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1.
PLoS One ; 17(5): e0266890, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35503765

RESUMO

Sodium-hydrogen exchangers (NHEs) tightly regulate intracellular pH (pHi), proliferation, migration and cell volume. Heterogeneity exists between pulmonary endothelial cells derived from different vascular segments, yet the activity and isoform expression of NHEs between these vascular segments has not been fully examined. Utilizing the ammonium-prepulse and recovery from acidification technique in a buffer lacking bicarbonate, pulmonary microvascular and pulmonary artery endothelial cells exhibited unique recovery rates from the acid load dependent upon the concentration of the sodium transport inhibitor, amiloride; further, pulmonary artery endothelial cells required a higher dose of amiloride to inhibit sodium-dependent acid recovery compared to pulmonary microvascular endothelial cells, suggesting a unique complement of NHEs between the different endothelial cell types. While NHE1 has been described in pulmonary endothelial cells, all NHE isoforms have not been accounted for. To address NHE expression in endothelial cells, qPCR was performed. Using a two-gene normalization approach, Sdha and Ywhag were identified for qPCR normalization and analysis of NHE isoforms between pulmonary microvascular and pulmonary artery endothelial cells. NHE1 and NHE8 mRNA were equally expressed between the two cell types, but NHE5 expression was significantly higher in pulmonary microvascular versus pulmonary artery endothelial cells, which was confirmed at the protein level. Thus, pulmonary microvascular and pulmonary artery endothelial cells exhibit unique NHE isoform expression and have a unique response to acid load revealed through recovery from cellular acidification.


Assuntos
Amilorida , Células Endoteliais , Ácidos/metabolismo , Amilorida/farmacologia , Células Endoteliais/metabolismo , Concentração de Íons de Hidrogênio , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Trocador 1 de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/metabolismo
2.
Physiol Rep ; 10(7): e15255, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35384364

RESUMO

The maintenance of endothelial health is required for normal vascular function and blood pressure regulation. The epithelial Na+ channel (ENaC) in endothelial cells has emerged as a new molecular player in the regulation of endothelial nitric oxide production and vascular stiffness. While ENaC expression in the kidney is negatively regulated by high [Na+ ], ENaC expression in isolated endothelial cells has been shown to increase in response to a high extracellular [Na+ ]. In culture, this increased expression leads to cellular stiffening and decreased nitric oxide release. In vivo, the effects of high salt diet on endothelial ENaC expression and activity have varied depending on the animal model utilized. Our aim in the present study was to examine the role of endothelial ENaC in mediating vasorelaxation in the C57Bl/6 mouse strain. We utilized pressure myography to test the responsiveness of thoracodorsal arteries to acetylcholine in mice with increased sodium consumption both in the presence and absence of increased aldosterone. ENaC's contribution was assessed with the use of the specific inhibitor amiloride. We found that while aldosterone had very little effect on ENaC's contribution to acetylcholine sensitivity, a high salt diet led to an amiloride-dependent shift in the acetylcholine response of vessels. However, the direction of this shift was dependent on the length of high salt diet administration. Overall, our studies reveal that ENaC's role in the endothelium may be more complicated than previously thought. The channel does not simply inhibit nitric oxide generation, but instead helps preserve a homeostatic response.


Assuntos
Amilorida , Vasodilatação , Acetilcolina/farmacologia , Amilorida/farmacologia , Animais , Dieta , Células Endoteliais/metabolismo , Canais Epiteliais de Sódio/metabolismo , Camundongos
3.
Acta Physiol (Oxf) ; 235(1): e13811, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35276025

RESUMO

Proteases are fundamental for a plethora of biological processes, including signalling and tissue remodelling, and dysregulated proteolytic activity can result in pathogenesis. In this review, we focus on a subclass of membrane-bound and soluble proteases that are defined as channel-activating proteases (CAPs), since they induce Na+ ion transport through an autocrine mechanism when co-expressed with the highly amiloride-sensitive epithelial sodium channel (ENaC) in Xenopus oocytes. These experiments first identified CAP1 (channel-activating protease 1, prostasin) followed by CAP2 (channel-activating protease 2, TMPRSS4) and CAP3 (channel-activating protease 3, matriptase) as in vitro mediators of ENaC current. Since then, more serine-, cysteine- and metalloproteases were confirmed as in vitro CAPs that potentially cleave and regulate ENaC, and thus this nomenclature was not further followed, but is accepted as functional term or alias. The precise mechanism of ENaC modulation by proteases has not been fully elucidated. Studies in organ-specific protease knockout models revealed evidence for their role in increasing ENaC activity, although the proteases responsible for ENaC activation are yet to be identified. We summarize recent findings in animal models of these CAPs with respect to their implication in ENaC activation. We discuss the consequences of dysregulated CAPs underlying epithelial phenotypes in pathophysiological conditions, and the role of selected protease inhibitors. We believe that these proteases may present interesting therapeutic targets for diseases with aberrant sodium homoeostasis.


Assuntos
Canais Epiteliais de Sódio , Peptídeo Hidrolases , Amilorida/farmacologia , Animais , Canais Epiteliais de Sódio/metabolismo , Transporte de Íons , Sódio/metabolismo
4.
Int J Mol Sci ; 23(4)2022 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-35216079

RESUMO

As inhabitants of soda lakes, Thioalkalivibrio versutus are halo- and alkaliphilic bacteria that have previously been shown to respire with the first demonstrated Na+-translocating cytochrome-c oxidase (CO). The enzyme generates a sodium-motive force (Δs) as high as -270 mV across the bacterial plasma membrane. However, in these bacteria, operation of the possible Δs consumers has not been proven. We obtained motile cells and used them to study the supposed Na+ energetic cycle in these bacteria. The resulting motility was activated in the presence of the protonophore 2-heptyl-4-hydroxyquinoline N-oxide (HQNO), in line with the same effect on cell respiration, and was fully blocked by amiloride-an inhibitor of Na+-motive flagella. In immotile starving bacteria, ascorbate triggered CO-mediated respiration and motility, both showing the same dependence on sodium concentration. We concluded that, in T. versutus, Na+-translocating CO and Na+-motive flagella operate in the Na+ energetic cycle mode. Our research may shed light on the energetic reason for how these bacteria are confined to a narrow chemocline zone and thrive in the extreme conditions of soda lakes.


Assuntos
Ectothiorhodospiraceae/metabolismo , Sódio/metabolismo , Amilorida/metabolismo , Membrana Celular/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Flagelos/metabolismo , Lagos/microbiologia
5.
Biochem Biophys Res Commun ; 592: 113-118, 2022 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-35042121

RESUMO

Human multidrug and toxin extrusion 1 (MATE1; SLC47A1) is highly expressed in the kidneys and the liver. It plays a significant role in drug and endogenous compound disposition, and therefore, a rapid evaluation of its inhibition is important for drug development and for the understanding of renal and hepatic physiology. Amiloride is a potassium-sparing diuretic used for treating hypertension; it also demonstrates strong fluorescence in organic solvent or detergent solutions. In this study, we investigated the transport characteristics of amiloride by human MATE1. Cellular accumulation of amiloride was evaluated in control vector- or MATE1-transfected HEK293 cells. Cells were lysed with 1% sodium dodecyl sulfate, and fluorescence was measured using a microplate reader at wavelengths of 364ex and 409em. With ammonium prepulse-induced intracellular acidification, MATE1 transported amiloride at an extracellular pH of 7.4. The uptake demonstrated an overshoot phenomenon and saturated, with the Km and Vmax being 23.5 µM and 1.01 nmol/mg/min, respectively. MATE1-mediated amiloride transport also presented with a bell-shaped pH profile that reached a maximum pH value of 7.4. The inhibitor sensitivity of MATE1-facilitated amiloride transport was similar to those of known substrates, such as tetraethylammonium and metformin. Among the tested inhibitors, pyrimethamine demonstrated the most potent inhibition with an IC50 value of 0.266 µM. Furthermore, MATE1 was found to be inhibited by fampridine, which was previously considered to be a non-inhibitor of MATE1. This study demonstrates that amiloride is a suitable fluorescent substrate for the in vitro study of the transport activity of MATE1.


Assuntos
Amilorida/metabolismo , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Preparações Farmacêuticas/metabolismo , Transporte Biológico , Células HEK293 , Humanos , Concentração Inibidora 50 , Prótons , Espectrometria de Fluorescência
6.
BMJ Case Rep ; 15(1)2022 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-34980640

RESUMO

Pseudohypoaldosteronism type 1B is a rare autosomal recessive disorder caused by dysfunction of amiloride-sensitive epithelial sodium channels (ENaCs). We present the case of a neonate with cardiogenic shock after cardiac arrest due to profound hyperkalaemia. Genetic testing revealed a novel homozygous variant in SCNNIA We review diagnostic considerations including the molecular mechanisms of disease, discuss treatment approaches and highlight the possible significance of the diversity of pulmonary ENaCs.


Assuntos
Hiperpotassemia , Pseudo-Hipoaldosteronismo , Amilorida , Canais Epiteliais de Sódio/genética , Homozigoto , Humanos , Hiperpotassemia/diagnóstico , Hiperpotassemia/etiologia , Recém-Nascido , Pseudo-Hipoaldosteronismo/complicações , Pseudo-Hipoaldosteronismo/diagnóstico , Pseudo-Hipoaldosteronismo/genética
7.
Blood Press Monit ; 27(3): 208-211, 2022 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-35044984

RESUMO

A genetic defect of 11 ß-hydroxysteroid dehydrogenase causes apparent mineralocorticoid excess syndrome. Since 50 days of life, our patient was hospitalized several times for various reasons including hypokalemia. At the age of 3.3 years, she was diagnosed with severe hypertension (160/120 mmHg). She also had left ventricular hypertrophy and hypertensive retinopathy and referred to our center. Her renal function and electrolytes were normal except for hypokalemia. She was on captopril treatment; nifedipine and propranolol were added. Plasma renin and aldosterone concentrations were 1.13 pg/ml (1-8.2 pg/ml) and 12.2 ng/dl (35-300 ng/dl), respectively. Severe hypertension, hypokalemia, low renin and aldosterone levels pointed to the diagnosis of apparent mineralocorticoid excess syndrome. Strict salt-restricted diet and potassium citrate were ordered. Genetic analysis of the HSD11B2 gene showed c.623G>A (p.Arg208His). Spironolactone was initiated. On follow-up, amiloride was added and her blood pressure was controlled. In patients with severe HSD11B2 mutation, combination therapy of spironolactone with amiloride could be effective in controlling blood pressure.


Assuntos
Hipertensão , Hipopotassemia , Síndrome de Excesso Aparente de Minerolocorticoides , Aldosterona , Amilorida , Pressão Sanguínea , Pré-Escolar , Feminino , Humanos , Hipertensão/tratamento farmacológico , Hipertensão/etiologia , Hipopotassemia/complicações , Hipopotassemia/etiologia , Síndrome de Excesso Aparente de Minerolocorticoides/complicações , Síndrome de Excesso Aparente de Minerolocorticoides/diagnóstico , Síndrome de Excesso Aparente de Minerolocorticoides/genética , Renina , Espironolactona/uso terapêutico
8.
Neurotoxicology ; 88: 1-13, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34656704

RESUMO

Cognitive impairment or "chemobrain" is a troublesome adverse effect which had been increasingly reported by cancer patients after doxorubicin (DOX) chemotherapy. Notably, Hypertension, a very common comorbidity in cancer patients, could pose a greater risk for negative cognitive outcomes. Amiloride (AML) is an antihypertensive, potassium-sparing diuretic that has been proven to be neuroprotective in different experimental models; this can be attributed to its ability to inhibit different ion transporters such as Na+/H+ exchanger (NHE), which upon excessive activation can result in intracellular cationic overload, followed by oxidative damage and cellular death. Accordingly, this study was designed to investigate the potential neuroprotective effect of AML against DOX-induced chemobrain and to elucidate possible underlying mechanisms. Briefly, Histopathological examination and neurobehavioral testing (Morris water maze, Y maze and passive avoidance test) showed that AML co-treatment (10 mg/kg/day) markedly attenuated DOX (2 mg/kg/week)-induced neurodegeneration and memory impairment after 4 weeks of treatments. We found that DOX administration up-regulated NHE expression and increased lactic acid content in the hippocampus which were markedly opposed by AML. Moreover, AML mitigated DOX-induced neuroinflammation and decreased hippocampal tumor necrosis factor-α level, nuclear factor kappa-B, and cyclooxygenase-2 expression. Additionally, AML counteracted DOX-induced hippocampal oxidative stress as indicated by normalized malondialdehyde and glutathione levels. Furthermore, AML halted DOX-induced hippocampal apoptosis as evidenced by decreased caspase-3 activity and lower cytochrome c immunoexpression. Our results in addition to the previously reported antitumor effects of AML and its ability to mitigate cancer resistance to DOX therapy could point toward possible new repositioning scenarios of the diuretic AML especially regarding hypertensive cancer patients.


Assuntos
Amilorida/farmacologia , Comprometimento Cognitivo Relacionado à Quimioterapia/tratamento farmacológico , Diuréticos/farmacologia , Doxorrubicina/toxicidade , Animais , Comprometimento Cognitivo Relacionado à Quimioterapia/etiologia , Doxorrubicina/antagonistas & inibidores , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Masculino , /tratamento farmacológico , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Wistar , Trocadores de Sódio-Hidrogênio/metabolismo
9.
J AOAC Int ; 105(2): 623-629, 2022 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-34519824

RESUMO

BACKGROUND: Determination of a multi-component mixture by HPLC requires many preliminary runs for method development which is both time-consuming and expensive due to the usage of large solvent volumes. OBJECTIVE: In the present study, the main objective was to reduce the preliminary runs that are required for optimizing the method conditions and also shorten the run time of analysis to be suitable for quality control laboratories where there are a large number of samples to be analyzed. METHODS: That was achieved using a two-factor, three-level response surface experiment which is a multivariate design that predicts the significant factors for optimizing the studied responses. RESULTS: The response surface design suggests that both acetonitrile ratio and flow rate are significant factors for full resolution of the studied mixture: atenolol, amiloride, and hydrochlorothiazide. The studied mixture was fully separated and determined in less than 5 min with perfect resolution. CONCLUSION: Experimental design is a very beneficial tool for optimization of the method conditions in HPLC, especially if the studied mixture ingredients have overlapping peaks. For atenolol, amiloride, and hydrochlorothiazide, acetonitrile and flow rate were found to be the significant factors that affected the resolution of the studied mixture. HIGHLIGHTS: Response surface design is a powerful tool that could be used for predicting the significant factors for separation in HPLC. Optimization of the method conditions was done using a limited number of preliminary runs. The studied ternary mixture was fully separated in less than 5 min with the aid of experimental design.


Assuntos
Anti-Hipertensivos , Hidroclorotiazida , Amilorida/análise , Amilorida/farmacologia , Cromatografia Líquida de Alta Pressão/métodos , Hidroclorotiazida/análise , Modelos Teóricos
10.
Cancer Med ; 11(1): 183-193, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34796694

RESUMO

Triple negative breast cancers (TNBCs) are very aggressive and have a poor prognosis due to lack of efficacious therapies. The only effective treatment is chemotherapy that however is frequently hindered by the occurrence of drug resistance. We approached this problem in vitro and in vivo on a triple negative and a hormone sensitive breast cancer cell lines: 4T1 and TS/A. A main defense mechanism of tumors is the extrusion of intracellular protons derived from the metabolic shift to glycolysis, and necessary to maintain an intracellular pH compatible with life. The resulting acidic extracellular milieu bursts the malignant behavior of tumors and impairs chemotherapy. Therefore, we investigated the efficacy of combined therapies that associate cisplatin (Cis) with proton exchanger inhibitors, such as esomeprazole (ESO) and 5-(N-ethyl-N-isopropyl)amiloride (EIPA). Our results demonstrate that in the 4T1 triple negative model the combined therapy Cis plus EIPA is significantly more effective than the other treatments. Instead, in the TS/A tumor the best therapeutic result is obtained with ESO alone. Remarkably, in both 4T1 and TS/A tumors these treatments correlate with increase of CD8+  T lymphocytes and dendritic cells, and a dramatic reduction of M2 macrophages and other suppressor myeloid cells (MDSC) in the tumor infiltrates.


Assuntos
Amilorida/análogos & derivados , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Cisplatino/uso terapêutico , Esomeprazol/uso terapêutico , Inibidores da Bomba de Prótons/uso terapêutico , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Amilorida/uso terapêutico , Animais , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Humanos , Concentração de Íons de Hidrogênio , Camundongos Endogâmicos BALB C , Trocador 1 de Sódio-Hidrogênio/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Macrófagos Associados a Tumor/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo
11.
Nephrol Dial Transplant ; 37(2): 298-303, 2022 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33914065

RESUMO

BACKGROUND: Amiloride is a competitive blocker of the epithelial sodium (Na) channel in the renal collecting duct. It is a less potent diuretic than thiazides or loop diuretics, but is often used in association with its potassium (K)-sparing profile. Whether amiloride has a hypocalciuric effect similar to thiazides remains unclear. Animal studies and experiments on cell lines suggested that amiloride increases calcium (Ca) reabsorption in the distal nephron, but human studies are scarce. METHODS: We performed a post hoc analysis of a study with 48 healthy males (mean ± standard deviation age, 23.2 ± 3.9 years) who were assigned to a high-Na/low-K diet for 7 days before receiving 20 mg of amiloride orally. Urinary excretions of electrolytes were measured at 3 and 6 h afterwards; we calculated the relative changes in urinary excretion rates after amiloride administration. RESULTS: The high-Na/low-K diet led to an expected suppression of plasma renin and aldosterone. Amiloride showed a mild natriuretic effect associated with a decreased kaliuresis. Urinary Ca excretion dropped substantially (by 80%) 3 h after amiloride administration and remained low at the sixth hour. At the same time, fractional excretion of lithium decreased by a third, reflecting an increased proximal tubular reabsorption. CONCLUSIONS: During a high-Na/low-K diet, amiloride had a strong acute hypocalciuric effect, most probably mediated by increased proximal Ca reabsorption, even though a distal effect cannot be excluded. Further studies should establish if chronic amiloride or combined amiloride/thiazide treatment may decrease calciuria more efficiently and be useful in preventing kidney stones.


Assuntos
Amilorida , Cálcio , Amilorida/farmacologia , Animais , Diuréticos/farmacologia , Voluntários Saudáveis , Humanos , Masculino , Potássio/metabolismo , Sódio/metabolismo
12.
13.
J Med Chem ; 65(3): 1933-1945, 2022 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-34898192

RESUMO

The urokinase plasminogen activator (uPA) plays a critical role in tumor cell invasion and migration and is a promising antimetastasis target. 6-Substituted analogues of 5-N,N-(hexamethylene)amiloride (HMA) are potent and selective uPA inhibitors that lack the diuretic and antikaliuretic properties of the parent drug amiloride. However, the compounds display pronounced selectivity for human over mouse uPA, thus confounding interpretation of data from human xenograft mouse models of cancer. Here, computational and experimental findings reveal that residue 99 is a key contributor to the observed species selectivity, whereby enthalpically unfavorable expulsion of a water molecule by the 5-N,N-hexamethylene ring occurs when residue 99 is Tyr (as in mouse uPA). Analogue 7 lacking the 5-N,N-hexamethylene ring maintained similar water networks when bound to human and mouse uPA and displayed reduced selectivity, thus supporting this conclusion. The study will guide further optimization of dual-potent human/mouse uPA inhibitors from the amiloride class as antimetastasis drugs.


Assuntos
Amilorida/análogos & derivados , Inibidores Enzimáticos/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Água/química , Amilorida/química , Amilorida/metabolismo , Animais , Inibidores Enzimáticos/química , Humanos , Camundongos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Molecular , Mutação , Ligação Proteica , Especificidade da Espécie , Termodinâmica , Ativador de Plasminogênio Tipo Uroquinase/genética , Ativador de Plasminogênio Tipo Uroquinase/metabolismo
14.
Commun Biol ; 5(1): 166, 2022 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-35210534

RESUMO

Increasing antimicrobial resistance compels the search for next-generation inhibitors with differing or multiple molecular targets. In this regard, energy conservation in Mycobacterium tuberculosis has been clinically validated as a promising new drug target for combatting drug-resistant strains of M. tuberculosis. Here, we show that HM2-16F, a 6-substituted derivative of the FDA-approved drug amiloride, is an anti-tubercular inhibitor with bactericidal properties comparable to the FDA-approved drug bedaquiline (BDQ; Sirturo®) and inhibits the growth of bedaquiline-resistant mutants. We show that HM2-16F weakly inhibits the F1Fo-ATP synthase, depletes ATP, and affects the entry of acetyl-CoA into the Krebs cycle. HM2-16F synergizes with the cytochrome bcc-aa3 oxidase inhibitor Q203 (Telacebec) and co-administration with Q203 sterilizes in vitro cultures in 14 days. Synergy with Q203 occurs via direct inhibition of the cytochrome bd oxidase by HM2-16F. This study shows that amiloride derivatives represent a promising discovery platform for targeting energy generation in drug-resistant tuberculosis.


Assuntos
Mycobacterium tuberculosis , Trifosfato de Adenosina , Amilorida/farmacologia , Antituberculosos/farmacologia , Citocromos , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Mycobacterium tuberculosis/metabolismo , Oxirredutases
15.
Behav Brain Res ; 416: 113565, 2022 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-34499933

RESUMO

Acid-sensing ion channels (ASICs) play significant roles in numerous neurological and pathological conditions, including pain. Although acid-induced nociception has been characterized previously in zebrafish, the contribution of ASICs in modulating pain-like behaviors is still unknown. Here, we investigated the role of amiloride, a nonselective ASICs blocker, in the negative modulation of specific behavioral responses in a zebrafish-based model of acute visceral pain. We verified that intraperitoneal injection (i.p.) of 0.25, 0.5, 1.0, and 2.0 mg/mL amiloride alone or vehicle did not change zebrafish behavior compared to saline-treated fish. Administration of 2.5% acetic acid (i.p.) elicited writhing-like response evidenced by the abnormal body curvature and impaired locomotion and motor activity. Attenuation of acetic acid-induced pain was verified at lower amiloride doses (0.25 and 0.5 mg/mL) whereas 1.0 and 2.0 mg/mL abolished pain-like responses. The protective effect of the highest amiloride dose tested was evident in preventing writhing-like responses and impaired locomotion and vertical activity. Collectively, amiloride antagonized abdominal writhing-like phenotype and aberrant behaviors, supporting the involvement of ASICs in a zebrafish-based model of acute visceral pain.


Assuntos
Canais Iônicos Sensíveis a Ácido , Amilorida/farmacologia , Locomoção/efeitos dos fármacos , Dor Visceral/tratamento farmacológico , Peixe-Zebra , Animais , Modelos Animais de Doenças , Injeções Intraperitoneais , Masculino , Nociceptividade/efeitos dos fármacos
16.
Mol Imaging Biol ; 24(3): 498-509, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34905147

RESUMO

PURPOSE: Macropinocytosis serves as a highly conserved endocytotic process that has recently been shown as a critical mechanism by which RAS-transformed cells transport extracellular protein into intracellular amino acid pathways to support their unique metabolic needs. We developed NIR fluorescently labeled molecular imaging probes to monitor macropinocytosis-mediated uptake of albumin in a K-RAS-dependent manner. PROCEDURES: Using western blot analysis, immunofluorescence, and flow cytometry, albumin retention was characterized in vitro across several RAS-activated lung and pancreatic cancer cell lines. AF790-albumin was synthesized and administered to mice bearing K-RAS mutant xenograft tumors of H460 (K-RAS p.Q61H) and H358 (K-RAS p.G12C) non-small cell lung cancers on each flank. Mice were treated daily with 2 mg/kg of ARS-1620, a targeted RAS p.G12C inhibitor, for 2 days and imaged following each treatment. Subsequently, the mice were then treated daily with 10 mg/kg of amiloride, a general inhibitor of macropinocytosis, for 2 days and imaged. Intratumoral distribution of AF790-albumin was assessed in vivo using near-infrared (NIR) fluorescence imaging. RESULTS: Albumin retention was observed as a function of K-RAS activity and macropinocytosis across several lung and pancreatic cancer cell lines. We documented that ARS-1620-induced inhibition of K-RAS activity or amiloride-mediated inhibition of macropinocytosis significantly reduced albumin uptake. Tumor retention in vivo of AF790-albumin was both RAS inhibition-dependent as well as abrogated by inhibition of macropinocytosis. CONCLUSIONS: These data provide a novel approach using NIR-labeled human serum albumin to identify and monitor RAS-driven tumors as well as evaluate the on-target efficacy in vivo of inhibitors, such as ARS-1620.


Assuntos
Antineoplásicos , Neoplasias Pulmonares , Neoplasias Pancreáticas , Albuminas/metabolismo , Albuminas/farmacologia , Amilorida , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Dextranos , Humanos , Camundongos , Mutação/genética , Imagem Óptica , Neoplasias Pancreáticas/diagnóstico por imagem , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/metabolismo , Piperazinas , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Quinazolinas
17.
Int J Mol Sci ; 22(23)2021 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-34884450

RESUMO

Few data exist about the presence and physiological role of Na+/H+ exchangers (NHEs) in the plasma membrane of mammalian sperm. In addition, the involvement of these channels in the ability of sperm to undergo capacitation and acrosomal reaction has not been investigated in any mammalian species. In the present study, we addressed whether these channels are implicated in these two sperm events using the pig as a model. We also confirmed the presence of NHE1 channels in the plasma membrane of ejaculated sperm by immunofluorescence and immunoblotting. The function of NHE channels during in vitro capacitation was analyzed by incubating sperm samples in capacitating medium for 300 min in the absence or presence of a specific blocker (DMA; 5-(N,N-dimethyl)-amiloride) at different concentrations (1, 5, and 10 µM); acrosome exocytosis was triggered by adding progesterone after 240 min of incubation. Sperm motility and kinematics, integrity of plasma and acrosome membranes, membrane lipid disorder, intracellular calcium and reactive oxygen species (ROS) levels, and mitochondrial membrane potential (MMP) were evaluated after 0, 60, 120, 180, 240, 250, 270, and 300 min of incubation. NHE1 localized in the connecting and terminal pieces of the flagellum and in the equatorial region of the sperm head and was found to have a molecular weight of 75 kDa. During the first 240 min of incubation, i.e., before the addition of progesterone, blocked and control samples did not differ significantly in any of the parameters analyzed. However, from 250 min of incubation, samples treated with DMA showed significant alterations in total motility and the amplitude of lateral head displacement (ALH), acrosomal integrity, membrane lipid disorder, and MMP. In conclusion, while NHE channels are not involved in the sperm ability to undergo capacitation, they could be essential for triggering acrosome exocytosis and hypermotility after progesterone stimulus.


Assuntos
Amilorida/análogos & derivados , Progesterona/farmacologia , Trocadores de Sódio-Hidrogênio/antagonistas & inibidores , Capacitação Espermática/efeitos dos fármacos , Reação Acrossômica/efeitos dos fármacos , Amilorida/farmacologia , Animais , Fenômenos Biomecânicos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Motilidade Espermática/efeitos dos fármacos , Suínos
18.
Commun Biol ; 4(1): 1347, 2021 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-34853399

RESUMO

The dire need for COVID-19 treatments has inspired strategies of repurposing approved drugs. Amantadine has been suggested as a candidate, and cellular as well as clinical studies have indicated beneficial effects of this drug. We demonstrate that amantadine and hexamethylene-amiloride (HMA), but not rimantadine, block the ion channel activity of Protein E from SARS-CoV-2, a conserved viroporin among coronaviruses. These findings agree with their binding to Protein E as evaluated by solution NMR and molecular dynamics simulations. Moreover, we identify two novel viroporins of SARS-CoV-2; ORF7b and ORF10, by showing ion channel activity in a X. laevis oocyte expression system. Notably, amantadine also blocks the ion channel activity of ORF10, thereby providing two ion channel targets in SARS-CoV-2 for amantadine treatment in COVID-19 patients. A screen of known viroporin inhibitors on Protein E, ORF7b, ORF10 and Protein 3a from SARS-CoV-2 revealed inhibition of Protein E and ORF7b by emodin and xanthene, the latter also blocking Protein 3a. This illustrates a general potential of well-known ion channel blockers against SARS-CoV-2 and specifically a dual molecular basis for the promising effects of amantadine in COVID-19 treatment. We therefore propose amantadine as a novel, cheap, readily available and effective way to treat COVID-19.


Assuntos
Amantadina/farmacologia , Amilorida/análogos & derivados , Antivirais/farmacologia , Rimantadina/farmacologia , SARS-CoV-2/efeitos dos fármacos , Proteínas Virais/fisiologia , Amilorida/farmacologia , Canais Iônicos/fisiologia
19.
Int J Mol Sci ; 22(20)2021 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-34681656

RESUMO

The Epithelial Sodium Channel/Degenerin (ENaC/DEG) family is a superfamily of sodium-selective channels that play diverse and important physiological roles in a wide variety of animal species. Despite their differences, they share a high homology in the pore region in which the ion discrimination takes place. Although ion selectivity has been studied for decades, the mechanisms underlying this selectivity for trimeric channels, and particularly for the ENaC/DEG family, are still poorly understood. This systematic review follows PRISMA guidelines and aims to determine the main components that govern ion selectivity in the ENaC/DEG family. In total, 27 papers from three online databases were included according to specific exclusion and inclusion criteria. It was found that the G/SxS selectivity filter (glycine/serine, non-conserved residue, serine) and other well conserved residues play a crucial role in ion selectivity. Depending on the ion type, residues with different properties are involved in ion permeability. For lithium against sodium, aromatic residues upstream of the selectivity filter seem to be important, whereas for sodium against potassium, negatively charged residues downstream of the selectivity filter seem to be important. This review provides new perspectives for further studies to unravel the mechanisms of ion selectivity.


Assuntos
Canais Epiteliais de Sódio/metabolismo , Amilorida/química , Amilorida/metabolismo , Animais , Canais Epiteliais de Sódio/química , Canais Epiteliais de Sódio/genética , Humanos , Transporte de Íons , Lítio/metabolismo , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Estrutura Quaternária de Proteína , Sódio/metabolismo
20.
Cell Rep ; 37(1): 109795, 2021 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-34610318

RESUMO

A controversial hypothesis pertaining to cystic fibrosis (CF) lung disease is that the CF transmembrane conductance regulator (CFTR) channel fails to inhibit the epithelial Na+ channel (ENaC), yielding increased Na+ reabsorption and airway dehydration. We use a non-invasive self-referencing Na+-selective microelectrode technique to measure Na+ transport across individual folds of distal airway surface epithelium preparations from CFTR-/- (CF) and wild-type (WT) swine. We show that, under unstimulated control conditions, WT and CF epithelia exhibit similar, low rates of Na+ transport that are unaffected by the ENaC blocker amiloride. However, in the presence of the cyclic AMP (cAMP)-elevating agents forskolin+IBMX (isobutylmethylxanthine), folds of WT tissues secrete large amounts of Na+, while CFTR-/- tissues absorb small, but potentially important, amounts of Na+. In cAMP-stimulated conditions, amiloride inhibits Na+ absorption in CFTR-/- tissues but does not affect secretion in WT tissues. Our results are consistent with the hypothesis that ENaC-mediated Na+ absorption may contribute to dehydration of CF distal airways.


Assuntos
AMP Cíclico/metabolismo , Canais Epiteliais de Sódio/metabolismo , Epitélio/metabolismo , Sódio/metabolismo , 1-Metil-3-Isobutilxantina/farmacologia , Amilorida/farmacologia , Animais , Animais Geneticamente Modificados/metabolismo , Colforsina/farmacologia , Fibrose Cística , Regulador de Condutância Transmembrana em Fibrose Cística/deficiência , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Modelos Animais de Doenças , Bloqueadores do Canal de Sódio Epitelial/farmacologia , Canais Epiteliais de Sódio/química , Transporte de Íons/efeitos dos fármacos , Masculino , Suínos
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