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1.
Int J Mol Sci ; 25(1)2024 Jan 03.
Artículo en Inglés | MEDLINE | ID: mdl-38203789

RESUMEN

The epithelial ion channel TRPV6 plays a pivotal role in calcium homeostasis. Channel function is intricately regulated at different stages, involving the lipid phosphatidylinositol-4,5-bisphosphate (PIP2). Given that dysregulation of TRPV6 is associated with various diseases, including different types of cancer, there is a compelling need for its pharmacological targeting. Structural studies provide insights on how TRPV6 is affected by different inhibitors, with some binding to sites else occupied by lipids. These include the small molecule cis-22a, which, however, also binds to and thereby blocks the pore. By combining calcium imaging, electrophysiology and optogenetics, we identified residues within the pore and the lipid binding site that are relevant for regulation by cis-22a and PIP2 in a bidirectional manner. Yet, mutation of the cytosolic pore exit reduced inhibition by cis-22a but preserved sensitivity to PIP2 depletion. Our data underscore allosteric communication between the lipid binding site and the pore and vice versa for most sites along the pore.


Asunto(s)
Calcio , Fosfatidilinositoles , Canales Catiónicos TRPV , Sitios de Unión , Citosol , Fosfatidilinositoles/metabolismo , Canales Catiónicos TRPV/metabolismo
2.
iScience ; 24(4): 102346, 2021 Apr 23.
Artículo en Inglés | MEDLINE | ID: mdl-33870140

RESUMEN

High expression levels of mitochondria-associated hexokinase-II (HKII) represent a hallmark of metabolically highly active cells such as fast proliferating cancer cells. Typically, the enzyme provides a crucial metabolic switch towards aerobic glycolysis. By imaging metabolic activities on the single-cell level with genetically encoded fluorescent biosensors, we here demonstrate that HKII activity requires intracellular K+. The K+ dependency of glycolysis in cells expressing HKII was confirmed in cell populations using extracellular flux analysis and nuclear magnetic resonance-based metabolomics. Reductions of intracellular K+ by gramicidin acutely disrupted HKII-dependent glycolysis and triggered energy stress pathways, while K+ re-addition promptly restored glycolysis-dependent adenosine-5'-triphosphate generation. Moreover, expression and activation of KV1.3, a voltage-gated K+ channel, lowered cellular K+ content and the glycolytic activity of HEK293 cells. Our findings unveil K+ as an essential cofactor of HKII and provide a mechanistic link between activities of distinct K+ channels and cell metabolism.

3.
Sci Rep ; 10(1): 13628, 2020 08 12.
Artículo en Inglés | MEDLINE | ID: mdl-32788621

RESUMEN

Mortality from ischemic heart disease (IHD) is significantly lower in Japan than in Western countries. The purpose of this study was to investigate differences in circulating microRNA (miRNA) levels related to IHD in Austrians and Japanese. Participants were middle-aged healthy male Austrians (n = 20) and Japanese (n = 20). Total miRNAs in serum from each participant were analyzed using the 3D-Gene miRNA Oligo chip. Twenty-one miRNAs, previously reported as associated with IHD, were compared between Austrians and Japanese. The expression levels of miR-106a-5p, miR-135a-3p, miR-150-3p, miR-16-5p, miR-17-5p. miR-191-5p, miR-320b, miR-451a, miR-486-5p, miR-663b, and miR-92a-3p were significantly higher, while the miR-2861 expression level was significantly lower in Austrians as compared to Japanese. Both in Austrians and Japanese, there were significant positive correlations between serum expression levels of each pair of the above miRNAs except for miR-2861. The expression level of miR-2861 showed significant positive correlations with the expression levels of miR-106a-5p, miR-150-3p, miR-17-5p, miR-486-5p, miR-663b and miR-92a-3p in Austrians but not in Japanese. In pathway analysis, proinflammatory cytokine production in foam cells and collagen synthesis in vascular smooth muscle cells were associated with differentially expressed miRNAs. Difference in miRNA levels may contribute to lower cardiovascular risk in Japan than in Western countries.


Asunto(s)
Biomarcadores de Tumor/genética , MicroARN Circulante/genética , Isquemia Miocárdica/diagnóstico , Austria/epidemiología , Biomarcadores de Tumor/análisis , Estudios de Casos y Controles , MicroARN Circulante/análisis , Perfilación de la Expresión Génica , Humanos , Japón/epidemiología , Masculino , Persona de Mediana Edad , Isquemia Miocárdica/sangre , Isquemia Miocárdica/epidemiología , Isquemia Miocárdica/genética , Proyectos Piloto , Pronóstico
4.
PLoS Biol ; 18(4): e3000700, 2020 04.
Artículo en Inglés | MEDLINE | ID: mdl-32330125

RESUMEN

Trimeric intracellular cation (TRIC) channels have been proposed to modulate Ca2+ release from the endoplasmic reticulum (ER) and determine oscillatory Ca2+ signals. Here, we report that TRIC-A-mediated amplitude and frequency modulation of ryanodine receptor 2 (RyR2)-mediated Ca2+ oscillations and inositol 1,4,5-triphosphate receptor (IP3R)-induced cytosolic signals is based on attenuating store-operated Ca2+ entry (SOCE). Further, TRIC-A-dependent delay in ER Ca2+ store refilling contributes to shaping the pattern of Ca2+ oscillations. Upon ER Ca2+ depletion, TRIC-A clusters with stromal interaction molecule 1 (STIM1) and Ca2+-release-activated Ca2+ channel 1 (Orai1) within ER-plasma membrane (PM) junctions and impairs assembly of the STIM1/Orai1 complex, causing a decrease in Orai1-mediated Ca2+ current and SOCE. Together, our findings demonstrate that TRIC-A is a negative regulator of STIM1/Orai1 function. Thus, aberrant SOCE could contribute to muscle disorders associated with loss of TRIC-A.


Asunto(s)
Canales Iónicos/metabolismo , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/metabolismo , Molécula de Interacción Estromal 1/metabolismo , Animales , Señalización del Calcio/fisiología , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Células HEK293 , Humanos , Masculino , Ratones Endogámicos C57BL , Complejos Multiproteicos/metabolismo , Proteínas de Neoplasias/genética , Proteína ORAI1/genética , Técnicas de Placa-Clamp , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Molécula de Interacción Estromal 1/genética
5.
Front Immunol ; 11: 613194, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33391284

RESUMEN

Canonical transient receptor potential (TRPC) channels are considered as elements of the immune cell Ca2+ handling machinery. We therefore hypothesized that TRPC photopharmacology may enable uniquely specific modulation of immune responses. Utilizing a recently established TRPC3/6/7 selective, photochromic benzimidazole agonist OptoBI-1, we set out to test this concept for mast cell NFAT signaling. RBL-2H3 mast cells were found to express TRPC3 and TRPC7 mRNA but lacked appreciable Ca2+/NFAT signaling in response to OptoBI-1 photocycling. Genetic modification of the cells by introduction of single recombinant TRPC isoforms revealed that exclusively TRPC6 expression generated OptoBI-1 sensitivity suitable for opto-chemical control of NFAT1 activity. Expression of any of three benzimidazole-sensitive TRPC isoforms (TRPC3/6/7) reconstituted plasma membrane TRPC conductances in RBL cells, and expression of TRPC6 or TRPC7 enabled light-mediated generation of temporally defined Ca2+ signaling patterns. Nonetheless, only cells overexpressing TRPC6 retained essentially low basal levels of NFAT activity and displayed rapid and efficient NFAT nuclear translocation upon OptoBI-1 photocycling. Hence, genetic modification of the mast cells' TRPC expression pattern by the introduction of TRPC6 enables highly specific opto-chemical control over Ca2+ transcription coupling in these immune cells.


Asunto(s)
Mastocitos/metabolismo , Factores de Transcripción NFATC/metabolismo , Transducción de Señal/fisiología , Canales Catiónicos TRPC/metabolismo , Animales , Calcio/metabolismo , Señalización del Calcio/fisiología , Línea Celular Tumoral , Inmunidad/fisiología , Optogenética/métodos , ARN Mensajero/metabolismo , Ratas
6.
Pharmacol Ther ; 200: 13-26, 2019 08.
Artículo en Inglés | MEDLINE | ID: mdl-30974125

RESUMEN

Non-selective cation conductances formed by transient receptor potential canonical (TRPC) proteins govern the function and fate of a wide range of human cell types. In the past decade, evidence has accumulated for a pivotal role of these channels in human diseases, raising substantial interest in their therapeutic targeting. As yet, an appreciable number of small molecules for block and modulation of recombinant TRPC conductances have been identified. However, groundbreaking progress in TRPC pharmacology towards therapeutic applications is lagging behind due to incomplete understanding of their molecular pharmacology and their exact role in disease. A major breakthrough that is expected to overcome these hurdles is the recent success in obtaining high-resolution structure information on TRPC channel complexes and the advent of TRP photopharmacology and optogenetics. Here, we summarize current concepts of enhancing the precision of therapeutic interference with TRPC signaling and TRPC-mediated pathological processes.


Asunto(s)
Fototerapia , Canales de Potencial de Receptor Transitorio/metabolismo , Animales , Humanos , Luz
7.
Cell Calcium ; 79: 57-67, 2019 05.
Artículo en Inglés | MEDLINE | ID: mdl-30831274

RESUMEN

Calcium signalling through store-operated calcium (SOC) entry is of crucial importance for T-cell activation and the adaptive immune response. This entry occurs via the prototypic Ca2+ release-activated Ca2+ (CRAC) channel. STIM1, a key molecular component of this process, is located in the membrane of the endoplasmic reticulum (ER) and is initially activated upon Ca2+ store depletion. This activation signal is transmitted to the plasma membrane via a direct physical interaction that takes place between STIM1 and the highly Ca2+-selective ion channel Orai1. The activation of STIM1 induces an extended cytosolic conformation. This, in turn, exposes the CAD/SOAR domain and leads to the formation of STIM1 oligomers. In this study, we focused on a small helical segment (STIM1 α3, aa 400-403), which is located within the CAD/SOAR domain. We determined this segment's specific functional role in terms of STIM1 activation and Orai1 gating. The STIM1 α3 domain appears not essential for STIM1 to interact with Orai1. Instead, it represents a key domain that conveys STIM1 interaction into Orai1 channel gating. The results of cysteine crosslinking experiments revealed the close proximity of STIM1 α3 to a region within Orai1, which was located at the cytosolic extension of transmembrane helix 3, forming a STIM1-Orai1 gating interface (SOGI). We suggest that the interplay between STIM1 α3 and Orai1 TM3 allows STIM1 coupling to be transmitted into physiological CRAC channel activation.


Asunto(s)
Canales de Calcio Activados por la Liberación de Calcio/metabolismo , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/metabolismo , Molécula de Interacción Estromal 1/metabolismo , Calcio/metabolismo , Células Cultivadas , Clonación Molecular , Células HEK293 , Humanos , Proteínas de Neoplasias/deficiencia , Proteínas de Neoplasias/genética , Proteína ORAI1/deficiencia , Proteína ORAI1/genética , Molécula de Interacción Estromal 1/deficiencia , Molécula de Interacción Estromal 1/genética
8.
Biochim Biophys Acta Mol Cell Res ; 1866(7): 1079-1091, 2019 07.
Artículo en Inglés | MEDLINE | ID: mdl-30408546

RESUMEN

Since calcium (Ca2+) regulates a large variety of cellular signaling processes in a cell's life, precise control of Ca2+ concentrations within the cell is essential. This enables the transduction of information via Ca2+ changes in a time-dependent and spatially defined manner. Here, we review molecular and functional aspects of how the store-operated Ca2+ channel Orai1 creates spatiotemporal Ca2+ microdomains. The architecture of this channel is unique, with a long helical pore and a six-fold symmetry. Energetic barriers within the Ca2+ channel pathway limit permeation to allow an extensive local Ca2+ increase in close proximity to the channel. The precise timing of the Orai1 channel function is controlled by direct binding to STIM proteins upon Ca2+ depletion in the endoplasmic reticulum. These induced Ca2+ microdomains are tailored to, and sufficient for, triggering long-term activation processes, such as transcription factor activation and subsequent gene regulation. We describe the principles of spatiotemporal activation of the transcription factor NFAT and compare its signaling characteristics to those of the autophagy regulating transcription factors, MITF and TFEB.


Asunto(s)
Autofagia/fisiología , Señalización del Calcio/fisiología , Calcio/metabolismo , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/metabolismo , Molécula de Interacción Estromal 1/metabolismo , Transcripción Genética/fisiología , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Humanos , Factor de Transcripción Asociado a Microftalmía/genética , Factor de Transcripción Asociado a Microftalmía/metabolismo , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/metabolismo , Proteínas de Neoplasias/genética , Proteína ORAI1/genética , Molécula de Interacción Estromal 1/genética
9.
Cells ; 7(7)2018 Jul 22.
Artículo en Inglés | MEDLINE | ID: mdl-30037143

RESUMEN

TRPC3 is one of the classical members of the mammalian transient receptor potential (TRP) superfamily of ion channels. TRPC3 is a molecule with intriguing sensory features including the direct recognition of and activation by diacylglycerols (DAG). Although TRPC3 channels are ubiquitously expressed, they appear to control functions of the cardiovascular system and the brain in a highly specific manner. Moreover, a role of TRPC3 in immunity, cancer, and tissue remodeling has been proposed, generating much interest in TRPC3 as a target for pharmacological intervention. Advances in the understanding of molecular architecture and structure-function relations of TRPC3 have been the foundations for novel therapeutic approaches, such as photopharmacology and optochemical genetics of TRPC3. This review provides an account of advances in therapeutic targeting of TRPC3 channels.

10.
Sci Signal ; 10(507)2017 Nov 28.
Artículo en Inglés | MEDLINE | ID: mdl-29184031

RESUMEN

The channel Orai1 requires Ca2+ store depletion in the endoplasmic reticulum and an interaction with the Ca2+ sensor STIM1 to mediate Ca2+ signaling. Alterations in Orai1-mediated Ca2+ influx have been linked to several pathological conditions including immunodeficiency, tubular myopathy, and cancer. We screened large-scale cancer genomics data sets for dysfunctional Orai1 mutants. Five of the identified Orai1 mutations resulted in constitutively active gating and transcriptional activation. Our analysis showed that certain Orai1 mutations were clustered in the transmembrane 2 helix surrounding the pore, which is a trigger site for Orai1 channel gating. Analysis of the constitutively open Orai1 mutant channels revealed two fundamental gates that enabled Ca2+ influx: Arginine side chains were displaced so they no longer blocked the pore, and a chain of water molecules formed in the hydrophobic pore region. Together, these results enabled us to identify a cluster of Orai1 mutations that trigger Ca2+ permeation associated with gene transcription and provide a gating mechanism for Orai1.


Asunto(s)
Membrana Celular/metabolismo , Activación del Canal Iónico/genética , Proteína ORAI1/genética , Activación Transcripcional/genética , Animales , Arginina/metabolismo , Calcio/metabolismo , Drosophila melanogaster , Genómica , Células HCT116 , Células HEK293 , Humanos , Simulación de Dinámica Molecular , Enfermedades Musculares/metabolismo , Mutación , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Proteína ORAI1/metabolismo , Técnicas de Placa-Clamp , Estructura Secundaria de Proteína/genética , Molécula de Interacción Estromal 1/genética , Molécula de Interacción Estromal 1/metabolismo
11.
Biochem Pharmacol ; 145: 64-80, 2017 12 01.
Artículo en Inglés | MEDLINE | ID: mdl-28859968

RESUMEN

Saxagliptin treatment has been associated with increased rate of hospitalization for heart failure in type 2 diabetic patients, though the underlying mechanism(s) remain elusive. To address this, we assessed the effects of saxagliptin on human atrial trabeculae, guinea pig hearts and cardiomyocytes. We found that the primary target of saxagliptin, dipeptidyl peptidase-4, is absent in cardiomyocytes, yet saxagliptin internalized into cardiomyocytes and impaired cardiac contractility via inhibition of the Ca2+/calmodulin-dependent protein kinase II-phospholamban-sarcoplasmic reticulum Ca2+-ATPase 2a axis and Na+-Ca2+ exchanger function in Ca2+ extrusion. This resulted in reduced sarcoplasmic reticulum Ca2+ content, diastolic Ca2+ overload, systolic dysfunction and impaired contractile force. Furthermore, saxagliptin reduced protein kinase C-mediated delayed rectifier K+ current that prolonged action potential duration and consequently QTc interval. Importantly, saxagliptin aggravated pre-existing cardiac dysfunction induced by ischemia/reperfusion injury. In conclusion, our novel results provide mechanisms for the off-target deleterious effects of saxagliptin on cardiac function and support the outcome of SAVOR-TIMI 53 trial that linked saxagliptin with the risk of heart failure.


Asunto(s)
Adamantano/análogos & derivados , Dipéptidos/toxicidad , Dipeptidil Peptidasa 4/metabolismo , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Atrios Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , Adamantano/toxicidad , Anciano , Animales , Línea Celular , Dipeptidil Peptidasa 4/genética , Inhibidores de la Dipeptidil-Peptidasa IV/farmacología , Femenino , Regulación Enzimológica de la Expresión Génica/fisiología , Cardiopatías/metabolismo , Humanos , Masculino , Ratones , Persona de Mediana Edad , Contracción Miocárdica/efectos de los fármacos , Miocitos Cardíacos/enzimología
12.
Biochem Biophys Res Commun ; 472(1): 40-5, 2016 Mar 25.
Artículo en Inglés | MEDLINE | ID: mdl-26902113

RESUMEN

We set out to determine the membrane potential (Vm) of the endothelial cell line EA.hy926 and its sensitivity to the antimycotic amphotericin B (AmB), a commonly used antifungal component in cell culture media. We measured the endothelial Vm under various experimental conditions by patch clamp technique and found that Vm of AmB-treated cells is (-12.1 ± 9.3) mV, while in AmB-untreated (control) cells it is (-57.1 ± 4.1) mV. In AmB-free extracellular solutions, Vm recovered toward control levels and this gain in Vm rapidly dissipated upon re-addition of AmB, demonstrating a rapid and reversible effect of AmB on endothelial Vm. The consequences of AmB dependent alterations in endothelial transmembrane potential were tested at the levels of Ca(2+) signaling, of nucleotide concentrations, and energy metabolism. In AmB-treated cells we found substantially reduced Ca(2+) entry (to about 60% of that in control cells) in response to histamine induced endoplasmic reticulum (ER) Ca(2+) depletion, and diminished the ATP-to-ADP ratio (by >30%). Our data demonstrate a marked and experimentally relevant dependence of basic functional parameters of cultured endothelial cells on the presence of the ionophoric antimycotic AmB. The profound and reversible effects of the widely used culture media component AmB need careful consideration when interpreting experimental data obtained under respective culture conditions.


Asunto(s)
Anfotericina B/toxicidad , Antifúngicos/toxicidad , Células Endoteliales/efectos de los fármacos , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Señalización del Calcio/efectos de los fármacos , Técnicas de Cultivo de Célula/métodos , Línea Celular , Medios de Cultivo/toxicidad , Células Endoteliales/metabolismo , Humanos , Potenciales de la Membrana/efectos de los fármacos , Técnicas de Placa-Clamp
13.
Sci Signal ; 9(412): ra10, 2016 Jan 26.
Artículo en Inglés | MEDLINE | ID: mdl-26814231

RESUMEN

STIM1 (stromal interaction molecule 1) and Orai proteins are the essential components of Ca(2+) release-activated Ca(2+) (CRAC) channels. We focused on the role of cholesterol in the regulation of STIM1-mediated Orai1 currents. Chemically induced cholesterol depletion enhanced store-operated Ca(2+) entry (SOCE) and Orai1 currents. Furthermore, cholesterol depletion in mucosal-type mast cells augmented endogenous CRAC currents, which were associated with increased degranulation, a process that requires calcium influx. Single point mutations in the Orai1 amino terminus that would be expected to abolish cholesterol binding enhanced SOCE to a similar extent as did cholesterol depletion. The increase in Orai1 activity in cells expressing these cholesterol-binding-deficient mutants occurred without affecting the amount in the plasma membrane or the coupling of STIM1 to Orai1. We detected cholesterol binding to an Orai1 amino-terminal fragment in vitro and to full-length Orai1 in cells. Thus, our data showed that Orai1 senses the amount of cholesterol in the plasma membrane and that the interaction of Orai1 with cholesterol inhibits its activity, thereby limiting SOCE.


Asunto(s)
Canales de Calcio/metabolismo , Calcio/metabolismo , Colesterol/metabolismo , Biotinilación , Línea Celular , Membrana Celular/metabolismo , Colesterol Oxidasa/metabolismo , Dicroismo Circular , Fenómenos Electrofisiológicos , Transferencia Resonante de Energía de Fluorescencia , Células HEK293 , Histamina/metabolismo , Humanos , Mastocitos/metabolismo , Mutación , Proteína ORAI1 , Péptidos/metabolismo , Mutación Puntual , Estructura Terciaria de Proteína , Transducción de Señal , Espectrometría de Fluorescencia
14.
Sci Rep ; 5: 8364, 2015 Feb 10.
Artículo en Inglés | MEDLINE | ID: mdl-25666479

RESUMEN

Mechanical interaction between cells - specifically distortion of tensional homeostasis-emerged as an important aspect of breast cancer genesis and progression. We investigated the biophysical characteristics of mechanosensitive ion channels (MSCs) in the malignant MCF-7 breast cancer cell line. MSCs turned out to be the most abundant ion channel species and could be activated by negative pressure at the outer side of the cell membrane in a saturable manner. Assessing single channel conductance (GΛ) for different monovalent cations revealed an increase in the succession: Li(+) < Na(+) < K(+) ≈Rb(+) ≈ Cs(+). Divalent cations permeated also with the order: Ca(2+) < Ba(2+). Comparison of biophysical properties enabled us to identify MSCs in MCF-7 as ion channels formed by the Piezo1 protein. Using patch clamp technique no functional MSCs were observed in the benign MCF-10A mammary epithelial cell line. Blocking of MSCs by GsMTx-4 resulted in decreased motility of MCF-7, but not of MCF-10A cells, underscoring a possible role of Piezo1 in invasion and metastatic propagation. The role of Piezo1 in biology and progression of breast cancer is further substantiated by markedly reduced overall survival in patients with increased Piezo1 mRNA levels in the primary tumor.


Asunto(s)
Neoplasias de la Mama/metabolismo , Movimiento Celular , Canales Iónicos/metabolismo , Mecanotransducción Celular , Proteínas de Neoplasias/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Femenino , Humanos , Canales Iónicos/genética , Transporte Iónico/genética , Células MCF-7 , Invasividad Neoplásica , Proteínas de Neoplasias/genética
15.
Am J Physiol Heart Circ Physiol ; 307(5): H689-700, 2014 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-25015964

RESUMEN

Urocortin 2 (Ucn2) is a cardioactive peptide exhibiting beneficial effects in normal and failing heart. In cardiomyocytes, it elicits cAMP- and Ca(2+)-dependent positive inotropic and lusitropic effects. We tested the hypothesis that, in addition, Ucn2 activates cardiac nitric oxide (NO) signaling and elucidated the underlying signaling pathways and mechanisms. In isolated rabbit ventricular myocytes, Ucn2 caused concentration- and time-dependent increases in phosphorylation of Akt (Ser473, Thr308), endothelial NO synthase (eNOS) (Ser1177), and ERK1/2 (Thr202/Tyr204). ERK1/2 phosphorylation, but not Akt and eNOS phosphorylation, was suppressed by inhibition of MEK1/2. Increased Akt phosphorylation resulted in increased Akt kinase activity and was mediated by corticotropin-releasing factor 2 (CRF2) receptors (astressin-2B sensitive). Inhibition of phosphatidylinositol 3-kinase (PI3K) diminished both Akt as well as eNOS phosphorylation mediated by Ucn2. Inhibition of protein kinase A (PKA) reduced Ucn2-induced phosphorylation of eNOS but did not affect the increase in phosphorylation of Akt. Conversely, direct receptor-independent elevation of cAMP via forskolin increased phosphorylation of eNOS but not of Akt. Ucn2 increased intracellular NO concentration ([NO]i), [cGMP], [cAMP], and cell shortening. Inhibition of eNOS suppressed the increases in [NO]i and cell shortening. When both PI3K-Akt and cAMP-PKA signaling were inhibited, the Ucn2-induced increases in [NO]i and cell shortening were attenuated. Thus, in rabbit ventricular myocytes, Ucn2 causes activation of cAMP-PKA, PI3K-Akt, and MEK1/2-ERK1/2 signaling. The MEK1/2-ERK1/2 pathway is not required for stimulation of NO signaling in these cells. The other two pathways, cAMP-PKA and PI3K-Akt, converge on eNOS phosphorylation at Ser1177 and result in pronounced and sustained cellular NO production with subsequent stimulation of cGMP signaling.


Asunto(s)
Ventrículos Cardíacos/metabolismo , Miocitos Cardíacos/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Óxido Nítrico/metabolismo , Urocortinas/metabolismo , Animales , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Ventrículos Cardíacos/citología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Conejos , Receptores de Hormona Liberadora de Corticotropina/metabolismo , Serina/metabolismo , Transducción de Señal
16.
Int J Cardiol ; 173(3): 472-80, 2014 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-24698234

RESUMEN

BACKGROUND: Prostaglandins (PGs), lipid autacoids derived from arachidonic acid, play a pivotal role during inflammation. PGD2 synthase is abundantly expressed in heart tissue and PGD2 has recently been found to induce cardiomyocyte apoptosis. PGD2 is an unstable prostanoid metabolite; therefore the objective of the present study was to elucidate whether its final dehydration product, 15-deoxy-Δ¹²,¹4-PGJ2 (15d-PGJ2, present at high levels in ischemic myocardium) might cause cardiomyocyte damage. METHODS AND RESULTS: Using specific (ant)agonists we show that 15d-PGJ2 induced formation of intracellular reactive oxygen species (ROS) and phosphorylation of p38 and p42/44 MAPKs via the PGD2 receptor DP2 (but not DP1 or PPARγ) in the murine atrial cardiomyocyte HL-1 cell line. Activation of the DP2-ROS-MAPK axis by 15d-PGJ2 enhanced transcription and translation of TNFα and induced apoptosis in HL-1 cardiomyocytes. Silencing of TNFα significantly attenuated the extrinsic (caspase-8) and intrinsic apoptotic pathways (bax and caspase-9), caspase-3 activation and downstream PARP cleavage and γH2AX activation. The apoptotic machinery was unaffected by intracellular calcium, transcription factor NF-κB and its downstream target p53. Of note, 9,10-dihydro-15d-PGJ2 (lacking the electrophilic carbon atom in the cyclopentenone ring) did not activate cellular responses. Selected experiments performed in primary murine cardiomyocytes confirmed data obtained in HL-1 cells namely that the intrinsic and extrinsic apoptotic cascades are activated via DP2/MAPK/TNFα signaling. CONCLUSIONS: We conclude that the reactive α,ß-unsaturated carbonyl group of 15d-PGJ2 is responsible for the pronounced upregulation of TNFα promoting cardiomyocyte apoptosis. We propose that inhibition of DP2 receptors could provide a possibility to modulate 15d-PGJ2-induced myocardial injury.


Asunto(s)
Apoptosis/fisiología , Sistema de Señalización de MAP Quinasas/fisiología , Miocitos Cardíacos/metabolismo , Prostaglandina D2/análogos & derivados , Receptores Inmunológicos/metabolismo , Receptores de Prostaglandina/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Apoptosis/efectos de los fármacos , Células Cultivadas , Inflamación/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Miocitos Cardíacos/efectos de los fármacos , Prostaglandina D2/farmacología , Receptores Inmunológicos/agonistas , Receptores de Prostaglandina/agonistas
17.
J Biol Chem ; 288(40): 29025-34, 2013 Oct 04.
Artículo en Inglés | MEDLINE | ID: mdl-23943619

RESUMEN

STIM1 and Orai1 represent the two molecular key components of the Ca(2+) release-activated Ca(2+) channels. Their activation involves STIM1 C terminus coupling to both the N terminus and the C terminus of Orai. Here we focused on the extended transmembrane Orai1 N-terminal (ETON, aa73-90) region, conserved among the Orai family forming an elongated helix of TM1 as recently shown by x-ray crystallography. To identify "hot spot" residues in the ETON binding interface for STIM1 interaction, numerous Orai1 constructs with N-terminal truncations or point mutations within the ETON region were generated. N-terminal truncations of the first four residues of the ETON region or beyond completely abolished STIM1-dependent Orai1 function. Loss of Orai1 function resulted from neither an impairment of plasma membrane targeting nor pore damage, but from a disruption of STIM1 interaction. In a complementary approach, we monitored STIM1-Orai interaction via Orai1 V102A by determining restored Ca(2+) selectivity as a consequence of STIM1 coupling. Orai1 N-terminal truncations that led to a loss of function consistently failed to restore Ca(2+) selectivity of Orai1 V102A in the presence of STIM1, demonstrating impairment of STIM1 binding. Hence, the major portion of the ETON region (aa76-90) is essential for STIM1 binding and Orai1 activation. Mutagenesis within the ETON region revealed several hydrophobic and basic hot spot residues that appear to control STIM1 coupling to Orai1 in a concerted manner. Moreover, we identified two basic residues, which protrude into the elongated pore to redound to Orai1 gating. We suggest that several hot spot residues in the ETON region contribute in aggregate to the binding of STIM1, which in turn is coupled to a conformational reorientation of the gate.


Asunto(s)
Canales de Calcio/química , Canales de Calcio/metabolismo , Activación del Canal Iónico , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Potenciales de Acción , Secuencia de Aminoácidos , Aminoácidos/metabolismo , Sitios de Unión , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Datos de Secuencia Molecular , Proteínas Mutantes/metabolismo , Proteína ORAI1 , Unión Proteica , Estructura Terciaria de Proteína , Eliminación de Secuencia/genética , Molécula de Interacción Estromal 1 , Relación Estructura-Actividad
18.
Cell Calcium ; 54(3): 175-85, 2013 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-23800762

RESUMEN

Utilizing a novel molecular model of TRPC3, based on the voltage-gated sodium channel from Arcobacter butzleri (Na(V)AB) as template, we performed structure-guided mutagenesis experiments to identify amino acid residues involved in divalent permeation and gating. Substituted cysteine accessibility screening within the predicted selectivity filter uncovered amino acids 629-631 as the narrowest part of the permeation pathway with an estimated pore diameter of < 5.8Å. E630 was found to govern not only divalent permeability but also sensitivity of the channel to block by ruthenium red. Mutations in a hydrophobic cluster at the cytosolic termini of transmembrane segment 6, corresponding to the S6 bundle crossing structure in Na(V)AB, distorted channel gating. Removal of a large hydrophobic residue (I667A or I667E) generated channels with approximately 60% constitutive activity, suggesting I667 as part of the dynamic structure occluding the permeation path. Destabilization of the gate was associated with reduced Ca2+ permeability, altered cysteine cross-linking in the selectivity filter and promoted channel block by ruthenium red. Collectively, we present a structural model of the TRPC3 permeation pathway and localize the channel's selectivity filter and the occluding gate. Moreover, we provide evidence for allosteric coupling between the gate and the selectivity filter in TRPC3.


Asunto(s)
Modelos Moleculares , Canales Catiónicos TRPC/metabolismo , Regulación Alostérica , Secuencia de Aminoácidos , Arcobacter/metabolismo , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Células HEK293 , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Técnicas de Placa-Clamp , Estructura Terciaria de Proteína , Rojo de Rutenio/farmacología , Electricidad Estática , Canales Catiónicos TRPC/química , Canales Catiónicos TRPC/genética
19.
Acta Biomater ; 8(8): 2953-62, 2012 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-22522133

RESUMEN

Control of endothelial phenotype involves a variety of signaling pathways and transcriptional regulators, including the junctional protein ß-catenin. This multifunctional signaling molecule is part of adhesion contacts in the endothelium and is able to translocate into the nucleus to activate genetic programs and control proliferation and the fate of the cells. We investigated the influence of laser-generated nanopatterns on polymeric cell culture substrates on endothelial tissue architecture, proliferation and ß-catenin signaling. For our experiments human microvascular endothelial cells or CD34(+) endothelial progenitor cells, isolated from human adipose tissue, were cultured on polyethylene terephthalate (PET) substrates with oriented nanostructures with lateral periodicities of 1.5 µm and 300 nm, respectively. The surface topography and chemistry of the PET substrates were characterized by electron microscopy, atomic force microscopy, water contact angle measurement and X-ray photoelectron spectroscopy. Analysis of cell phenotype markers as well as ß-catenin signaling revealed that short-term culture of endothelial cells on nanostructured substrates generates a proliferative cell phenotype associated with nuclear accumulation of ß-catenin and activation of specific ß-catenin target genes. The effects of the nanostructures were not directly correlated with nanostructure-induced alignment of cells and were also clearly distinguishable from the effects of altered PET surface chemistry due to photomodification. In summary, we present a novel mechanism of surface topology-dependent control of transcriptional programs in mature endothelium and endothelial progenitor cells.


Asunto(s)
Células Endoteliales/citología , Células Endoteliales/metabolismo , Nanoestructuras/química , Tereftalatos Polietilenos/farmacología , Transducción de Señal/genética , Transcripción Genética/efectos de los fármacos , beta Catenina/genética , Adhesión Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Ciclina D1/metabolismo , Células Endoteliales/efectos de los fármacos , Células Endoteliales/ultraestructura , Humanos , Microscopía de Fuerza Atómica , Microvasos/citología , Nanoestructuras/ultraestructura , Espectroscopía de Fotoelectrones , Tereftalatos Polietilenos/química , Transporte de Proteínas/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Agua/química , beta Catenina/metabolismo
20.
BJU Int ; 110(10): 1455-62, 2012 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-22508007

RESUMEN

OBJECTIVES: To examine the acute effects of sunitinib on inotropic function, intracellular Ca(2+) transients, myofilament Ca(2+) sensitivity and generation of reactive oxygen species (ROS) in human multicellular myocardium and isolated mouse cardiomyocytes. To search for microRNAs as suitable biomarkers for indicating toxic cardiac effects. PATIENTS AND METHODS: After exposure to sunitinib (0.1-10 µg/mL) developed force, diastolic tension and kinetic variables were assessed in isolated human myocardium. Changes in myocyte sarcomere length, whole-cell calcium transients, myofilament force-Ca(2+) relationship, and ROS generation were examined in isolated ventricular mouse cardiomyocytes. Microarray and realtime-PCR were used to screen for differentially expressed microRNAs in cultured cardiomyocytes that were exposed for 24 h to sunitinib. RESULTS: We found that higher concentrations of sunitinib (1 and 10 µg/mL) decreased developed force at 30 minutes 76.9 + 2.8 and 54.5 + 6.3%, compared to 96.1 + 2.6% in controls (P < 0.01). Sunitinib exposure significantly decreased sarcomere shortening and Ca2+ transients. Myofilament Ca(2+) sensitivity was not altered, while ROS levels were significantly increased after exposure to the drug. MicroRNA expression patterns were not altered by sunitinib. CONCLUSIONS: Sunitinib elicits a dose-dependent negative inotropic effect in myocardium, accompanied by a decline in intracellular Ca(2+) and increased ROS generation. In clinical practice, these cardiotoxic effects should be considered in cases where cardiac concentrations of sunitinib could be increased.


Asunto(s)
Antineoplásicos/efectos adversos , Indoles/efectos adversos , Contracción Miocárdica/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Inhibidores de Proteínas Quinasas/efectos adversos , Pirroles/efectos adversos , Anciano , Animales , Calcio/metabolismo , Relación Dosis-Respuesta a Droga , Espectroscopía de Resonancia por Spin del Electrón , Femenino , Corazón/efectos de los fármacos , Humanos , Técnicas In Vitro , Masculino , Ratones , Ratones Endogámicos C57BL , Miocitos Cardíacos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Sarcómeros/efectos de los fármacos , Sunitinib
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