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1.
Int J Mol Sci ; 20(10)2019 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-31091671

RESUMO

The human Na+/H+ exchanger isoform 1 (NHE1) is a plasma membrane transport protein that plays an important role in pH regulation in mammalian cells. Because of the generation of protons by intermediary metabolism as well as the negative membrane potential, protons accumulate within the cytosol. Extracellular signal-regulated kinase (ERK)-mediated regulation of NHE1 is important in several human pathologies including in the myocardium in heart disease, as well as in breast cancer as a trigger for growth and metastasis. NHE1 has a N-terminal, a 500 amino acid membrane domain, and a C-terminal 315 amino acid cytosolic domain. The C-terminal domain regulates the membrane domain and its effects on transport are modified by protein binding and phosphorylation. Here, we discuss the physiological regulation of NHE1 by ERK, with an emphasis on the critical effects on structure and function. ERK binds directly to the cytosolic domain at specific binding domains. ERK also phosphorylates NHE1 directly at multiple sites, which enhance NHE1 activity with subsequent downstream physiological effects. The NHE1 cytosolic regulatory tail possesses both ordered and disordered regions, and the disordered regions are stabilized by ERK-mediated phosphorylation at a phosphorylation motif. Overall, ERK pathway mediated phosphorylation modulates the NHE1 tail, and affects the activity, structure, and function of this membrane protein.


Assuntos
Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Trocador 1 de Sódio-Hidrogênio/metabolismo , Animais , Humanos , Fosforilação , Domínios Proteicos , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Trocador 1 de Sódio-Hidrogênio/química , Trocador 1 de Sódio-Hidrogênio/genética
2.
Nitric Oxide ; 87: 1-9, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30849492

RESUMO

We explored possibility that sodium/calcium exchanger 1 (NCX1) is involved in pH modulation and apoptosis induction in GYY4137 treated cells. We have shown that although 10 days treatment with GYY4137 did not significantly decreased volume of tumors induced by colorectal cancer DLD1 cells in nude mice, it already induced apoptosis in these tumors. Treatment of DLD1 and ovarian cancer A2780 cells with GYY4137 resulted in intracellular acidification in a concentration-dependent manner. We observed increased mRNA and protein expression of both, NCX1 and sodium/hydrogen exchanger 1 (NHE1) in DLD1-induced tumors from GYY4137-treated mice. NCX1 was coupled with NHE1 in A2780 and DLD1 cells and this complex partially disintegrated after GYY4137 treatment. We proposed that intracellular acidification is due to uncoupling of NCX1/NHE1 complex rather than blocking of the reverse mode of NCX1, probably due to internalization of NHE1. Results might contribute to understanding molecular mechanism of H2S-induced apoptosis in tumor cells.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Sulfeto de Hidrogênio/metabolismo , Morfolinas/farmacologia , Compostos Organotiofosforados/farmacologia , Trocador de Sódio e Cálcio/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Camundongos Nus , Trocador 1 de Sódio-Hidrogênio/metabolismo
3.
Cell Physiol Biochem ; 52(2): 172-185, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30816666

RESUMO

BACKGROUND/AIMS: Myocardial stretch increases cardiac force in two consecutive phases: The first one due to Frank-Starling mechanism, followed by the gradually developed slow force response (SFR). The latter is the mechanical counterpart of an autocrine/paracrine mechanism involving the release of angiotensin II (Ang II) and endothelin (ET) leading to Na⁺/H⁺ exchanger 1 (NHE-1) phosphorylation and activation. Since previous evidence indicates that p38-MAP kinase (p38-MAPK) negatively regulates the Ang II-induced NHE1 activation in vascular smooth muscle and the positive inotropic effect of ET in the heart, we hypothesized that this kinase might modulate the magnitude of the SFR to stretch. METHODS: Experiments were performed in isolated rat papillary muscles subjected to sudden stretch from 92 to 98% of its maximal length, in the absence or presence of the p38-MAPK inhibitor SB202190, or its inactive analogous SB202474. Western blot technique was used to determine phosphorylation level of p38-MAPK, ERK1/2, p90RSK and NHE-1 (previously immunoprecipitated with NHE-1 polyclonal antibody). Dual specificity phosphatase 6 (DUSP6) expression was evaluated by RT-PCR and western blot. Additionally, the Na⁺-dependent intracellular pH recovery from an ammonium prepulse-induced acid load was used to asses NHE-1 activity. RESULTS: The SFR was larger under p38-MAPK inhibition (SB202190), effect that was not observed in the presence of an inactive analogous (SB202474). Myocardial stretch activated p38-MAPK, while pre-treatment with SB202190 precluded this effect. Inhibition of p38-MAPK increased stretched-induced NHE-1 phosphorylation and activity, key event in the SFR development. Consistently, p38-MAPK inhibition promoted a greater increase in ERK1/2-p90RSK phosphorylation/activation after myocardial stretch, effect that may certainly be responsible for the observed increase in NHE-1 phosphorylation under this condition. Myocardial stretch induced up-regulation of the DUSP6, which specifically dephosphorylates ERK1/2, effect that was blunted by SB202190. CONCLUSION: Taken together, our data support the notion that p38-MAPK activation after myocardial stretch restricts the SFR by limiting ERK1/2-p90RSK phosphorylation, and consequently NHE-1 phosphorylation/activity, through a mechanism that involves DUSP6 up-regulation.


Assuntos
Fosfatase 6 de Especificidade Dupla/biossíntese , Regulação Enzimológica da Expressão Gênica , Sistema de Sinalização das MAP Quinases , Contração Miocárdica , Miocárdio/enzimologia , Regulação para Cima , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Imidazóis/farmacologia , Fosforilação/efeitos dos fármacos , Piridinas/farmacologia , Ratos , Ratos Wistar , Trocador 1 de Sódio-Hidrogênio/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores
4.
Eur J Pharmacol ; 849: 96-105, 2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30721701

RESUMO

Since the original description as potent antianginal compounds, phosphodiesterase 5A inhibitors have continuously increased their possible therapeutic applications. In the heart, Sildenafil was shown to protect against an ischemic insult by decreasing cardiac Na+/H+ exchanger (NHE1) activity, action that was mediated by protein kinase G. p38 mitogen activated protein kinase (p38MAPK) activation was described in cardiac ischemia, but its precise role remains elusive. It has been shown that p38MAPK is activated by protein kinase G (PKG) in certain non-cardiac tissues, while in others modulates NHE1 activity. Current study was aimed to seek the role of p38MAPK in the Sildenafil-triggered pathway leading to NHE1 inhibition in myocardium. Rat isolated papillary muscles were used to evaluate NHE1 activity during intracellular pH recovery from an acidic load. Protein kinases phosphorylation (activation) was determined by western blot. Sustained acidosis promoted NHE1 hyperactivity by enhancing Ser703 phosphorylation, effect that was blunted by Sildenafil. p38MAPK inhibition reversed the effect of Sildenafil on NHE1. Activation of p38MAPK, by Sodium Arsenite or Anisomycin, mimicked the inhibitory effect of Sildenafil on the exchanger. Consistently, Sildenafil induced p38MAPK phosphorylation/activation during acidosis. Neither Sildenafil nor p38MAPK inhibition affected extracellular signal-regulated kinases 1/2 phosphorylation, kinases upstream NHE1. Furthermore, inhibition of NHE1 after p38MAPK activation was precluded by preventing the activation of protein phosphatase 2A with Okadaic Acid. Taken together, these results suggest that activation of p38MAPK is a necessary step to trigger the inhibitory effect of Sildenafil on cardiac NHE1 activity, thorough a mechanism that involves protein phosphatase 2A-mediated exchanger dephosphorylation.


Assuntos
Coração/efeitos dos fármacos , Miocárdio/metabolismo , Citrato de Sildenafila/farmacologia , Trocadores de Sódio-Hidrogênio/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Acidose/enzimologia , Acidose/metabolismo , Acidose/patologia , Animais , Ativação Enzimática/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Miocárdio/citologia , Miocárdio/patologia , Fosforilação/efeitos dos fármacos , Proteína Fosfatase 2/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Wistar , Trocador 1 de Sódio-Hidrogênio/metabolismo
5.
Vascul Pharmacol ; 115: 26-32, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30695730

RESUMO

AIM: Selenium, a trace element involved in important enzymatic activities inside the body, has protective effects against cardiovascular diseases including atherosclerosis. The safe dose of selenium in the organism is very narrow, limiting the supplementation of selenium in diet. The aim of this study is to explore whether selenium quantum dots (SeQDs) prevent atherosclerosis and to investigate the potential mechanisms. METHODS: An amorphous form of SeQDs (A-SeQDs) and a crystalline form of SeQDs (C-SeQDs) were prepared through self-redox decomposition of selenosulfate precursor. Endothelial dysfunction was induced by balloon injury plus high fat diet (HFD) in rats. Atherosclerotic model was established by feeding Apoe-/- mice with HFD. RESULTS: Administrations of A-SeQDs but not C-SeQDs dramatically improved endothelium-dependent relaxation, and accelerated would healing in primary endothelial cells isolated from rats, which was comprised by co-treatment of LiCl. Lentivirus-mediated knockdown of Na+/H+ exchanger 1 (NHE1) abolished LiCl-induced endothelial dysfunction in rats. In cultured endothelial cells, A-SeQDs, as well as cariporide, inhibited NHE1 activities, decreased intracellular pH value and Ca2+ concentration, and reduced calpain activity increased by ox-LDL. These protective effects of A-SeQDs were reversed by LiCl treatment in endothelial cells. In Apoe-/- mice feeding with HFD, A-SeQDs prevented endothelial dysfunction and reduced the size of atherosclerotic plaque in aortic arteries. Further, lentivirus-mediated NHE1 gene overexpression abolished the protective effects of A-SeQDs against endothelial dysfunction and atherosclerosis in Apoe-/- mice. CONCLUSION: A-SeQDs prevents endothelial dysfunction and the growth of atherosclerotic plaque through NHE1 inhibition and subsequent inactivation of Ca2+/calpain signaling. Clinically, the administration of A-SeQDs is an effective approach to treat atherosclerosis.


Assuntos
Aterosclerose/prevenção & controle , Células Endoteliais/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Pontos Quânticos , Selênio/farmacologia , Trocador 1 de Sódio-Hidrogênio/antagonistas & inibidores , Lesões do Sistema Vascular/terapia , Vasodilatação/efeitos dos fármacos , Animais , Aterosclerose/metabolismo , Aterosclerose/patologia , Aterosclerose/fisiopatologia , Sinalização do Cálcio/efeitos dos fármacos , Calpaína/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Endotélio Vascular/fisiopatologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Concentração de Íons de Hidrogênio , Masculino , Camundongos Knockout para ApoE , Ratos , Ratos Sprague-Dawley , Trocador 1 de Sódio-Hidrogênio/metabolismo , Lesões do Sistema Vascular/metabolismo , Lesões do Sistema Vascular/patologia , Lesões do Sistema Vascular/fisiopatologia , Cicatrização/efeitos dos fármacos
6.
Exp Mol Pathol ; 107: 110-117, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30594602

RESUMO

BACKGROUND: Renal cell carcinoma (RCC) is the most common form of kidney cancer. Recent studies reported that Tescalcin was overexpressed in various tumor types. However, the status of Tescalcin protein expression in RCC and its biological function is uncertain. This study was designed to investigate the expression of Tescalcin in human RCC and its biological function. METHODS: shRNA transfection was performed to abrogates the expression of Tescalcin. Quantitative real time PCR and western blotting assays were used to determine mRNA and protein expression levels, respectively. The cell viability was analyzed by MTT and colony formation. Cell flow cytometry was used to assess pHi value and cell apoptosis. Cell invasive and migratory ability was measured with modified Boyden chamber assay. Xenograft model was setup to evaluate tumor growth. RESULTS: Tescalcin was overexpressed in RCC tissues compared with matched normal tissues. It was also overexpressed in RCC cell lines relative that of normal cells. Suppression Tescalcin with specific shRNA resulted in the inhibition of cell proliferation, migration, invasion and apoptosis of RCC cells. Additionally, silencing of Tescalcin also caused the inhibition of the tumor growth in nude mice. Mechanistic study showed that Tescalcin regulated cell proliferation, migration and invasion via NHE1/pHi axis as well as AKT/NF-κB signaling pathway. CONCLUSIONS: These findings demonstrate that atopic expression of Tescalcin facilitates the survival, migration and invasion of RCC cells via NHE1/pHi axis as well as AKT/ NF-κB signaling pathway, providing new perspectives for the future study of Tescalcin as a therapeutic target for RCC.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Carcinoma de Células Renais/patologia , Neoplasias Renais/patologia , NF-kappa B/metabolismo , Trocador 1 de Sódio-Hidrogênio/metabolismo , Animais , Proliferação de Células/fisiologia , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica/fisiologia , Xenoenxertos , Humanos , Camundongos , Camundongos Nus , Invasividade Neoplásica/fisiopatologia , Transdução de Sinais/fisiologia
7.
Biochim Biophys Acta Mol Basis Dis ; 1865(1): 26-37, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30326259

RESUMO

Metastatic cancer cells are highly plastic for the expression of different tumor phenotype hallmarks and organotropism. This plasticity is highly regulated but the dynamics of the signaling processes orchestrating the shift from one cell phenotype and metastatic organ pattern to another are still largely unknown. The scaffolding protein NHERF1 has been shown to regulate the expression of different neoplastic phenotypes through its PDZ domains, which forms the mechanistic basis for metastatic organotropism. This reprogramming activity was postulated to be dependent on its differential phosphorylation patterns. Here, we show that NHERF1 phosphorylation on S279/S301 dictates several tumor phenotypes such as in vivo invasion, NHE1-mediated matrix digestion, growth and vasculogenic mimicry. Remarkably, injecting mice with cells having differential NHERF1 expression and phosphorylation drove a shift from the predominantly lung colonization (WT NHERF1) to predominately bone colonization (double S279A/S301A mutant), indicating that NHERF1 phosphorylation also acts as a signaling switch in metastatic organotropism.


Assuntos
Neoplasias da Mama/metabolismo , Fenótipo , Fosfoproteínas/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Animais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Proteínas Mutantes/metabolismo , Invasividade Neoplásica , Metástase Neoplásica , Fosfoproteínas/genética , Fosforilação , Transdução de Sinais , Trocador 1 de Sódio-Hidrogênio/metabolismo , Trocadores de Sódio-Hidrogênio/genética , Ensaios Antitumorais Modelo de Xenoenxerto , Peixe-Zebra
8.
Am J Chin Med ; 46(8): 1915-1931, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30525897

RESUMO

Na + /H + exchanger 1 (NHE1) plays a vital role in the oncogenesis and development of hepatocellular carcinoma (HCC) and has been regarded as a promising target for the treatment of HCC. Ginsenoside Rg3 (Rg3), a bioactive ginseng compound, is suggested to possess pleiotropic antitumor effects on HCC. However, the underlying mechanisms of Rg3 suppressing HCC remain unclear. In the present study, we uncovered a novel antitumor mechanism of Rg3 on HCC by decreasing NHE1 expression through in vivo and in vitro studies. Mechanistically, we demonstrated that epidermal growth factor (EGF) could dramatically upregulate NHE1 expression, while increasing the phosphorylated extracellular signal-regulated protein kinase (ERK1/2) level and hypoxia-inducible factor 1 alpha (HIF-1 α) expression. In the presence of ERK1/2-specific inhibitor PD98059, EGF stimulated HIF-1 α and NHE1 expression was obviously blocked in addition, the presence of HIF-1 α -specific inhibitor 2-methoxyestradiol (2-MeOE2) blocked EGF stimulated NHE1 expression. Moreover, results from in vivo and in vitro studies indicate that Rg3 treatment markedly decreased the expression of EGF, EGF receptor (EGFR), phosphorylated ERK1/2 and HIF-1 α . Conclusively, these findings suggested that NHE1 was stimulated by EGF, and Rg3 could decrease NHE1 expression by integrally inhibiting EGF-EGFR-ERK1/2-HIF- α signal axis in HCC. Together, our evidence indicated that Rg3 was an effective multi-targets antitumor agent for the treatment of HCC.


Assuntos
Antineoplásicos Fitogênicos , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Expressão Gênica/efeitos dos fármacos , Ginsenosídeos/farmacologia , Ginsenosídeos/uso terapêutico , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Terapia de Alvo Molecular , Fitoterapia , Trocador 1 de Sódio-Hidrogênio/genética , Trocador 1 de Sódio-Hidrogênio/metabolismo , Linhagem Celular Tumoral , Humanos
9.
Life Sci ; 209: 197-201, 2018 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-30089233

RESUMO

Cardiac remodeling, characterized by excessive extracellular matrix (ECM) remodeling, predisposes the heart to failure if left unresolved. Understanding the signaling mechanisms involved in excessive extracellular matrix (ECM) remodeling is necessary to identify the means to regress the development of cardiac remodeling and heart failure. Recently, hyaluronan (HA), a ubiquitously expressed glycosaminoglycan in the ECM, was shown to participate in tissue fibrosis and myofibroblast proliferation through interacting with its ubiquitously expressed cell-surface receptor, CD44. CD44 is a multifunctional transmembrane glycoprotein that serves as a cell-surface receptor for a number of ECM proteins. The mechanism by which the interaction between CD44-HA contributes to ECM and cardiac remodeling remains unknown. A previous study performed on a non-cardiac model showed that CD44-HA enhances Na+/H+ exchanger isoform-1 (NHE1) activity, causing ECM remodeling, HA metabolism and tumor invasion. Interestingly, NHE1 has been demonstrated to be involved in cardiac remodeling and myocardial fibrosis. In addition, it has previously been demonstrated that CD44 is upregulated in transgenic mouse hearts expressing active NHE-1. The role of CD44, HA and NHE1 and the cellular interplay of these factors in the ECM and cardiac remodeling is the focus of this review.


Assuntos
Remodelamento Atrial , Insuficiência Cardíaca/fisiopatologia , Receptores de Hialuronatos/metabolismo , Ácido Hialurônico/metabolismo , Miócitos Cardíacos/fisiologia , Trocador 1 de Sódio-Hidrogênio/metabolismo , Remodelação Ventricular , Humanos , Miócitos Cardíacos/citologia , Transdução de Sinais
10.
In Vitro Cell Dev Biol Anim ; 54(4): 311-320, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29532321

RESUMO

Short-chain fatty acids (SCFAs) produced by microbial fermentation of dietary fibers are utilized by intestinal epithelial cells to provide an energy source for the ruminant. Although the regulation of mRNA expression and inflammatory response involved in SCFAs is established in other animals and tissues, the underlying mechanisms of the inflammatory response by SCFAs in goat jejunum epithelial cells (GJECs) have not been understood. Therefore, the objective of the study is to investigate the underlying mechanisms of the effects of SCFAs on SCFA transporters and inflammatory response in GJECs. These results showed that the acetate, butyrate, and SCFA concentration were markedly reduced in GJECs (p < 0.01). In addition, the propionate concentration was significantly decreased in GJECs (p < 0.05). The mRNA abundance of monocarboxylate transporter 1 (MCT1), MCT4, NHE1, and putative anion transporter 1 (PAT1) was elevated (p < 0.05) by 20 mM SCFAs at pH 7.4 compared with exposure to the pH group. The anion exchanger 2 (AE2) was increased (p < 0.05) by 20 mM SCFAs at pH 6.2. The mRNA abundance of vH+ ATPase B subunit (vH+ ATPase) was attenuated by SCFAs. For inflammatory responses, IL-1ß and TNF-α were increased with SCFAs (p < 0.05). In addition, IκBα involved in NF-κB signaling pathways was disrupted by SCFAs. Consistently, p-p65 signaling molecule was enhanced by adding SCFAs. However, IL-6 was attenuated by adding SCFAs (p < 0.05). Furthermore, p-ErK1/2 mitogen-activated protein kinase (MAPK) signaling pathway was downregulated by adding SCFAs. In conclusion, these novel findings demonstrated that mRNA abundance involved in SCFA absorption is probably associated to SCFAs and pH value, and mechanism of the inflammatory response by SCFAs may be involved in NF-κB and p-ErK1/2 MAPK signaling pathways in GJECs. These pathways may mediate protective inflammation response in GJECs.


Assuntos
Ácidos Graxos Voláteis/farmacologia , Cabras/metabolismo , Animais , Células Cultivadas , Células Epiteliais/metabolismo , Ácidos Graxos Voláteis/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Cabras/genética , Interleucina-6/genética , Interleucina-6/metabolismo , Jejuno/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , RNA Mensageiro/metabolismo , Trocador 1 de Sódio-Hidrogênio/genética , Trocador 1 de Sódio-Hidrogênio/metabolismo , Simportadores/genética , Simportadores/metabolismo
11.
Reproduction ; 155(5): 433-445, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29491124

RESUMO

Sperm motility, a feature essential for in vivo fertilization, is influenced by intracellular pH (pHi) homeostasis. Several mechanisms are involved in pHi regulation, among which sodium-hydrogen exchangers (NHEs), a family of integral transmembrane proteins that catalyze the exchange of Na+ for H+ across lipid bilayers. A preliminary characterization of NHE activity and kinetic parameters, followed by analysis of the expression and localization of the protein in ram spermatozoa was performed. NHE activity showed an apparent Km for external Na+ of 17.61 mM. Immunoblotting revealed a molecular mass of 85 kDa. Immunolocalization pattern showed some species-specific aspects, such as positive labeling at the equatorial region of the sperm head. Cariporide, a selective NHE1 inhibitor, significantly reduced pHi recovery (85%). Similarly, exposure to cariporide significantly inhibited different motility parameters, including those related to sperm capacitation. In vitro fertilization (IVF) was not affected by cariporide, possibly due to the non-dramatic, although significant, drop in motility and velocity parameters or due to prolonged exposure during IVF, which may have caused progressive loss of its inhibitory effect. In conclusion, this is the first study documenting, in a large animal model (sheep) of well-known translational relevance, a direct functional role of NHE on sperm pHi and motility. The postulated specificity of cariporide toward isoform 1 of the Na+/H+ exchanger seems to suggest that NHE1 may contribute to the observed effects on sperm cell functionality.


Assuntos
Guanidinas/farmacologia , Trocador 1 de Sódio-Hidrogênio/metabolismo , Motilidade Espermática/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Sulfonas/farmacologia , Animais , Concentração de Íons de Hidrogênio , Masculino , Ovinos , Capacitação Espermática/efeitos dos fármacos , Capacitação Espermática/fisiologia , Motilidade Espermática/fisiologia , Espermatozoides/metabolismo
12.
Cell Physiol Biochem ; 45(6): 2560-2576, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29558744

RESUMO

BACKGROUND/AIMS: The peptide hormone angiotensin II (ATII) plays a prominent role in regulating vasoconstriction and blood pressure. Its primary target is the angiotensin II receptor type 1 (AT1), the stimulation of which induces an increase in cytosolic [Ca2+] and calmodulin activation. Ca2+-bound (activated) calmodulin stimulates the activity of the Na+/ H+ exchanger isoform 1 (NHE1); and increased NHE1 activity is known to promote melanoma cell motility. The competitive AT1 receptor inhibitor losartan is often used to lower blood pressure in hypertensive patients. Since AT1 mediates ATII-stimulated NHE1 activity, we set out to investigate whether ATII and losartan have an impact on NHE1-dependent behavior of human melanoma (MV3) cells. METHODS: ATII receptor expression was verified by PCR, F-actin was visualized using fluorescently labeled phalloidin, and cytosolic [Ca2+] and pH were determined ratiometrically using Fura-2 and BCECF, respectively. MV3 cell behavior was analyzed using migration, adhesion, invasion and proliferation assays. RESULTS: MV3 cells express both AT1 and the angiotensin II receptor type 2 (AT2). Stimulation of MV3 cells with ATII increased NHE1 activity which could be counteracted by both losartan and the Ca2+/ calmodulin inhibitor ophiobolin-A. ATII stimulation induced a decrease in MV3 cell migration and a more spherical cell morphology accompanied by an increase in the density of F-actin. Independently of the presence of ATII, both NHE1 and migratory activity were reduced when AT1 was blocked by losartan. On the other hand, losartan clearly increased cell adhesion to, and the invasion of, a collagen type I substrate. The AT2 inhibitor PD123319 did not affect NHE1 activity, proliferation and migration, but increased adhesion and invasion. CONCLUSION: Losartan inhibits NHE1 activity and the migration of human melanoma cells. At the same time, losartan promotes MV3 cell adhesion and invasion. The therapeutic use of AT1 antagonists (sartans) in hypertensive cancer patients should therefore be given critical consideration.


Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Movimento Celular/efeitos dos fármacos , Losartan/farmacologia , Melanoma/tratamento farmacológico , Invasividade Neoplásica/prevenção & controle , Trocador 1 de Sódio-Hidrogênio/metabolismo , Linhagem Celular Tumoral , Humanos , Melanoma/metabolismo , Melanoma/patologia , Invasividade Neoplásica/patologia , Metástase Neoplásica/patologia , Metástase Neoplásica/prevenção & controle
13.
Arch Oral Biol ; 90: 19-26, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29524788

RESUMO

OBJECTIVE: Homeostasis of intracellular pH (pHi) plays vital roles in many cell functions, such as proliferation, apoptosis, differentiation and metastasis. Thus far, Na+-H+ exchanger (NHE), Na+-HCO3- co-transporter (NBC), Cl-/HCO3- exchanger (AE) and Cl-/OH- exchanger (CHE) have been identified to co-regulate pHi homeostasis. However, functional and biological pHi-regulators in human dental pulp stem cells (hDPSCs) have yet to be identified. DESIGN: Microspectrofluorimetry technique with pH-sensitive fluorescent dye, BCECF, was used to detect pHi changes. NH4Cl and Na+-acetate pre-pulse were used to induce intracellular acidosis and alkalosis, respectively. Isoforms of pHi-regulators were detected by Western blot technique. RESULTS: The resting pHi was no significant difference between that in HEPES-buffered (nominal HCO3--free) solution or CO2/HCO3-buffered system (7.42 and 7.46, respectively). The pHi recovery following the induced-intracellular acidosis was blocked completely by removing [Na+]o, while only slowed (-63%) by adding HOE694 (a NHE1 specific inhibitor) in HEPES-buffered solution. The pHi recovery was inhibited entirely by removing [Na+]o, while adding HOE 694 pulse DIDS (an anion-transporter inhibitor) only slowed (-55%) the acid extrusion. Both in HEPES-buffered and CO2/HCO3-buffered system solution, the pHi recovery after induced-intracellular alkalosis was entirely blocked by removing [Cl-]o. Western blot analysis showed the isoforms of pHi regulators, including NHE1/2, NBCe1/n1, AE1/2/3/4 and CHE in the hDPSCs. CONCLUSIONS: We demonstrate for the first time that resting pHi is significantly higher than 7.2 and meditates functionally by two Na+-dependent acid extruders (NHE and NBC), two Cl--dependent acid loaders (CHE and AE) and one Na+-independent acid extruder(s) in hDPSCs. These findings provide novel insight for basic and clinical treatment of dentistry.


Assuntos
Equilíbrio Ácido-Base/fisiologia , Citoplasma/metabolismo , Polpa Dentária/metabolismo , Homeostase/fisiologia , Células-Tronco/metabolismo , Desequilíbrio Ácido-Base , Ácidos/farmacologia , Cloreto de Amônio , Antiporters/metabolismo , Apoptose , Tampões (Química) , Diferenciação Celular , Proliferação de Células , Citoplasma/efeitos dos fármacos , Guanidinas/farmacologia , Humanos , Concentração de Íons de Hidrogênio , Bombas de Íon/efeitos dos fármacos , Bombas de Íon/metabolismo , Metástase Neoplásica , Isoformas de Proteínas , Sódio/farmacologia , Simportadores de Sódio-Bicarbonato/metabolismo , Trocador 1 de Sódio-Hidrogênio/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Células-Tronco/efeitos dos fármacos , Sulfonas/farmacologia
14.
Cell Rep ; 21(4): 1009-1020, 2017 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-29069584

RESUMO

Sodium accumulates in the interstitium and promotes inflammation through poorly defined mechanisms. We describe a pathway by which sodium enters dendritic cells (DCs) through amiloride-sensitive channels including the alpha and gamma subunits of the epithelial sodium channel and the sodium hydrogen exchanger 1. This leads to calcium influx via the sodium calcium exchanger, activation of protein kinase C (PKC), phosphorylation of p47phox, and association of p47phox with gp91phox. The assembled NADPH oxidase produces superoxide with subsequent formation of immunogenic isolevuglandin (IsoLG)-protein adducts. DCs activated by excess sodium produce increased interleukin-1ß (IL-1ß) and promote T cell production of cytokines IL-17A and interferon gamma (IFN-γ). When adoptively transferred into naive mice, these DCs prime hypertension in response to a sub-pressor dose of angiotensin II. These findings provide a mechanistic link between salt, inflammation, and hypertension involving increased oxidative stress and IsoLG production in DCs.


Assuntos
Citocinas/metabolismo , Células Dendríticas/metabolismo , Canais Epiteliais de Sódio/metabolismo , Hipertensão/metabolismo , Amilorida/farmacologia , Animais , Células Cultivadas , Citocinas/genética , Bloqueadores do Canal de Sódio Epitelial/farmacologia , Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NADPH Oxidases/metabolismo , Estresse Oxidativo , Prostaglandinas E/metabolismo , Proteína Quinase C/metabolismo , Sódio/metabolismo , Trocador 1 de Sódio-Hidrogênio/metabolismo , Superóxidos/metabolismo
15.
Vet Immunol Immunopathol ; 191: 68-73, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28895869

RESUMO

Cytoplasmic pH homeostasis is required for an appropriate response in polymorphonuclear neutrophils (PMNs). In these cells, chemotaxis and reactive oxygen species (ROS) production are reduced by the use of Na+/H+ exchanger (NHE-1) inhibitors, but these results are mainly obtained using amiloride, a non-selective NHE-1 inhibitor. In bovine PMNs, the role of NHE-1 in functional responses has not been confirmed yet. The aim of this study was to determine the role of NHE-1 using amiloride and zoniporide in pH regulation, ROS production, matrix metalloproteinase 9 (MMP-9) release and calcium flux in bovine PMNs induced by the platelet activation factor (PAF), additionally we evaluated the presence of NHE-1 and NHE-2 mRNA Our data show the presence only of NHE-1 but not NHE-2 in bovine PMNs. Amiloride or zoniporide inhibited the intracellular alkalization induced by PAF without affecting calcium flux. Amiloride diminished ROS production and MMP-9 release, while zoniporide enhanced ROS production without change the MMP-9 release induced by PAF. Our work led us to conclude that changes in intracellular pH induced by PAF are regulated by NHE-1 in bovine neutrophils, but the effects of amiloride on ROS production and MMP-9 release induced by PAF are not NHE-1 dependent.


Assuntos
Amilorida/farmacologia , Metaloproteinase 9 da Matriz/metabolismo , Neutrófilos/efeitos dos fármacos , Fator de Ativação de Plaquetas/antagonistas & inibidores , Explosão Respiratória/efeitos dos fármacos , Trocador 1 de Sódio-Hidrogênio/antagonistas & inibidores , Animais , Cálcio/metabolismo , Bovinos , Feminino , Guanidinas/farmacologia , Neutrófilos/enzimologia , Neutrófilos/metabolismo , Fator de Ativação de Plaquetas/farmacologia , Reação em Cadeia da Polimerase/veterinária , Pirazóis/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Trocador 1 de Sódio-Hidrogênio/metabolismo
16.
Can J Physiol Pharmacol ; 95(10): 1213-1223, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28727929

RESUMO

Cardiomyopathy is found in patients with Duchenne (DMD) and Becker (BMD) muscular dystrophies, which are linked muscle diseases caused by mutations in the dystrophin gene. Dystrophin defects are not limited to DMD but are also present in mild BMD. The hereditary cardiomyopathic hamster of the UM-X7.1 strain is a particular experimental model of heart failure (HF) leading to early death in muscular dystrophy (dystrophin deficiency and sarcoglycan mutation) and heart disease (δ-sarcoglycan deficiency and dystrophin mutation) in human DMD. Using this model, our previous work showed a defect in intracellular sodium homeostasis before the appearance of any apparent biochemical and histological defects. This was attributed to the continual presence of the fetal slow sodium channel, which was also found to be active in human DMD. Due to muscular intracellular acidosis, the intracellular sodium overload in DMD and BMD was also due to sodium influx through the sodium-hydrogen exchanger NHE-1. Lifetime treatment with an NHE-1 inhibitor prevented intracellular Na+ overload and early death due to HF. Our previous work also showed that another proton transporter, the voltage-gated proton channel (Hv1), exists in many cell types including heart cells and skeletal muscle fibers. The Hv1 could be indirectly implicated in the beneficial effect of blocking NHE-1.


Assuntos
Cardiomiopatias/etiologia , Insuficiência Cardíaca/etiologia , Canais Iônicos/metabolismo , Distrofia Muscular de Duchenne/complicações , Miocárdio/metabolismo , Trocador 1 de Sódio-Hidrogênio/metabolismo , Animais , Cálcio/metabolismo , Cardiomiopatias/tratamento farmacológico , Cardiomiopatias/genética , Cardiomiopatias/metabolismo , Fármacos Cardiovasculares/uso terapêutico , Modelos Animais de Doenças , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Canais Iônicos/genética , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Transdução de Sinais , Sódio/metabolismo , Trocador 1 de Sódio-Hidrogênio/antagonistas & inibidores , Trocador 1 de Sódio-Hidrogênio/genética
17.
Cell Mol Biol (Noisy-le-grand) ; 63(5): 11-18, 2017 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-28719339

RESUMO

Diabetes is known to alter both oxidative and glycolytic pathways in a fiber type-dependent manner. The aim of present study was to investigate the effects of endurance training on muscle NHE1 and NBC1 genes and proteins expression in type 2 diabetic rats. Male wistar rats (n=30), 4 weeks old and 95.7±10.8g, were randomly selected and divided into control, diabetic without training and diabetic with training groups. Diabetes was induced by injection of low dose of streptotozin and feeding with high-fat diet. The Endurance training was performed for 7 weeks that started with relatively low speed and duration of 20 m min-1 for 20 min in the first week and gradually reached to 30 m min-1 for 35min in the last week. NHE1 and NBC1 genes and proteins expression were determined by Real time-PCR and western blotting techniques, respectively, in Soleus as an oxidative and EDL (Extensor digitorum longus) as a glycolytic muscle preparation. NHE1 mRNA and protein expression reduced significantly in EDL and Soleus in the diabetic without training group compared with the control group. However, reduction in the expression of NBC1 gene and protein in the diabetic without training group compared to controls did not significant. Endurance training increased NHE1 and NBC1 genes and proteins expression in both EDL and Soleus in the diabetic training group compared to control groups. In conclusion, endurance training may improve the capacity of pHi regulation in muscles by lactate-independent pathway.


Assuntos
Diabetes Mellitus Experimental/genética , Dieta Hiperlipídica , Regulação da Expressão Gênica , Glicólise , Fibras Musculares de Contração Rápida/metabolismo , Simportadores de Sódio-Bicarbonato/genética , Trocador 1 de Sódio-Hidrogênio/genética , Animais , Biomarcadores/metabolismo , Glicemia/metabolismo , Peso Corporal , Glicólise/genética , Insulina/sangue , Resistência à Insulina , Masculino , Oxirredução , Condicionamento Físico Animal , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Wistar , Simportadores de Sódio-Bicarbonato/metabolismo , Trocador 1 de Sódio-Hidrogênio/metabolismo , Estreptozocina
18.
Cell Signal ; 36: 145-153, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28483634

RESUMO

Na+/H+ exchanger isoform one (NHE1) is a pH regulatory protein that is present in renal tissues and serves to remove protons from within cells and protect against intracellular acidification. NHE1 has a large 315 amino acid cytosolic regulatory domain that regulates the catalytic membrane domain. We examined protein-mediated regulation of NHE1 through the cytosolic domain. Affinity chromatography with the C-terminus of NHE1 yielded a number of NHE1 binding proteins including 14-3-3 protein, heat shock proteins (Hsp90 and Hsp70) and Na+/K+ ATPase. We confirmed that 14-3-3 and heat shock proteins bind to or regulate NHE1 but could not confirm that Na+/K+ ATPase binds to the intact protein. The Hsp90 inhibitor 17-AAG decreased NHE1 activity and NHE1 phosphorylation in MDCK cells but did not decrease protein levels. Additionally, 17-AAG decreased phospho-AKT levels. Direct inhibition of AKT with MK2206 decreased NHE1 activity, though this effect was not additive with the effect of 17-AAG. The results demonstrate that in renal cells, NHE1 is associated with several regulatory proteins including Hsp90, and that Hsp90 affects its function possibly through altered phosphorylation of the protein via the AKT kinase.


Assuntos
Proteínas de Choque Térmico HSP90/metabolismo , Rim/citologia , Rim/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Trocador 1 de Sódio-Hidrogênio/metabolismo , Proteínas 14-3-3/metabolismo , Animais , Benzoquinonas/farmacologia , Cromatografia de Afinidade , Cães , Compostos Heterocíclicos com 3 Anéis/farmacologia , Lactamas Macrocíclicas/farmacologia , Células Madin Darby de Rim Canino , Camundongos , Ligação Proteica/efeitos dos fármacos , Isoformas de Proteínas/metabolismo
19.
Oncotarget ; 8(26): 42857-42875, 2017 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-28476026

RESUMO

Hypoxic cancer cells exhibit resistance to many therapies. This study compared the therapeutic effect of targeting the pH regulatory proteins (CAIX, NHE1 and V-ATPase) that permit cancer cells to adapt to hypoxic conditions, using both 2D and 3D culture models. Drugs targeting CAIX, NHE1 and V-ATPase exhibited anti-proliferative effects in MCF-7, MDA-MB-231 and HBL-100 breast cancer cell lines in 2D. Protein and gene expression analysis in 2D showed that CAIX was the most hypoxia-inducible protein of the 3 targets. However, the expression of CAIX differed between the 3 cell lines. This difference in CAIX expression in hypoxia was consistent with a varying activity of FIH-1 between the cell lines. 3D expression analysis demonstrated that both CAIX and NHE1 were up-regulated in the hypoxic areas of multicellular tumor spheroids. However, the induction of CAIX expression in hypoxia was again cell line dependent. 3D invasion assays conducted with spheroids showed that CAIX inhibition significantly reduced the invasion of cells. Finally, the capability of both NHE1 and CAIX inhibitors to combine effectively with irradiation was exhibited in clonogenic assays. Proteomic-mass-spectrometric analysis indicated that CAIX inhibition might be combining with irradiation through stimulating apoptotic cell death. Of the three proteins, CAIX represents the target with the most promise for the treatment of breast cancer.


Assuntos
Neoplasias da Mama/metabolismo , Concentração de Íons de Hidrogênio , Hipóxia/metabolismo , Neoplasias da Mama/genética , Anidrase Carbônica IX/antagonistas & inibidores , Anidrase Carbônica IX/metabolismo , Hipóxia Celular , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Hipóxia/genética , Fator 1 Induzível por Hipóxia/metabolismo , Espectrometria de Massas , Oxigênio/metabolismo , Proteômica/métodos , Trocador 1 de Sódio-Hidrogênio/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo
20.
Acta Physiol (Oxf) ; 221(2): 129-141, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28319329

RESUMO

AIM: The electroneutral Na+ , HCO3- cotransporter NBCn1 and Na+ /H+ exchanger NHE1 regulate acid-base balance in vascular smooth muscle cells (VSMCs) and modify artery function and structure. Pathological conditions - notably ischaemia - can dramatically perturb intracellular (i) and extracellular (o) pH and [Na+ ]. We examined effects of low [Na+ ]o and pHo on NBCn1 and NHE1 activity in VSMCs of small arteries. METHODS: We measured pHi by 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein-based fluorescence microscopy of mouse mesenteric arteries and induced intracellular acidification by NH4+ prepulse technique. RESULTS: NBCn1 activity - defined as Na+ -dependent, amiloride-insensitive net base uptake with CO2 /HCO3- present - was inhibited equally when pHo decreased from 7.4 (22 mm HCO3-/5% CO2 ) by metabolic (pHo 7.1/11 mm HCO3-: 22 ± 8%; pHo 6.8/5.5 mm HCO3-: 61 ± 7%) or respiratory (pHo 7.1/10% CO2 : 35 ± 11%; pHo 6.8/20% CO2 : 56 ± 7%) acidosis. Extracellular acidosis more prominently inhibited NHE1 activity - defined as Na+ -dependent net acid extrusion without CO2 /HCO3- present - at both pHo 7.1 (45 ± 9%) and 6.8 (85 ± 5%). Independently of pHo , lowering [Na+ ]o from 140 to 70 mm reduced NBCn1 and NHE1 activity <20% whereas transport activities declined markedly (25-50%) when [Na+ ]o was reduced to 35 mm. Steady-state pHi decreased more during respiratory (ΔpHi /ΔpHo  = 71 ± 4%) than metabolic (ΔpHi /ΔpHo  = 30 ± 7%) acidosis. CONCLUSION: Extracellular acidification inhibits NBCn1 and NHE1 activity in VSMCs. NBCn1 is equivalently inhibited when pCO2 is raised or [HCO3-]o decreased. Lowering [Na+ ]o inhibits NBCn1 and NHE1 markedly only below the typical physiological and pathophysiological range. We propose that inhibition of Na+ -dependent net acid extrusion at low pHo protects against cellular Na+ overload at the cost of intracellular acidification.


Assuntos
Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/fisiologia , Simportadores de Sódio-Bicarbonato/metabolismo , Trocador 1 de Sódio-Hidrogênio/metabolismo , Sódio/sangue , Acidose , Animais , Transporte Biológico Ativo , Células Cultivadas , Concentração de Íons de Hidrogênio , Masculino , Camundongos , Simportadores de Sódio-Bicarbonato/genética , Trocador 1 de Sódio-Hidrogênio/genética
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