K+ secretion. We identify here a signaling mechanism for this local effect of K+. We show that an increase in extracellular [K+] leads to a rise in intracellular chloride (Cl-), which stimulates a previously unknown scaffolding activity of the protein 'with no lysine-1' (WNK1) kinase. WNK1 interacts selectively with SGK1 and recruits it to mTORC2, resulting in enhanced SGK1 phosphorylation and SGK1-dependent activation of ENaC. This scaffolding effect of WNK1 is independent of its own kinase activity and does not cause a generalized stimulation of mTORC2 kinase activity. These findings establish a novel WNK1-dependent regulatory mechanism that harnesses mTORC2 kinase activity selectively toward SGK1 to control epithelial ion transport and electrolyte homeostasis.


Assuntos
Proteínas Imediatamente Precoces , Animais , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Cloretos/metabolismo , Canais Epiteliais de Sódio/genética , Canais Epiteliais de Sódio/metabolismo , Proteínas Serina-Treonina Quinases , Serina-Treonina Quinases TOR/metabolismo , Transporte de Íons , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/metabolismo , Mamíferos/metabolismo
2.
J Am Soc Nephrol ; 34(6): 1019-1038, 2023 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-36890646

RESUMO

SIGNIFICANCE STATEMENT: Rapid renal responses to ingested potassium are essential to prevent hyperkalemia and also play a central role in blood pressure regulation. Although local extracellular K + concentration in kidney tissue is increasingly recognized as an important regulator of K + secretion, the underlying mechanisms that are relevant in vivo remain controversial. To assess the role of the signaling kinase mTOR complex-2 (mTORC2), the authors compared the effects of K + administered by gavage in wild-type mice and knockout mice with kidney tubule-specific inactivation of mTORC2. They found that mTORC2 is rapidly activated to trigger K + secretion and maintain electrolyte homeostasis. Downstream targets of mTORC2 implicated in epithelial sodium channel regulation (SGK1 and Nedd4-2) were concomitantly phosphorylated in wild-type, but not knockout, mice. These findings offer insight into electrolyte physiologic and regulatory mechanisms. BACKGROUND: Increasing evidence implicates the signaling kinase mTOR complex-2 (mTORC2) in rapid renal responses to changes in plasma potassium concentration [K + ]. However, the underlying cellular and molecular mechanisms that are relevant in vivo for these responses remain controversial. METHODS: We used Cre-Lox-mediated knockout of rapamycin-insensitive companion of TOR (Rictor) to inactivate mTORC2 in kidney tubule cells of mice. In a series of time-course experiments in wild-type and knockout mice, we assessed urinary and blood parameters and renal expression and activity of signaling molecules and transport proteins after a K + load by gavage. RESULTS: A K + load rapidly stimulated epithelial sodium channel (ENaC) processing, plasma membrane localization, and activity in wild-type, but not in knockout, mice. Downstream targets of mTORC2 implicated in ENaC regulation (SGK1 and Nedd4-2) were concomitantly phosphorylated in wild-type, but not knockout, mice. We observed differences in urine electrolytes within 60 minutes, and plasma [K + ] was greater in knockout mice within 3 hours of gavage. Renal outer medullary potassium (ROMK) channels were not acutely stimulated in wild-type or knockout mice, nor were phosphorylation of other mTORC2 substrates (PKC and Akt). CONCLUSIONS: The mTORC2-SGK1-Nedd4-2-ENaC signaling axis is a key mediator of rapid tubule cell responses to increased plasma [K + ] in vivo . The effects of K + on this signaling module are specific, in that other downstream mTORC2 targets, such as PKC and Akt, are not acutely affected, and ROMK and Large-conductance K + (BK) channels are not activated. These findings provide new insight into the signaling network and ion transport systems that underlie renal responses to K +in vivo .


Assuntos
Proteínas Imediatamente Precoces , Potássio , Camundongos , Animais , Fosforilação , Potássio/metabolismo , Canais Epiteliais de Sódio/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Potássio na Dieta , Serina-Treonina Quinases TOR/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Imediatamente Precoces/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Rim/metabolismo , Proteínas de Transporte/metabolismo , Camundongos Knockout , Transporte de Íons
3.
Int J Mol Sci ; 22(4)2021 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-33673381

RESUMO

Tumor necrosis factor (TNF) is known to activate the epithelial Na+ channel (ENaC) in A549 cells. A549 cells are widely used model for ENaC research. The role of δ-ENaC subunit in TNF-induced activation has not been studied. In this study we hypothesized that δ-ENaC plays a major role in TNF-induced activation of ENaC channel in A549 cells which are widely used model for ENaC research. We used CRISPR/Cas 9 approach to knock down (KD) the δ-ENaC in A549 cells. Western blot and immunofluorescence assays were performed to analyze efficacy of δ-ENaC protein KD. Whole-cell patch clamp technique was used to analyze the TNF-induced activation of ENaC. Overexpression of wild type δ-ENaC in the δ-ENaC KD of A549 cells restored the TNF-induced activation of whole-cell Na+ current. Neither N-linked glycosylation sites nor carboxyl terminus domain of δ-ENaC was necessary for the TNF-induced activation of whole-cell Na+ current in δ-ENaC KD of A549 cells. Our data demonstrated that in A549 cells the δ-ENaC plays a major role in TNF-induced activation of ENaC.


Assuntos
Sistemas CRISPR-Cas , Canais Epiteliais de Sódio , Fator de Necrose Tumoral alfa/metabolismo , Células A549 , Canais Epiteliais de Sódio/genética , Canais Epiteliais de Sódio/metabolismo , Humanos , Fator de Necrose Tumoral alfa/genética
4.
Int J Mol Sci ; 22(14)2021 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-34298871

RESUMO

Autistic spectrum disorder (ASD) refers to a group of neurodevelopmental disorders characterized by impaired social interaction and cognitive deficit, restricted repetitive behaviors, altered immune responses, and imbalanced oxidative stress status. In recent years, there has been a growing interest in studying the role of nicotinic acetylcholine receptors (nAChRs), specifically α7-nAChRs, in the CNS. Influence of agonists for α7-nAChRs on the cognitive behavior, learning, and memory formation has been demonstrated in neuro-pathological condition such as ASD and attention-deficit hyperactivity disorder (ADHD). Curcumin (CUR), the active compound of the spice turmeric, has been shown to act as a positive allosteric modulator of α7-nAChRs. Here we hypothesize that CUR, acting through α7-nAChRs, influences the neuropathology of ASD. In patch clamp studies, fast inward currents activated by choline, a selective agonist of α7-nAChRs, were significantly potentiated by CUR. Moreover, choline induced enhancement of spontaneous inhibitory postsynaptic currents was markedly increased in the presence of CUR. Furthermore, CUR (25, 50, and 100 mg/kg, i.p.) ameliorated dose-dependent social deficits without affecting locomotor activity or anxiety-like behaviors of tested male Black and Tan BRachyury (BTBR) mice. In addition, CUR (50 and 100 mg/kg, i.p.) mitigated oxidative stress status by restoring the decreased levels of superoxide dismutase (SOD) and catalase (CAT) in the hippocampus and the cerebellum of treated mice. Collectively, the observed results indicate that CUR potentiates α7-nAChRs in native central nervous system neurons, mitigates disturbed oxidative stress, and alleviates ASD-like features in BTBR mice used as an idiopathic rodent model of ASD, and may represent a promising novel pharmacological strategy for ASD treatment.


Assuntos
Transtorno do Espectro Autista/tratamento farmacológico , Transtorno do Espectro Autista/metabolismo , Transtorno Autístico/tratamento farmacológico , Curcumina/farmacologia , Hipocampo/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Regulação Alostérica/efeitos dos fármacos , Animais , Transtorno Autístico/metabolismo , Colina/farmacologia , Modelos Animais de Doenças , Hipocampo/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Agonistas Nicotínicos/farmacologia , Comportamento Social
5.
Pflugers Arch ; 470(11): 1615-1631, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30088081

RESUMO

The epithelial Na channel (ENaC) plays an essential role in lung physiology by modulating the amount of liquid lining the respiratory epithelium. Here, we tested the effect of breaking alveolar epithelial cell barrier integrity on ENaC expression and function. We found that either mechanical wounding by scratching the monolayer or disruption of tight junction with EDTA induced a ~ 50% decrease of α,ß and γENaC mRNA expression and an 80% reduction of ENaC short-circuit current (Isc) at 6 h. Scratching the cell monolayer generated a Ca2+ wave that spread from the margin of the scratch to distant cells. Pretreatment with BAPTA-AM, an intracellular Ca2+ chelator, abolished the effect of mechanical wounding and EDTA on αENaC mRNA expression, suggesting that [Ca2+]i is important for this modulation. We tested the hypothesis that a mechanosensitive channel such as TRPV4, a cationic channel known to increase [Ca2+]i, could mediate this effect. Activation of the channel with the TRPV4 specific agonist GSK-1016790A (GSK) decreased αENAC mRNA expression and almost completely abolished ENaC Isc. Pretreatment of alveolar epithelial cells with HC-067047 (HC0), a specific TRPV4 antagonist, reduced the extent of αENAC mRNA downregulation by mechanical wounding and EDTA. Altogether, our results suggest that mechanical stress induced by wounding or TRPV4-mediated loss of tight junction increases [Ca2+]i and elicits a Ca2+ wave that affects ENaC expression and function away from the site of injury. These data are important to better understand how Ca2+ signaling affects lung liquid clearance in injured lungs.


Assuntos
Células Epiteliais Alveolares/metabolismo , Cálcio/metabolismo , Canais Epiteliais de Sódio/genética , Lesão Pulmonar/metabolismo , Animais , Células Cultivadas , Regulação para Baixo , Canais Epiteliais de Sódio/metabolismo , Masculino , Mecanotransdução Celular , Ratos , Ratos Sprague-Dawley , Canais de Cátion TRPV/metabolismo
6.
J Biol Chem ; 291(45): 23440-23451, 2016 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-27645999

RESUMO

Regulation of the epithelial sodium channel (ENaC), which regulates fluid homeostasis and blood pressure, is complex and remains incompletely understood. The TIP peptide, a mimic of the lectin-like domain of TNF, activates ENaC by binding to glycosylated residues in the extracellular loop of ENaC-α, as well as to a hitherto uncharacterized internal site. Molecular docking studies suggested three residues, Val567, Glu568, and Glu571, located at the interface between the second transmembrane and C-terminal domains of ENaC-α, as a critical site for binding of the TIP peptide. We generated Ala replacement mutants in this region of ENaC-α and examined its interaction with TIP peptide (3M, V567A/E568A/E571A; 2M, V567A/E568A; and 1M, E571A). 3M and 2M ENaC-α, but not 1M ENaC-α, displayed significantly reduced binding capacity to TIP peptide and to TNF. When overexpressed in H441 cells, 3M mutant ENaC-α formed functional channels with similar gating and density characteristics as the WT subunit and efficiently associated with the ß and γ subunits in the plasma membrane. We subsequently assayed for increased open probability time and membrane expression, both of which define ENaC activity, following addition of TIP peptide. TIP peptide increased open probability time in H441 cells overexpressing wild type and 1M ENaC-α channels, but not 3M or 2M ENaC-α channels. On the other hand, TIP peptide-mediated reduction in ENaC ubiquitination was similar in cells overexpressing either WT or 3M ENaC-α subunits. In summary, this study has identified a novel site in ENaC-α that is crucial for activation of the open probability of the channel, but not membrane expression, by the lectin-like domain of TNF.


Assuntos
Agonistas do Canal de Sódio Epitelial/farmacologia , Canais Epiteliais de Sódio/metabolismo , Peptídeos Cíclicos/farmacologia , Linhagem Celular Tumoral , Canais Epiteliais de Sódio/química , Canais Epiteliais de Sódio/genética , Células HEK293 , Humanos , Simulação de Acoplamento Molecular , Mutação Puntual , Domínios Proteicos/efeitos dos fármacos , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Ubiquitinação/efeitos dos fármacos
7.
J Biol Chem ; 289(43): 30144-60, 2014 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-25217642

RESUMO

Membrane attachment via a C-terminal glycosylphosphatidylinositol anchor is critical for conversion of PrP(C) into pathogenic PrP(Sc). Therefore the effects of the anchor on PrP structure and function need to be deciphered. Three PrP variants, including full-length PrP (residues 23-231, FL_PrP), N-terminally truncated PrP (residues 90-231, T_PrP), and PrP missing its central hydrophobic region (Δ105-125, ΔCR_PrP), were equipped with a C-terminal membrane anchor via a semisynthesis strategy. Analyses of the interactions of lipidated PrPs with phospholipid membranes demonstrated that C-terminal membrane attachment induces a different binding mode of PrP to membranes, distinct from that of non-lipidated PrPs, and influences the biochemical and conformational properties of PrPs. Additionally, fluorescence-based assays indicated pore formation by lipidated ΔCR_PrP, a variant that is known to be highly neurotoxic in transgenic mice. This finding was supported by using patch clamp electrophysiological measurements of cultured cells. These results provide new evidence for the role of the membrane anchor in PrP-lipid interactions, highlighting the importance of the N-terminal and the central hydrophobic domain in these interactions.


Assuntos
Glicosilfosfatidilinositóis/metabolismo , Lipídeos de Membrana/metabolismo , Príons/química , Príons/metabolismo , 4-Cloro-7-nitrobenzofurazano/metabolismo , Animais , Fenômenos Eletrofisiológicos , Endopeptidase K/metabolismo , Fluoresceínas/metabolismo , Fluorescência , Células HEK293 , Humanos , Cinética , Lipossomos/metabolismo , Lipossomos/ultraestrutura , Camundongos , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Peptídeos/metabolismo , Fosfolipídeos/metabolismo , Príons/ultraestrutura , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Triptofano/metabolismo
8.
Am J Respir Crit Care Med ; 190(5): 522-32, 2014 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-25029038

RESUMO

RATIONALE: Alveolar liquid clearance is regulated by Na(+) uptake through the apically expressed epithelial sodium channel (ENaC) and basolaterally localized Na(+)-K(+)-ATPase in type II alveolar epithelial cells. Dysfunction of these Na(+) transporters during pulmonary inflammation can contribute to pulmonary edema. OBJECTIVES: In this study, we sought to determine the precise mechanism by which the TIP peptide, mimicking the lectin-like domain of tumor necrosis factor (TNF), stimulates Na(+) uptake in a homologous cell system in the presence or absence of the bacterial toxin pneumolysin (PLY). METHODS: We used a combined biochemical, electrophysiological, and molecular biological in vitro approach and assessed the physiological relevance of the lectin-like domain of TNF in alveolar liquid clearance in vivo by generating triple-mutant TNF knock-in mice that express a mutant TNF with deficient Na(+) uptake stimulatory activity. MEASUREMENTS AND MAIN RESULTS: TIP peptide directly activates ENaC, but not the Na(+)-K(+)-ATPase, upon binding to the carboxy-terminal domain of the α subunit of the channel. In the presence of PLY, a mediator of pneumococcal-induced pulmonary edema, this binding stabilizes the ENaC-PIP2-MARCKS complex, which is necessary for the open probability conformation of the channel and preserves ENaC-α protein expression, by means of blunting the protein kinase C-α pathway. Triple-mutant TNF knock-in mice are more prone than wild-type mice to develop edema with low-dose intratracheal PLY, correlating with reduced pulmonary ENaC-α subunit expression. CONCLUSIONS: These results demonstrate a novel TNF-mediated mechanism of direct ENaC activation and indicate a physiological role for the lectin-like domain of TNF in the resolution of alveolar edema during inflammation.


Assuntos
Agonistas do Canal de Sódio Epitelial/metabolismo , Canais Epiteliais de Sódio/metabolismo , Peptídeos Cíclicos/metabolismo , Alvéolos Pulmonares/metabolismo , Edema Pulmonar/metabolismo , Estreptolisinas , Fator de Necrose Tumoral alfa/metabolismo , Animais , Proteínas de Bactérias , Agonistas do Canal de Sódio Epitelial/química , Canais Epiteliais de Sódio/química , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Peptídeos Cíclicos/química , Alvéolos Pulmonares/microbiologia , Edema Pulmonar/microbiologia , Fator de Necrose Tumoral alfa/química
9.
Mol Pharmacol ; 84(6): 899-910, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24077967

RESUMO

AP301 [Cyclo(CGQRETPEGAEAKPWYC)], a cyclic peptide comprising the human tumor necrosis factor lectin-like domain (TIP domain) sequence, is currently being developed as a treatment for lung edema and has been shown to reduce extravascular lung water and improve lung function in mouse, rat, and pig models. The current paradigm for liquid homeostasis in the adult mammalian lung is that passive apical uptake of sodium via the amiloride-sensitive epithelial Na⁺ channel (ENaC) and nonselective cyclic-nucleotide-gated cation channels creates the major driving force for reabsorption of water through the alveolar epithelium in addition to other ion channels such as potassium and chloride channels. AP301 can increase amiloride-sensitive current in A549 cells as well as in freshly isolated type II alveolar epithelial cells from different species. ENaC is expressed endogenously in all of these cell types. Consequently, this study was undertaken to determine whether ENaC is the specific target of AP301. The effect of AP301 in A549 cells as well as in human embryonic kidney cells and Chinese hamster ovary cells heterologously expressing human ENaC subunits (α, ß, γ, and δ) was measured in patch clamp experiments. The congener TIP peptide AP318 [Cyclo(4-aminobutanoic acid-GQRETPEGAEAKPWYD)] activated ENaC by increasing single-channel open probability. AP301 increased current in proteolytically activated (cleaved) but not near-silent (uncleaved) ENaC in a reversible manner. αßγ- or δßγ-ENaC coexpression was required for maximal activity. No increase in current was observed after deglycosylation of extracellular domains of ENaC. Thus, our data suggest that the specific interaction of AP301 with both endogenously and heterologously expressed ENaC requires precedent binding to glycosylated extracellular loop(s).


Assuntos
Canais Epiteliais de Sódio/metabolismo , Peptídeos Cíclicos/farmacologia , Edema Pulmonar/tratamento farmacológico , Animais , Células CHO , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Glicosilação , Células HEK293 , Humanos , Técnicas de Patch-Clamp , Subunidades Proteicas/metabolismo
10.
Pulm Pharmacol Ther ; 26(3): 356-63, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23313096

RESUMO

Pulmonary permeability oedema is a frequent complication in a number of life-threatening lung conditions, such as ALI and ARDS. Apart from ventilation strategies, no specific therapy yet exists for treatment of these potentially fatal illnesses. The oedema-reducing capacity of the lectin-like domain of TNF (TIP) and of synthetic peptides, mTIP and hTIP, which mimic the TIP domain of mouse and human TNF, have been demonstrated in various studies in rodents. Cell-based electrophysiological studies have revealed that the alveolar fluid clearing capacity of TNF and the TIP peptides is due to activation of the amiloride-sensitive Na(+) current in alveolar epithelial cells and that the primary site of action is on the apical side of these cells. AP301, a synthetic cyclic peptide mimicking the TIP domain of human TNF is currently undergoing clinical trials as a therapy for pulmonary permeability oedema. AP301 has been shown to improve alveolar liquid clearance and lung function in a porcine model of ALI. For non-clinical regulatory assessment, dog, pig and rat are standard animal models; accordingly, pre-clinical toxicological and pharmacological safety studies have been conducted with AP301 in dogs and rats. Hitherto, no studies have assessed the pharmacodynamic effect of AP301 on primary canine or porcine type II AEC. The current study describes the effect of AP301 on the amiloride-sensitive Na(+) current in type II AEC isolated from dog, pig and rat lungs. In whole cell patch clamp experiments with dog type II AEC, an increase in the amiloride-sensitive Na(+) current from 3.7 pA to 49.4 pA was observed in the presence of AP301; in pig type II AEC, an increase from 10.0 pA to 159.6 pA was observed, and in rat AEC, from 6.9 pA to 62.4 pA. In whole cell patch clamp experiments in A549 cells, AP301-induced enhancement of the amiloride-sensitive current was eliminated when Na(+) in the bath solution was replaced with N-methyl-d-glucamine (NMDG), and when the cells were pre-incubated with 5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside (AICAR), an inhibitor of ENaC, but enhancement was unaffected by addition of cyclic nucleotide-gated (CNG) channel inhibitors Zn(2+) or l-cis-diltiazem prior to AP301. These results provide strong evidence that AP301 activates the amiloride-sensitive Na(+) current through ENaC in type II AEC from dog, pig and rat. To our knowledge, this is the first cell-based analysis of the oedema-clearing effect of AP301 observed in the porcine model of pulmonary oedema. Furthermore, the results validate the dog and pig models in non-clinical assessment of AP301.


Assuntos
Canais Epiteliais de Sódio/metabolismo , Peptídeos Cíclicos/farmacologia , Alvéolos Pulmonares/metabolismo , Amilorida/farmacologia , Animais , Linhagem Celular Tumoral , Diltiazem/farmacologia , Cães , Relação Dose-Resposta a Droga , Lectinas/farmacologia , Peptídeos Cíclicos/metabolismo , Ratos , Suínos , Fator de Necrose Tumoral alfa/farmacologia , Zinco/farmacologia
11.
Curr Mol Pharmacol ; 16(3): 411-418, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-35894469

RESUMO

BACKGROUND: The activity of the amiloride-sensitive epithelial sodium channel (ENaC) in the tight epithelia of the lung is regulated by proteolytic activation and ubiquitination. Pathophysiology of lung diseases is directly related to changes in one or both of these mechanisms. METHODS: In this study, we investigated the impact of ubiquitination and cathepsin-mediated proteolytic activation mechanisms on the functional regulation of ENaC in lung cancer A549 cells using the patch-clamp technique. RESULTS: Our findings suggest that inhibiting the proteasome (polyubiquitination) with MG132 improves ENaC activity, whereas altering the pH of the lysosome (monoubiquitination inhibition) with NH4Cl has no effect on ENaC activity. In A549 cells, inhibition of cathepsin B (CSTB) decreased the ENaC current, open probabilities (NPo and Po), and the number of active channels. CONCLUSION: These findings delineate novel modes of ENaC degradation and proteolytic activation of functional channels in A549 cells. Our findings indicate that both proteolytic activation and ubiquitination of ENaC significantly affect channel function and add new insights into the endogenous ENaC processing which might help to further understand the pathophysiology of the lung disease.


Assuntos
Canais Epiteliais de Sódio , Ubiquitina-Proteína Ligases , Humanos , Canais Epiteliais de Sódio/genética , Canais Epiteliais de Sódio/metabolismo , Células A549 , Ubiquitinação , Transdução de Sinais
12.
Front Pharmacol ; 13: 821758, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35185573

RESUMO

Cannabidiol (CBD), a major non-psychotropic cannabinoid found in the Cannabis plant, has been shown to exert anti-nociceptive, anti-psychotic, and anti-convulsant effects and to also influence the cardiovascular system. In this study, the effects of CBD on major ion currents were investigated using the patch-clamp technique in rabbit ventricular myocytes. CBD inhibited voltage-gated Na+ and Ca2+ channels with IC50 values of 5.4 and 4.8 µM, respectively. In addition, CBD, at lower concentrations, suppressed ion currents mediated by rapidly and slowly activated delayed rectifier K+ channels with IC50 of 2.4 and 2.1 µM, respectively. CBD, up to 10 µM, did not have any significant effect on inward rectifier I K1 and transient outward I to currents. The effects of CBD on these currents developed gradually, reaching steady-state levels within 5-8 min, and recoveries were usually slow and partial. Hill coefficients higher than unity in concentration-inhibition curves suggested multiple CBD binding sites on these channels. These findings indicate that CBD affects cardiac electrophysiology by acting on a diverse range of ion channels and suggest that caution should be exercised when CBD is administered to carriers of cardiac channelopathies or to individuals using drugs known to affect the rhythm or the contractility of the heart.

13.
Pharmaceuticals (Basel) ; 15(10)2022 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-36297299

RESUMO

Capsaicin is a naturally occurring alkaloid derived from chili pepper which is responsible for its hot, pungent taste. It exerts multiple pharmacological actions, including pain-relieving, anti-cancer, anti-inflammatory, anti-obesity, and antioxidant effects. Previous studies have shown that capsaicin significantly affects the contractility and automaticity of the heart and alters cardiovascular functions. In this study, the effects of capsaicin were investigated on voltage-gated ion currents in rabbit ventricular myocytes. Capsaicin inhibited rapidly activated (IKr) and slowly activated (IKs) K+ currents and transient outward (Ito) K+ current with IC50 values of 3.4 µM,14.7 µM, and 9.6 µM, respectively. In addition, capsaicin, at higher concentrations, suppressed voltage-gated Na+ and Ca2+ currents and inward rectifier IK1 current with IC50 values of 42.7 µM, 34.9 µM, and 38.8 µM, respectively. Capsaicin inhibitions of INa, IL-Ca, IKr, IKs, Ito, and IK1 were not reversed in the presence of capsazepine (3 µM), a TRPV1 antagonist. The inhibitory effects of capsaicin on these currents developed gradually, reaching steady-state levels within 3 to 6 min, and the recoveries were usually incomplete during washout. In concentration-inhibition curves, apparent Hill coefficients higher than unity suggested multiple interaction sites of capsaicin on these channels. Collectively, these findings indicate that capsaicin affects cardiac electrophysiology by acting on a diverse range of ion channels and suggest that caution should be exercised when capsaicin is administered to carriers of cardiac channelopathies or to individuals with arrhythmia-prone conditions, such as ischemic heart diseases.

14.
Pflugers Arch ; 461(1): 53-63, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20924598

RESUMO

Single point mutations in pore-forming S6 segments of calcium channels may transform a high-voltage-activated into a low-voltage-activated channel, and resulting disturbances in calcium entry may cause channelopathies (Hemara-Wahanui et al., Proc Natl Acad Sci U S A 102(21):7553-7558, 16). Here we ask the question how physicochemical properties of amino acid residues in gating-sensitive positions on S6 segments determine the threshold of channel activation of Ca(V)1.2. Leucine in segment IS6 (L434) and a newly identified activation determinant in segment IIIS6 (G1193) were mutated to a variety of amino acids. The induced leftward shifts of the activation curves and decelerated current activation and deactivation suggest a destabilization of the closed and a stabilisation of the open channel state by most mutations. A selection of 17 physicochemical parameters (descriptors) was calculated for these residues and examined for correlation with the shifts of the midpoints of the activation curve (ΔV (act)). ΔV (act) correlated with local side-chain flexibility in position L434 (IS6), with the polar accessible surface area of the side chain in position G1193 (IIIS6) and with hydrophobicity in position I781 (IIS6). Combined descriptor analysis for positions I781 and G1193 revealed that additional amino acid properties may contribute to conformational changes during the gating process. The identified physicochemical properties in the analysed gating-sensitive positions (accessible surface area, side-chain flexibility, and hydrophobicity) predict the shifts of the activation curves of Ca(V)1.2.


Assuntos
Canais de Cálcio Tipo L/fisiologia , Ativação do Canal Iônico/fisiologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Canais de Cálcio Tipo L/genética , Células Cultivadas , Análise Mutacional de DNA , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ativação do Canal Iônico/genética , Termodinâmica
15.
Breast Cancer Res Treat ; 129(2): 387-400, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21046231

RESUMO

Resistance against first and second generation (irreversible) ErbB inhibitors is an unsolved problem in clinical oncology. The purpose of this study was to examine the effects of the irreversible ErbB inhibitors pelitinib and canertinib on growth of breast and ovarian cancer cells. Although in vitro growth-inhibitory effects of both drugs exceeded by far the effects of all reversible ErbB blockers tested (lapatinib, erlotinib, and gefitinib), complete growth inhibition was usually not reached. To define the mechanism of resistance, we examined downstream signaling pathways in drug-exposed cells by Western blot analysis. Although ErbB phosphorylation was reduced by pelitinib and canertinib, activation of the AKT/mTOR pathway remained essentially unaltered in drug-resistant cells. Correspondingly, transfection of tumor cells with constitutively activated AKT was found to promote resistance against all ErbB inhibitors tested, whereas dominant negative AKT reinstalled sensitivity in drug-resistant cells. In a next step, we applied PI3K/AKT/mTOR blockers including the dual PI3K/mTOR kinase inhibitor NVP-BEZ235. These agents were found to cooperate with pelitinib and canertinib in producing in vitro growth inhibition in cancer cells resistant against ErbB-targeting drugs. In conclusion, our data show that ErbB drug-refractory activation of the PI3K/AKT/mTOR pathway plays a crucial role in resistance against classical and second-generation irreversible ErbB inhibitors, and NVP-BEZ235 can override this form of resistance against pelitinib and canertinib.


Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/enzimologia , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/antagonistas & inibidores , Imidazóis/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Quinolinas/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Aminoquinolinas/farmacologia , Compostos de Anilina/farmacologia , Western Blotting , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ativação Enzimática , Receptores ErbB/metabolismo , Feminino , Humanos , Terapia de Alvo Molecular , Morfolinas/farmacologia , Neoplasias Ovarianas/enzimologia , Neoplasias Ovarianas/patologia , Fosfatidilinositol 3-Quinase/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Fatores de Tempo , Transfecção
16.
Eur J Pharmacol ; 895: 173889, 2021 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-33482177

RESUMO

Careful analysis of previously published reports and some new insights into the structure activity studies revealed an important role of Threonine 1143 in drug binding. Substituting T1143 by alanine and other residues significantly reduced channel inhibition by qDil and Dil. Mutation T1143A did not affect channel activation or inactivation while almost completely diminishing channel block by Dil or qDil. These findings support the view that T1143 serves as drug binding determinant. Other mutations in this position than T1143A (T1143L/Y/S/N/C/V/E) diminished channel inhibition by qDil but additionally affected channel activation and inactivation and may therefore affect channel block allosterically. Collectively, our data suggest that T1143 is an essential diltiazem binding determinant.


Assuntos
Bloqueadores dos Canais de Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/efeitos dos fármacos , Diltiazem/farmacologia , Sequência de Aminoácidos , Sítios de Ligação , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo L/metabolismo , Diltiazem/metabolismo , Células HEK293 , Humanos , Ligação de Hidrogênio , Cinética , Potenciais da Membrana , Mutação Puntual , Ligação Proteica , Relação Estrutura-Atividade , Treonina
17.
Eur J Pharmacol ; 901: 174090, 2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-33831414

RESUMO

The mineralocorticoid hormone aldosterone stimulates sodium reabsorption in the collecting ducts by increasing the activity of the epithelial sodium channel (ENaC). Being a rate-liming channel the loss of function mutations caused Pseudohypoaldosteronism 1 (PHA1). Despite elevated plasma aldosterone in PHA 1 patients the modulation of PHA 1 causing ENaC mutants with hormone has never been studied. After recording control ENaC current in PHA1 causing ENaC stop codon mutants we demonstrated the activation of aldosterone in the whole cell as well as single channel patch clamp assays. Single channel recoding experiments demonstrated that aldosterone can increase the open probability of all analyzed PHA 1 stop codon mutants and WT. Additionally, we demonstrated by western blot experiments that aldosterone can increase the expression of WT and PHA 1 stop codon mutants. Extensive whole cell patch clamp experiments demonstrated that C-terminal γ ENaC domain is necessary for aldosterone to activate whole cell current in HEK-293 cells. This novel finding of γ ENaC C-terminus dependent activation of whole cell current by aldosterone could alter our understanding of ENaC-mediated sodium reabsorption in the aldosterone-sensitive distal nephron (ASDN).


Assuntos
Aldosterona/farmacologia , Canais Epiteliais de Sódio/efeitos dos fármacos , Pseudo-Hipoaldosteronismo/genética , Pseudo-Hipoaldosteronismo/metabolismo , Agonistas de Canais de Sódio/farmacologia , Códon de Terminação/efeitos dos fármacos , Células HEK293 , Humanos , Túbulos Renais Distais/efeitos dos fármacos , Mutação , Néfrons/efeitos dos fármacos , Técnicas de Patch-Clamp
18.
J Interv Card Electrophysiol ; 60(3): 387-394, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32328860

RESUMO

PURPOSE: Cellular changes occurring in diabetic cardiomyopathy include disturbances of calcium and sodium homeostasis. Voltage-gated sodium channels are responsible for the initiation of cardiac action potentials, and the excitability would create relevance. The effect of ranolazine as a sodium channel blocker on atrium electromechanical parameters is investigated and compared with lidocaine in streptozocin-treated diabetic rats. METHODS: After an 8-week induction of diabetes type I, the effect of cumulative concentrations of ranolazine and lidocaine on the electrophysiology of isolated atrium was studied. Ranolazine's effects were evaluated on cardiac sodium current in normal- and high-glucose medium, with whole-cell patch-clamp technique. RESULTS: Ranolazine at therapeutic concentrations had no significant statistical effect on refractory period in normal and diabetic isolated heart. Ranolazine (10 µM) caused a hyperpolarizing shift of V1/2 for steady-state inactivation in normal media, while it significantly elicited a depolarizing shift in high-glucose media (p < 0.05). CONCLUSION: It is concluded that in the isolated rat atrium preparation, ranolazine and lidocaine have no beneficial on diabetic cardiomyopathy. Although refractoriness and contractility were not much different in normal and diabetic atria, there was a definite effect of ranolazine and lidocaine on sodium current in varying concentrations. This may have significance in future therapeutics.


Assuntos
Diabetes Mellitus Experimental , Lidocaína , Acetanilidas/farmacologia , Potenciais de Ação , Animais , Diabetes Mellitus Experimental/tratamento farmacológico , Lidocaína/farmacologia , Piperazinas/farmacologia , Ranolazina/farmacologia , Ratos , Bloqueadores dos Canais de Sódio/farmacologia
19.
Cells ; 10(5)2021 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-34062982

RESUMO

Phytochemicals, such as monoterpenes, polyphenols, curcuminoids, and flavonoids, are known to have anti-inflammatory, antioxidant, neuroprotective, and procognitive effects. In this study, the effects of several polyhydroxy flavonoids, as derivatives of differently substituted 5,7-dihydroxy-4H-chromen-4-one including apigenin, genistein, luteolin, kaempferol, quercetin, gossypetin, and phloretin with different lipophilicities (cLogP), as well as topological polar surface area (TPSA), were tested for induction of Ca2+ transients by α7 human nicotinic acetylcholine (α7 nACh) receptors expressed in SH-EP1 cells. Apigenin (10 µM) caused a significant potentiation of ACh (30 µM)-induced Ca2+ transients, but did not affect Ca2+ transients induced by high K+ (60 mM) containing solutions. Co-application of apigenin with ACh was equally effective as apigenin preincubation. However, the effect of apigenin significantly diminished by increasing ACh concentrations. The flavonoids tested also potentiated α7 nACh mediated Ca2+ transients with descending potency (highest to lowest) by genistein, gossypetin, kaempferol, luteolin, phloretin, quercetin, and apigenin. The specific binding of α7 nACh receptor antagonist [125I]-bungarotoxin remained unchanged in the presence of any of the tested polyhydroxy flavonoids, suggesting that these compounds act as positive allosteric modulators of the α7-nACh receptor in SH-EP1 cells. These findings suggest a clinical potential for these phytochemicals in the treatment of various human diseases from pain to inflammation and neural disease.


Assuntos
Apigenina/farmacologia , Compostos Fitoquímicos/farmacologia , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Regulação Alostérica , Apigenina/química , Sinalização do Cálcio/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Compostos Fitoquímicos/química , Potássio/metabolismo , Ligação Proteica , Receptor Nicotínico de Acetilcolina alfa7/efeitos dos fármacos
20.
Respir Physiol Neurobiol ; 259: 104-110, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30171906

RESUMO

While alveolar liquid clearance (ALC) mediated by the ß2-adrenergic receptor (ß2-AR) plays an important role in lung edema resolution in certain models of lung injury, in more severe lung injury models, this response might disappear. Indeed, we have shown that in an ischemia-reperfusion-induced lung injury model, ß2-agonists do not enhance ALC. The objective of this study was to determine if downregulation of the ß2-AR could explain the lack of response to ß2-agonists in this lung injury model. In an in vivo canine model of lung transplantation, we observed no change in ß2-AR concentration or affinity in the injured transplanted lungs compared to the native lungs. Furthermore, we could not enhance ALC in transplanted lungs with dcAMP + aminophylline, a treatment that bypasses the ß2-adrenergic receptor and is known to stimulate ALC in normal lungs. However, transplantation decreased αENaC expression in the lungs by 50%. We conclude that the lack of response to ß2-agonists in ischemia-reperfusion-induced lung injury is not associated with significant downregulation of the ß2-adrenergic receptors but is attributable to decreased expression of the ENaC channel, which is essential for sodium transport and alveolar liquid clearance in the lung.


Assuntos
Lesão Pulmonar , Alvéolos Pulmonares/fisiopatologia , Receptores Adrenérgicos beta 2/uso terapêutico , Traumatismo por Reperfusão/complicações , Aminofilina/farmacologia , Animais , Pressão Sanguínea/efeitos dos fármacos , Broncodilatadores/farmacologia , AMP Cíclico/farmacologia , Modelos Animais de Doenças , Cães , Relação Dose-Resposta a Droga , Canais Epiteliais de Sódio/metabolismo , Feminino , Frequência Cardíaca/efeitos dos fármacos , Imidazóis/uso terapêutico , Radioisótopos do Iodo/farmacocinética , Iodocianopindolol/farmacocinética , Lesão Pulmonar/etiologia , Lesão Pulmonar/fisiopatologia , Masculino , Propanolaminas/uso terapêutico , Ligação Proteica/efeitos dos fármacos , Edema Pulmonar/etiologia , RNA Mensageiro
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