RESUMEN
Cardiotoxicity, characterized by adverse impacts on normal heart function due to drug exposure, is a significant concern due to the potentially serious side effects associated with various pharmaceuticals. It is essential to detect the cardiotoxicity of a drug as early as possible in the testing phase of a medical composite. Therefore, there is a pressing need for more reliable in vitro models that accurately mimic the in vivo conditions of cardiac biopsies. In a functional beating heart, cardiac muscle cells are under the effect of static and cyclic stretches. It has been demonstrated that cultured cardiac biopsies can benefit from external mechanical loads that resemble the in vivo condition, increasing the probability of cardiotoxicity detection in the early testing stages. In this work, a biosensor is designed and fabricated to allow for stretch monitoring in biopsies and tissue cultures using an innovative sensing mechanism. The detection setup is based on a biocompatible, thin, flexible membrane-where the samples are attached-which is used as an optical waveguide to detect pressure-caused shape changes and stretches. Various prototypes have been fabricated with a cost-effective process, and different measurements have been carried out to experimentally validate the proposed measurement technique. From these evaluations, stretches of up to 1.5% have been measured, but the performed simulations point towards the possibility of expanding the considered technique up to 10-30% stretches.
Asunto(s)
Cardiotoxicidad , Corazón , HumanosRESUMEN
We investigated whether the substrate for nitric oxide (NO) production, extracellular l-arginine, contributes to relaxations induced by activating small (SKCa) conductance Ca2+-activated potassium channels. In endothelial cells, acetylcholine increased 3H-l-arginine uptake, while blocking the SKCa and the intermediate (IKCa) conductance Ca2+-activated potassium channels reduced l-arginine uptake. A blocker of the y+ transporter system, l-lysine also blocked 3H-l-arginine uptake. Immunostaining showed co-localization of endothelial NO synthase (eNOS), SKCa3, and the cationic amino acid transporter (CAT-1) protein of the y+ transporter system in the endothelium. An opener of SKCa channels, cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine (CyPPA) induced large currents in endothelial cells, and concentration-dependently relaxed porcine retinal arterioles. In the presence of l-arginine, concentration-response curves for CyPPA were leftward shifted, an effect unaltered in the presence of low sodium, but blocked by l-lysine in the retinal arterioles. Our findings suggest that SKCa channel activity regulates l-arginine uptake through the y+ transporter system, and we propose that in vasculature affected by endothelial dysfunction, l-arginine administration requires the targeting of additional mechanisms such as SKCa channels to restore endothelium-dependent vasodilatation.
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Arginina/farmacología , Arteriolas/fisiología , Espacio Extracelular/química , Activación del Canal Iónico/efectos de los fármacos , Vasos Retinianos/fisiología , Vasodilatación/efectos de los fármacos , Animales , Arteriolas/efectos de los fármacos , Transportador de Aminoácidos Catiónicos 1/metabolismo , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Óxido Nítrico Sintasa de Tipo III/metabolismo , Pirazoles/farmacología , Pirimidinas/farmacología , Ratas , Vasos Retinianos/efectos de los fármacos , Canales de Potasio de Pequeña Conductancia Activados por el Calcio , PorcinosRESUMEN
Abstract: The epithelial intermediate-conductance calcium/calmodulin-regulated KCa3.1 channel is considered to be a regulator of intestine function by controlling chloride secretion and water/salt balance. Yet, little is known about the functional importance of KCa3.1 in the intestinal epithelium in vivo. Our objective was to determine the impact of epithelial-specific inducible overexpression of a KCa3.1 transgene (KCa3.1+) and of inducible suppression (KCa3.1-) on intestinal homeostasis and function in mice. KCa3.1 overexpression in the duodenal epithelium of doxycycline (DOX)-treated KCa3.1+ mice was 40-fold above the control levels. Overexpression caused an inflated duodenum and doubling of the chyme content. Histology showed conserved architecture of crypts, villi, and smooth muscle. Unaltered proliferating cell nuclear antigen (PCNA) immune reactivity and reduced amounts of terminal deoxynucleotide transferase mediated X-dUTP nick end labeling (TUNEL)-positive apoptotic cells in villi indicated lower epithelial turnover. Myography showed a reduction in the frequency of spontaneous propulsive muscle contractions with no change in amplitude. The amount of stool in the colon was increased and the frequency of colonic contractions was reduced in KCa3.1+ animals. Senicapoc treatment prevented the phenotype. Suppression of KCa3.1 in DOX-treated KCa3.1- mice caused no overt intestinal phenotype. In conclusion, inducible KCa3.1 overexpression alters intestinal functions by increasing the chyme content and reducing spontaneous contractions and epithelial apoptosis. Induction of epithelial KCa3.1 can play a mechanistic role in the process of adaptation of the intestine.
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Duodeno/fisiología , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/metabolismo , Mucosa Intestinal/fisiología , Animales , Digestión , Duodeno/ultraestructura , Eliminación de Gen , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/genética , Mucosa Intestinal/ultraestructura , Ratones , Ratones Endogámicos C57BL , Contracción Muscular , Transgenes , Regulación hacia ArribaRESUMEN
Small/intermediate conductance KCa channels (KCa2/3) are Ca(2+)/calmodulin regulated K(+) channels that produce membrane hyperpolarization and shape neurologic, epithelial, cardiovascular, and immunologic functions. Moreover, they emerged as therapeutic targets to treat cardiovascular disease, chronic inflammation, and some cancers. Here, we aimed to generate a new pharmacophore for negative-gating modulation of KCa2/3 channels. We synthesized a series of mono- and dibenzoates and identified three dibenzoates [1,3-phenylenebis(methylene) bis(3-fluoro-4-hydroxybenzoate) (RA-2), 1,2-phenylenebis(methylene) bis(3-fluoro-4-hydroxybenzoate), and 1,4-phenylenebis(methylene) bis(3-fluoro-4-hydroxybenzoate)] with inhibitory efficacy as determined by patch clamp. Among them, RA-2 was the most drug-like and inhibited human KCa3.1 with an IC50 of 17 nM and all three human KCa2 subtypes with similar potencies. RA-2 at 100 nM right-shifted the KCa3.1 concentration-response curve for Ca(2+) activation. The positive-gating modulator naphtho[1,2-d]thiazol-2-ylamine (SKA-31) reversed channel inhibition at nanomolar RA-2 concentrations. RA-2 had no considerable blocking effects on distantly related large-conductance KCa1.1, Kv1.2/1.3, Kv7.4, hERG, or inwardly rectifying K(+) channels. In isometric myography on porcine coronary arteries, RA-2 inhibited bradykinin-induced endothelium-derived hyperpolarization (EDH)-type relaxation in U46619-precontracted rings. Blood pressure telemetry in mice showed that intraperitoneal application of RA-2 (≤100 mg/kg) did not increase blood pressure or cause gross behavioral deficits. However, RA-2 decreased heart rate by ≈145 beats per minute, which was not seen in KCa3.1(-/-) mice. In conclusion, we identified the KCa2/3-negative-gating modulator, RA-2, as a new pharmacophore with nanomolar potency. RA-2 may be of use to generate structurally new types of negative-gating modulators that could help to define the physiologic and pathomechanistic roles of KCa2/3 in the vasculature, central nervous system, and during inflammation in vivo.
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Bradicardia/inducido químicamente , Vasos Coronarios/efectos de los fármacos , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/antagonistas & inhibidores , Vasodilatación/efectos de los fármacos , Animales , Benzoatos/química , Benzoatos/farmacología , Bradicardia/fisiopatología , Vasos Coronarios/fisiología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/fisiología , Femenino , Células HEK293 , Frecuencia Cardíaca/efectos de los fármacos , Frecuencia Cardíaca/fisiología , Humanos , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/antagonistas & inhibidores , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/fisiología , Activación del Canal Iónico , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Técnicas de Cultivo de Órganos , Bloqueadores de los Canales de Potasio/química , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/fisiología , Vasodilatación/fisiologíaRESUMEN
Small-conductance (KCa2) and intermediate-conductance (KCa3.1) calcium-activated K(+) channels are voltage-independent and share a common calcium/calmodulin-mediated gating mechanism. Existing positive gating modulators like EBIO, NS309, or SKA-31 activate both KCa2 and KCa3.1 channels with similar potency or, as in the case of CyPPA and NS13001, selectively activate KCa2.2 and KCa2.3 channels. We performed a structure-activity relationship (SAR) study with the aim of optimizing the benzothiazole pharmacophore of SKA-31 toward KCa3.1 selectivity. We identified SKA-111 (5-methylnaphtho[1,2-d]thiazol-2-amine), which displays 123-fold selectivity for KCa3.1 (EC50 111 ± 27 nM) over KCa2.3 (EC50 13.7 ± 6.9 µM), and SKA-121 (5-methylnaphtho[2,1-d]oxazol-2-amine), which displays 41-fold selectivity for KCa3.1 (EC50 109 nM ± 14 nM) over KCa2.3 (EC50 4.4 ± 1.6 µM). Both compounds are 200- to 400-fold selective over representative KV (KV1.3, KV2.1, KV3.1, and KV11.1), NaV (NaV1.2, NaV1.4, NaV1.5, and NaV1.7), as well as CaV1.2 channels. SKA-121 is a typical positive-gating modulator and shifts the calcium-concentration response curve of KCa3.1 to the left. In blood pressure telemetry experiments, SKA-121 (100 mg/kg i.p.) significantly lowered mean arterial blood pressure in normotensive and hypertensive wild-type but not in KCa3.1(-/-) mice. SKA-111, which was found in pharmacokinetic experiments to have a much longer half-life and to be much more brain penetrant than SKA-121, not only lowered blood pressure but also drastically reduced heart rate, presumably through cardiac and neuronal KCa2 activation when dosed at 100 mg/kg. In conclusion, with SKA-121, we generated a KCa3.1-specific positive gating modulator suitable for further exploring the therapeutical potential of KCa3.1 activation.
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Benzotiazoles/farmacología , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/metabolismo , Oxazoles/farmacología , Animales , Benzotiazoles/química , Benzotiazoles/farmacocinética , Presión Sanguínea/efectos de los fármacos , Bradiquinina/farmacología , Células COS , Chlorocebus aethiops , Vasos Coronarios/efectos de los fármacos , Vasos Coronarios/fisiología , Cristalografía por Rayos X , Células HEK293 , Humanos , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/genética , Activación del Canal Iónico , Contracción Isométrica , Masculino , Ratones Endogámicos C57BL , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/fisiología , Oxazoles/química , Oxazoles/farmacocinética , Relación Estructura-Actividad , Porcinos , Vasodilatadores/farmacologíaRESUMEN
This work provides a comprehensive characterization of porcine myocardial tissue, combining true biaxial (TBx), simple triaxial shear (STS) and confined compression (CC) tests to analyze its elastic behavior under cyclic loads. We expanded this study to different zones of the ventricular free wall, providing insights into the local behavior along the longitudinal and radial coordinates. The aging impact was also assessed by comparing two age groups (4 and 8 months). Resulting data showed that the myocardium exhibits a highly nonlinear hyperelastic and incompressible behavior. We observed an anisotropy ratio of 2-2.4 between averaged peak stresses in TBx tests and 1-0.59-0.40 orthotropy ratios for normalised fiber-sheet-normal peak stresses in STS tests. We obtained a highly incompressible response, reaching volumetric pressures of 2-7 MPa for perfused tissue in CC tests, with notable differences when fluid drainage was allowed, suggesting a high permeability. Regional analysis showed reduced stiffness and anisotropy (20-25%) at the apical region compared to the medial, which we attributed to differences in the fiber field dispersion. Compressibility also increased towards the epicardium and apical regions. Regarding age-related variations, 8-month animals showed stiffer response (at least 25% increase), particularly in directions where the mechanical stress is absorbed by collagenous fibers (more than 90%), as supported by a histological analysis. Although compressibility of perfused tissue remained unchanged, permeability significantly reduced in 8-month-old animals. Our findings offer new insights into myocardial properties, emphasizing on local variations, which can help to get a more realistic understanding of cardiac mechanics in this common animal model. STATEMENT OF SIGNIFICANCE: In this work, we conducted a comprehensive analysis of the passive mechanical behavior of porcine myocardial tissue through biaxial, triaxial shear, and confined compression tests. Unlike previous research, we investigated the variation in mechanical response across the left ventricular free wall, conventionally assumed homogeneous, revealing differences in terms of stiffness and compressibility. Additionally, we evaluated agerelated effects on mechanical properties by comparing two age groups, observing significant variations in stiffness and permeability. To date, there has been no such indepth exploration of myocardial elastic response and compressibility considering regional variations along the wall and may contribute to a better understanding of the cardiac tissue's passive mechanical response.
RESUMEN
Engineered heart tissues (EHTs) built from human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) showed promising results for cardiac function restoration following myocardial infarction. Nevertheless, human iPSC-CMs have longer action potential and lower cell-to-cell coupling than adult-like CMs. These immature electrophysiological properties favor arrhythmias due to the generation of electrophysiological gradients when hiPSC-CMs are injected in the cardiac tissue. Culturing hiPSC-CMs on three-dimensional (3D) scaffolds can promote their maturation and influence their alignment. However, it is still uncertain how on-scaffold culturing influences the overall electrophysiology of the in vitro and implanted EHTs, as it requires expensive and time consuming experimentation. Here, we computationally investigated the impact of the scaffold design on the EHT electrical depolarization and repolarization before and after engraftment on infarcted tissue. We first acquired and processed electrical recordings from in vitro EHTs, which we used to calibrate the modeling and simulation of in silico EHTs to replicate experimental outcomes. Next, we built in silico EHT models for a range of scaffold pore sizes, shapes (square, rectangular, auxetic, hexagonal) and thicknesses. In this setup, we found that scaffolds made of small (0.2 mm2), elongated (30° half-angle) hexagons led to faster EHT activation and better mimicked the cardiac anisotropy. The scaffold thickness had a marginal role on the not engrafted EHT electrophysiology. Moreover, EHT engraftment on infarcted tissue showed that the EHT conductivity should be at least 5% of that in healthy tissue for bidirectional EHT-myocardium electrical propagation. For conductivities above such threshold, the scaffold made of small elongated hexagons led to the lowest activation time (AT) in the coupled EHT-myocardium. If the EHT conductivity was further increased and the hiPSC-CMs were uniformly oriented parallel to the epicardial cells, the total AT and the repolarization time gradient decreased substantially, thus minimizing the likelihood for arrhythmias after EHT transplantation.
Asunto(s)
Células Madre Pluripotentes Inducidas , Infarto del Miocardio , Humanos , Ingeniería de Tejidos/métodos , Miocitos Cardíacos/fisiología , Miocardio , Arritmias CardíacasRESUMEN
Age-related fibrosis in the left ventricle (LV) has been mainly studied in animals by assessing collagen content. Using second-harmonic generation microscopy and image processing, we evaluated amount, aggregation and spatial distribution of LV collagen in young to old pigs, and middle-age and elder living donors. All collagen features increased when comparing adult and old pigs with young ones, but not when comparing adult with old pigs or middle-age with elder individuals. Remarkably, all collagen parameters strongly correlated with lipofuscin, a biological age marker, in humans. By building patient-specific models of human ventricular tissue electrophysiology, we confirmed that amount and organization of fibrosis modulated arrhythmia vulnerability, and that distribution should be accounted for arrhythmia risk assessment. In conclusion, we characterize the age-associated changes in LV collagen and its potential implications for ventricular arrhythmia development. Consistency between pig and human results substantiate the pig as a relevant model of age-related LV collagen dynamics.
RESUMEN
Aging is the main risk factor for cardiovascular diseases. In humans, cardiac aging remains poorly characterized. Most studies are based on chronological age (CA) and disregard biological age (BA), the actual physiological age (result of the aging rate on the organ structure and function), thus yielding potentially imperfect outcomes. Deciphering the molecular basis of ventricular aging, especially by BA, could lead to major progresses in cardiac research. We aim to describe the transcriptome dynamics of the aging left ventricle (LV) in humans according to both CA and BA and characterize the contribution of microRNAs, key transcriptional regulators. BA is measured using two CA-associated transcriptional markers: CDKN2A expression, a cell senescence marker, and apparent age (AppAge), a highly complex transcriptional index. Bioinformatics analysis of 132 LV samples shows that CDKN2A expression and AppAge represent transcriptomic changes better than CA. Both BA markers are biologically validated in relation to an aging phenotype associated with heart dysfunction, the amount of cardiac fibrosis. BA-based analyses uncover depleted cardiac-specific processes, among other relevant functions, that are undetected by CA. Twenty BA-related microRNAs are identified, and two of them highly heart-enriched that are present in plasma. We describe a microRNA-gene regulatory network related to cardiac processes that are partially validated in vitro and in LV samples from living donors. We prove the higher sensitivity of BA over CA to explain transcriptomic changes in the aging myocardium and report novel molecular insights into human LV biological aging. Our results can find application in future therapeutic and biomarker research.
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Envejecimiento/genética , Biomarcadores/metabolismo , Ventrículos Cardíacos/metabolismo , MicroARNs/genética , Femenino , Humanos , MasculinoRESUMEN
Ion channels have recently attracted attention as potential mediators of skin disease. Here, we explored the consequences of genetically encoded induction of the cell volume-regulating Ca2+-activated KCa3.1 channel (Kcnn4) for murine epidermal homeostasis. Doxycycline-treated mice harboring the KCa3.1+-transgene under the control of the reverse tetracycline-sensitive transactivator (rtTA) showed 800-fold channel overexpression above basal levels in the skin and solid KCa3.1-currents in keratinocytes. This overexpression resulted in epidermal spongiosis, progressive epidermal hyperplasia and hyperkeratosis, itch and ulcers. The condition was accompanied by production of the pro-proliferative and pro-inflammatory cytokines, IL-ß1 (60-fold), IL-6 (33-fold), and TNFα (26-fold) in the skin. Treatment of mice with the KCa3.1-selective blocker, Senicapoc, significantly suppressed spongiosis and hyperplasia, as well as induction of IL-ß1 (-88%) and IL-6 (-90%). In conclusion, KCa3.1-induction in the epidermis caused expression of pro-proliferative cytokines leading to spongiosis, hyperplasia and hyperkeratosis. This skin condition resembles pathological features of eczematous dermatitis and identifies KCa3.1 as a regulator of epidermal homeostasis and spongiosis, and as a potential therapeutic target.
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Eccema/genética , Epidermis/patología , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/genética , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/metabolismo , Queratosis/genética , Piel/metabolismo , Transgenes , Acetamidas/farmacología , Animales , Citocinas/metabolismo , Doxiciclina/farmacología , Eccema/tratamiento farmacológico , Femenino , Homeostasis/genética , Hiperplasia/tratamiento farmacológico , Hiperplasia/genética , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/antagonistas & inhibidores , Queratinocitos/metabolismo , Queratosis/tratamiento farmacológico , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Transactivadores/metabolismo , Compuestos de Tritilo/farmacologíaRESUMEN
Cardiac tissue slices preserve the heterogeneous structure and multicellularity of the myocardium and allow its functional characterization. However, access to human ventricular samples is scarce. We aim to demonstrate that slices from small transmural core biopsies collected from living donors during routine cardiac surgery preserve structural and functional properties of larger myocardial specimens, allowing accurate electrophysiological characterization. In pigs, we compared left ventricular transmural core biopsies with transmural tissue blocks from the same ventricular region. In humans, we analyzed transmural biopsies and papillary muscles from living donors. All tissues were vibratome-sliced. By histological analysis of the transmural biopsies, we showed that tissue architecture and cellular organization were preserved. Enzymatic and vital staining methods verified viability. Optically mapped transmembrane potentials confirmed that action potential duration and morphology were similar in pig biopsies and tissue blocks. Action potential morphology and duration in human biopsies and papillary muscles agreed with published ranges. In both pigs and humans, responses to increasing pacing frequencies and ß-adrenergic stimulation were similar in transmural biopsies and larger tissues. We show that it is possible to successfully collect and characterize tissue slices from human myocardial biopsies routinely extracted from living donors, whose behavior mimics that of larger myocardial preparations both structurally and electrophysiologically.
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Potenciales de Acción , Electrofisiología Cardíaca , Fenómenos Electrofisiológicos , Ventrículos Cardíacos/fisiopatología , Donadores Vivos , Potenciales de la Membrana , Animales , Humanos , PorcinosRESUMEN
BACKGROUND: TRPV4 channels are calcium-permeable cation channels that are activated by several physicochemical stimuli. Accordingly, TRPV4 channels have been implicated in the regulation of osmosensing, mechanotransduction, thermosensation, and epithelial/endothelial barrier functions. Whether TRPV4 is also mechanistically implicated in melanoma cell proliferation is not clear. Here, we hypothesized that TRPV4 is expressed in human melanoma and that pharmacological activation interferes with cell proliferation. METHODOLOGY/PRINCIPAL FINDINGS: TRPV4 functions were studied in melanoma cell lines (A375, SK-MEL-28, MKTBR), immortalized non-cancer keratinocytes (HaCaT), and murine 3T3 fibroblasts by patch-clamp, qRT-PCR, intracellular calcium measurements, cell proliferation, and flow cytometric assays of apoptosis and cell cycle. The selective TRPV4-activator, GSK1016790A, elicited non-selective cation currents with TRPV4-typical current-voltage-relationship in all cell lines. GSK1016790A-induced currents were blocked by the TRPV4-blocker, HC067047. TRPV4 mRNA expression was demonstrated by qRT-PCR. In A375 cells, TRPV4 activation was frequently paralleled by co-activation of calcium/calmodulin-regulated KCa3.1 channels. Light microscopy showed that TRPV4-activation produced rapid cellular disarrangement, nuclear densification, and detachment of a large fraction of all melanoma cell lines and HaCaT cells. TRPV4-activation induced apoptosis and drastically inhibited A375 and HaCaT proliferation that could be partially prevented by HC067047. CONCLUSIONS/SIGNIFICANCE: Our study showed that TRPV4 channels were functionally expressed in human melanoma cell lines and in human keratinocytes. Pharmacological TRPV4 activation in human melanoma cells and keratinocytes caused severe cellular disarrangement, necrosis and apoptosis. Pharmacological targeting of TRPV4 could be an alternative or adjuvant therapeutic strategy to treat melanoma progression and other proliferative skin disorders.
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Apoptosis/efectos de los fármacos , Queratinocitos/patología , Melanoma/patología , Canales Catiónicos TRPV/agonistas , Células 3T3 , Animales , Calcio/metabolismo , Ciclo Celular , Línea Celular , Línea Celular Tumoral , Citometría de Flujo , Humanos , Queratinocitos/metabolismo , Leucina/análogos & derivados , Leucina/farmacología , Melanoma/metabolismo , Ratones , Técnicas de Placa-Clamp , Sulfonamidas/farmacologíaRESUMEN
Protein kinase CK2α, one of the two catalytic isoforms of the protein kinase CK2 has been shown to contribute to tumor development, tumor proliferation and suppression of apoptosis in various malignancies. We conducted this study to investigate CK2 expression in different subtypes of Renal Cell Carcinoma (RCC) and in the benign oncocytoma. qRT-PCR, immunohistochemistry and Western blot analyses revealed that CK2α expression was significantly increased at the mRNA and protein levels in clear cell RCC (ccRCC). Also the kinase activity of CK2 was significantly increased in ccRCC compared to normal renal cortex. Nuclear protein expression of CK2α correlated in univariate analysis with poor Progression Free Survival (HR = 8.11, p = 0.016). Functional analyses (cell proliferation assay) revealed an inhibitory effect of Caki-2 cell growth following CK2 inhibition with CX-4945. Our results suggest that CK2α promotes migration and invasion of ccRCC and therefore could serve as a novel prognostic biomarker and molecular therapeutic target in this type of cancer.
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Adenoma Oxifílico/patología , Carcinoma de Células Renales/patología , Quinasa de la Caseína II/genética , Quinasa de la Caseína II/metabolismo , Neoplasias Renales/patología , Adenoma Oxifílico/mortalidad , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/genética , Carcinoma de Células Renales/mortalidad , Quinasa de la Caseína II/antagonistas & inhibidores , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Femenino , Humanos , Neoplasias Renales/mortalidad , Masculino , Persona de Mediana Edad , Naftiridinas/farmacología , Invasividad Neoplásica/genética , Fenazinas , ARN Mensajero/genéticaRESUMEN
The calcium/calmodulin-gated KCa3.1 channel regulates normal and abnormal mitogenesis by controlling K+-efflux, cell volume, and membrane hyperpolarization-driven calcium-entry. Recent studies suggest modulation of KCa3.1 by omega-3 fatty acids as negative modulators and impaired KCa3.1 functions in the inherited lysosomal storage disorder (LSD), Fabry disease (FD). In the first part of present study, we characterize KCa3.1 in murine and human fibroblasts and test the impact of omega-3 fatty acids on fibroblast proliferation. In the second, we study whether KCa3.1 is altered in the LSDs, FD, and Niemann-Pick disease type C (NPC). Our patch-clamp and mRNA-expression studies on murine and human fibroblasts show functional expression of KCa3.1. KCa currents display the typical pharmacological fingerprint of KCa3.1: Ca2+-activation, potentiation by the positive-gating modulators, SKA-31 and SKA-121, and inhibition by TRAM-34, Senicapoc (ICA-17043), and the negative-gating modulator, 13b. Considering modulation by omega-3 fatty acids we found that α-linolenic acid (α-LA) and docosahexanenoic acid (DHA) inhibit KCa3.1 currents and strongly reduce fibroblast growth. The α-LA-rich linseed oil and γ-LA-rich borage oil at 0.5% produce channel inhibition while α-LA/γ-LA-low oils has no anti-proliferative effect. Concerning KCa3.1 in LSD, mRNA expression studies, and patch-clamp on primary fibroblasts from FD and NPC patients reveal lower KCa3.1-gene expression and membrane expression than in control fibroblasts. In conclusion, the omega-3 fatty acid, α-LA, and α-LA/γ-LA-rich plant oils, inhibit fibroblast KCa3.1 channels and mitogenesis. Reduced fibroblast KCa3.1 functions are a feature and possible biomarker of cell dysfunction in FD and NPC and supports the concept that biased lipid metabolism is capable of negatively modulating KCa3.1 expression.
RESUMEN
Opening of intermediate-conductance calcium-activated potassium channels (KC a 3.1) produces membrane hyperpolarization in the vascular endothelium. Here, we studied the ability of two new KC a 3.1-selective positive-gating modulators, SKA-111 and SKA-121, to (1) evoke porcine endothelial cell KC a 3.1 membrane hyperpolarization, (2) induce endothelium-dependent and, particularly, endothelium-derived hyperpolarization (EDH)-type relaxation in porcine coronary arteries (PCA) and (3) influence coronary artery tone in isolated rat hearts. In whole-cell patch-clamp experiments on endothelial cells of PCA (PCAEC), KC a currents evoked by bradykinin (BK) were potentiated ≈7-fold by either SKA-111 or SKA-121 (both at 1 µM) and were blocked by a KC a 3.1 blocker, TRAM-34. In membrane potential measurements, SKA-111 and SKA-121 augmented bradykinin-induced hyperpolarization. Isometric tension measurements in large- and small-calibre PCA showed that SKA-111 and SKA-121 potentiated endothelium-dependent relaxation with intact NO synthesis and EDH-type relaxation to BK by ≈2-fold. Potentiation of the BK response was prevented by KC a 3.1 inhibition. In Langendorff-perfused rat hearts, SKA-111 potentiated coronary vasodilation elicited by BK. In conclusion, our data show that positive-gating modulation of KC a 3.1 channels improves BK-induced membrane hyperpolarization and endothelium-dependent relaxation in small and large PCA as well as in the coronary circulation of rats. Positive-gating modulators of KC a 3.1 could be therapeutically useful to improve coronary blood flow and counteract impaired coronary endothelial dysfunction in cardiovascular disease.
Asunto(s)
Vasos Coronarios/citología , Células Endoteliales/metabolismo , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/efectos de los fármacos , Animales , Bradiquinina/farmacología , Células Cultivadas , Circulación Coronaria/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Regulación de la Expresión Génica , Corazón/efectos de los fármacos , Corazón/fisiología , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/fisiología , Masculino , Óxido Nítrico Sintasa de Tipo III/genética , Óxido Nítrico Sintasa de Tipo III/metabolismo , Oxazoles/farmacología , Técnicas de Placa-Clamp , Pirazoles/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Porcinos , Vasodilatación/efectos de los fármacos , Vasodilatadores/farmacologíaRESUMEN
In traditional herbal medicine, Rock Tea (Jasonia glutinosa) is known for its prophylactic and therapeutic value in various disorders including arterial hypertension. However, the mechanism by which Rock Tea exerts blood pressure-lowering actions has not been elucidated yet. Our aim was to demonstrate vasorelaxing effects of Rock Tea extract and to reveal its possible action mechanism. Isometric myography was conducted on high-K+-precontracted rings from rat thoracic aorta and tested extracts at concentrations of 0.5-5 mg/ml. Whole-cell patch-clamp experiments were performed in rat aortic vascular smooth muscle cells (line A7r5) to determine blocking effects on L-type Ca(2+) channels. Rock Tea extract relaxed the aorta contracted by high [K+] concentration dependently with an EC50 of ≈2.4 mg/ml and produced ≈75 % relaxation at the highest concentration tested. The L-type Ca(2+) channel blocker, verapamil (10(-6) M), had similar effects. Rock Tea extract had no effect in nominally Ca(2+)-free high-K(+) buffer but significantly inhibited contractions to re-addition of Ca(2+). Rock Tea extract inhibited the contractions induced by the L-type Ca(2+) channel activator Bay K 8644 (10(-5) M) and by phenylephrine (10(-6) M). Rock Tea extract and Y-27632 (10(-6) M), Rho-kinase inhibitor, had similar effects and the respective effects were not additive. Patch-clamp experiments demonstrated that Rock Tea extract (2.5 mg/ml) virtually abolished L-type Ca(2+) currents in A7r5. We conclude that Rock Tea extract produced vasorelaxation of rat aorta and that this relaxant effect is mediated by inhibition of L-type Ca(2+) channels. Rock Tea extracts may be of phytomedicinal value for prevention and adjuvant treatment of hypertension and other cardiovascular diseases.
Asunto(s)
Antihipertensivos/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Extractos Vegetales/farmacología , Vasodilatadores/farmacología , Animales , Aorta/efectos de los fármacos , Aorta/fisiopatología , Asteraceae/química , Canales de Calcio Tipo L/metabolismo , Señalización del Calcio , Evaluación Preclínica de Medicamentos , Masculino , Relajación Muscular , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/fisiopatología , Técnicas de Placa-Clamp , Fenilefrina/farmacología , Ratas Wistar , Tés de Hierbas , Vasodilatación , Verapamilo/farmacologíaRESUMEN
BACKGROUND AND PURPOSE: The intermediate conductance calcium/calmodulin-regulated K+ channel KCa 3.1 produces hyperpolarizing K+ currents that counteract depolarizing currents carried by transient receptor potential (TRP) channels, and provide the electrochemical driving force for Cl- and fluid movements. We investigated whether a deficiency in KCa 3.1 (KCa 3.1-/- ) protects against fatal pulmonary circulatory collapse in mice after pharmacological activation of the calcium-permeable TRP subfamily vanilloid type 4 (TRPV4) channels. EXPERIMENTAL APPROACH: An opener of TRPV4 channels, GSK1016790A, was infused in wild-type (wt) and KCa 3.1-/- mice; haemodynamic parameters, histology and pulmonary vascular reactivity were measured; and patch clamp was performed on pulmonary arterial endothelial cells (PAEC). KEY RESULTS: In wt mice, GSK1016790A decreased right ventricular and systemic pressure leading to a fatal circulatory collapse that was accompanied by increased protein permeability, lung haemorrhage and fluid extravasation. In contrast, KCa 3.1-/- mice exhibited a significantly smaller drop in pressure to GSK1016790A infusion, no haemorrhage and fluid water extravasation, and the mice survived. Moreover, the GSK1016790A-induced relaxation of pulmonary arteries of KCa 3.1-/- mice was significantly less than that of wt mice. GSK1016790A induced TRPV4 currents in PAEC from wt and KCa 3.1-/- mice, which co-activated KCa 3.1 and disrupted membrane resistance in wt PAEC, but not in KCa 3.1-/- PAEC. CONCLUSIONS AND IMPLICATIONS: Our findings show that a genetic deficiency of KCa 3.1 channels prevented fatal pulmonary circulatory collapse and reduced lung damage caused by pharmacological activation of calcium-permeable TRPV4 channels. Therefore, inhibition of KCa 3.1channels may have therapeutic potential in conditions characterized by abnormal high endothelial calcium signalling, barrier disruption, lung oedema and pulmonary circulatory collapse.
RESUMEN
BACKGROUND: Ca2+-activated K+ channels have been implicated in cancer cell growth, metastasis, and tumor angiogenesis. Here we hypothesized that high mRNA and protein expression of the intermediate-conductance Ca2+-activated K+ channel, KCa3.1, is a molecular marker of clear cell Renal Cell Carcinoma (ccRCC) and metastatic potential and survival. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed channel expression by qRT-PCR, immunohistochemistry, and patch-clamp in ccRCC and benign oncocytoma specimens, in primary ccRCC and oncocytoma cell lines, as well as in two ccRCC cell lines (Caki-1 and Caki-2). CcRCC specimens contained 12-fold higher mRNA levels of KCa3.1 than oncocytoma specimens. The large-conductance channel, KCa1.1, was 3-fold more highly expressed in ccRCC than in oncocytoma. KCa3.1 mRNA expression in ccRCC was 2-fold higher than in the healthy cortex of the same kidney. Disease specific survival trended towards reduction in the subgroup of high-KCa3.1-expressing tumors (p<0.08 vs. low-KCa3.1-expressing tumors). Progression-free survival (time to metastasis/recurrence) was reduced significantly in the subgroup of high-KCa3.1-expressing tumors (p<0.02, vs. low-KCa3.1-expressing tumors). Immunohistochemistry revealed high protein expression of KCa3.1 in tumor vessels of ccRCC and oncocytoma and in a subset of ccRCC cells. Oncocytoma cells were devoid of KCa3.1 protein. In a primary ccRCC cell line and Caki-1/2-ccRCC cells, we found KCa3.1-protein as well as TRAM-34-sensitive KCa3.1-currents in a subset of cells. Furthermore, Caki-1/2-ccRCC cells displayed functional Paxilline-sensitive KCa1.1 currents. Neither KCa3.1 nor KCa1.1 were found in a primary oncocytoma cell line. Yet KCa-blockers, like TRAM-34 (KCa3.1) and Paxilline (KCa1.1), had no appreciable effects on Caki-1 proliferation in-vitro. CONCLUSIONS/SIGNIFICANCE: Our study demonstrated expression of KCa3.1 in ccRCC but not in benign oncocytoma. Moreover, high KCa3.1-mRNA expression levels were indicative of low disease specific survival of ccRCC patients, short progression-free survival, and a high metastatic potential. Therefore, KCa3.1 is of prognostic value in ccRCC.
Asunto(s)
Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/mortalidad , Regulación Neoplásica de la Expresión Génica , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/biosíntesis , Neoplasias Renales/metabolismo , Neoplasias Renales/mortalidad , Proteínas de Neoplasias/biosíntesis , Adulto , Anciano , Carcinoma de Células Renales/patología , Línea Celular Tumoral , Supervivencia sin Enfermedad , Femenino , Humanos , Neoplasias Renales/patología , Masculino , Persona de Mediana Edad , Metástasis de la Neoplasia , Tasa de SupervivenciaRESUMEN
Despite the substantial knowledge on the antidiabetic, antiobesity and antihypertensive actions of tungstate, information on its primary target/s is scarce. Tungstate activates both the ERK1/2 pathway and the vascular voltage- and Ca2+-dependent large-conductance BKαß1 potassium channel, which modulates vascular smooth muscle cell (VSMC) proliferation and function, respectively. Here, we have assessed the possible involvement of BKαß1 channels in the tungstate-induced ERK phosphorylation and its relevance for VSMC proliferation. Western blot analysis in HEK cell lines showed that expression of vascular BKαß1 channels potentiates the tungstate-induced ERK1/2 phosphorylation in a Gi/o protein-dependent manner. Tungstate activated BKαß1 channels upstream of G proteins as channel activation was not altered by the inhibition of G proteins with GDPßS or pertussis toxin. Moreover, analysis of Gi/o protein activation measuring the FRET among heterologously expressed Gi protein subunits suggested that tungstate-targeting of BKαß1 channels promotes G protein activation. Single channel recordings on VSMCs from wild-type and ß1-knockout mice indicated that the presence of the regulatory ß1 subunit was essential for the tungstate-mediated activation of BK channels in VSMCs. Moreover, the specific BK channel blocker iberiotoxin lowered tungstate-induced ERK phosphorylation by 55% and partially reverted (by 51%) the tungstate-produced reduction of platelet-derived growth factor (PDGF)-induced proliferation in human VSMCs. Our observations indicate that tungstate-targeting of BKαß1 channels promotes activation of PTX-sensitive Gi proteins to enhance the tungstate-induced phosphorylation of ERK, and inhibits PDGF-stimulated cell proliferation in human vascular smooth muscle.
Asunto(s)
Señalización del Calcio/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Compuestos de Tungsteno/farmacología , Animales , Calcio/metabolismo , Señalización del Calcio/genética , Quinasas MAP Reguladas por Señal Extracelular/genética , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/genética , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Células HEK293 , Humanos , Canales de Potasio de Gran Conductancia Activados por el Calcio/genética , Sistema de Señalización de MAP Quinasas/genética , Ratones , Ratones Noqueados , Músculo Liso Vascular/citología , Fosforilación/efectos de los fármacos , Factor de Crecimiento Derivado de Plaquetas/farmacologíaRESUMEN
BACKGROUND: Cytochrome P450- and ω-hydrolase products (epoxyeicosatrienoic acids (EETs), hydroxyeicosatetraeonic acid (20-HETE)), natural omega-3 fatty acids (ω3), and pentacyclic triterpenes have been proposed to contribute to a wide range of vaso-protective and anti-fibrotic/anti-cancer signaling pathways including the modulation of membrane ion channels. Here we studied the modulation of intermediate-conductance Ca(2+)/calmodulin-regulated K(+) channels (K(Ca)3.1) by EETs, 20-HETE, ω3, and pentacyclic triterpenes and the structural requirements of these fatty acids to exert channel blockade. METHODOLOGY/PRINCIPAL FINDINGS: We studied modulation of cloned human hK(Ca)3.1 and the mutant hK(Ca)3.1(V275A) in HEK-293 cells, of rK(Ca)3.1 in aortic endothelial cells, and of mK(Ca)3.1 in 3T3-fibroblasts by inside-out and whole-cell patch-clamp experiments, respectively. In inside-out patches, Ca(2+)-activated hK(Ca)3.1 were inhibited by the ω3, DHA and α-LA, and the ω6, AA, in the lower µmolar range and with similar potencies. 5,6-EET, 8,9-EET, 5,6-DiHETE, and saturated arachidic acid, had no appreciable effects. In contrast, 14,15-EET, its stable derivative, 14,15-EEZE, and 20-HETE produced channel inhibition. 11,12-EET displayed less inhibitory activity. The K(Ca)3.1(V275A) mutant channel was insensitive to any of the blocking EETs. Non-blocking 5,6-EET antagonized the inhibition caused by AA and augmented cloned hK(Ca)3.1 and rK(Ca)3.1 whole-cell currents. Pentacyclic triterpenes did not modulate K(Ca)3.1 currents. CONCLUSIONS/SIGNIFICANCE: Inhibition of K(Ca)3.1 by EETs (14,15-EET), 20-HETE, and ω3 critically depended on the presence of electron double bonds and hydrophobicity within the 10 carbons preceding the carboxyl-head of the molecules. From the physiological perspective, metabolism of AA to non-blocking 5,6,- and 8,9-EET may cause AA-de-blockade and contribute to cellular signal transduction processes influenced by these fatty acids.