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INTRODUCTION: Limited data were published on the management of direct oral anticoagulants in the insertion of pacemaker and cardiac monitoring devices. This study describes the management and outcomes of edoxaban, a direct oral factor Xa inhibitor, in patients undergoing pacemaker or monitoring device implantation in routine clinical practice. METHODS AND RESULTS: EMIT-AF/VTE collected data of patients from Europe, Korea, and Taiwan. Timing and duration of periprocedural interruption of edoxaban were at the treating physician's discretion. Pacemakers or monitoring devices were implanted into 136 patients who were evaluated from 5 days pre- until 30 days post-procedure. The primary outcomes were the incidences of acute thromboembolic events (ATE), ischemic events, and International Society on Thrombosis and Haemostasis-defined Major Bleeding; secondary outcomes included incidence of clinically relevant non-major bleeding (CRNMB) and perioperative edoxaban interruption times. Conformance with European Heart Rhythm Association (EHRA) Guidance on interruption of direct oral anticoagulant therapy was variable: of the cardiac monitoring device patients, where no interruption of therapy would be expected, nonetheless, 62.5% had interruption of treatment, whereas in pacemaker procedures, where interruption would be expected, 23.4% had no interruption. No ATE or ischemic events occurred. One case of CRNMB and two of minor bleeding occurred. All bleedings occurred more than 3 days after the procedure. CONCLUSION/RELEVANCE: The periprocedural complication risk for edoxaban treated patients undergoing pacemaker or invasive cardiac monitoring implantation was low. This population of patients was well managed in routine practice.
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Dispositivos de Terapia de Resincronización Cardíaca , Inhibidores del Factor Xa/administración & dosificación , Hemorragia/epidemiología , Isquemia/epidemiología , Marcapaso Artificial , Piridinas/administración & dosificación , Tiazoles/administración & dosificación , Tromboembolia/epidemiología , Anciano , Anticoagulantes/administración & dosificación , Europa (Continente)/epidemiología , Femenino , Humanos , Masculino , Estudios Prospectivos , República de Corea/epidemiología , Factores de Riesgo , Taiwán/epidemiologíaRESUMEN
Xin Su Ning (XSN), a China patented and certified multi-herbal medicine, has been available in China since 2005 for treating cardiac ventricular arrhythmia including arrhythmia induced by ischemic heart diseases and viral myocarditis, without adverse reactions being reported. It is vitally important to discover pharmacologically how XSN as a multicomponent medicine exerts its clinical efficacy, and whether the therapeutic effect of XSN can be verified by standard clinical trial studies. In this paper we report our discoveries in a cellular electrophysiological study and in a three-armed, randomized, double-blind, placebo-controlled, parallel-group, multicenter trial. Conventional electrophysiological techniques were used to study the cellular antiarrhythmic mechanism of XSN. Data was then modeled with computational simulation of human action potential (AP) of the cardiac ventricular myocytes. The clinical trial was conducted with a total of 861 eligible participants randomly assigned in a ratio of 2:2:1 to receive XSN, mexiletine, or the placebo for 4 weeks. The primary and secondary endpoint was the change of premature ventricular contraction (PVC) counts and PVC-related symptoms, respectively. This trial was registered in the Chinese Clinical Trial Register Center (ChiCTR-TRC-14004180). We found that XSN prolonged AP duration of the ventricular myocytes in a dose-dependent, reversible manner and blocked potassium channels. Patients in XSN group exhibited significant total effective responses in the reduction of PVCs compared to those in the placebo group (65.85% vs. 27.27%, P < 0.0001). No severe adverse effects attributable to XSN were observed. In conclusion, XSN is an effective multicomponent antiarrhythmic medicine to treat PVC without adverse effect in patients, which is convincingly supported by its class I & III pharmacological antiarrhythmic mechanism of blocking hERG potassium channels and hNaV1.5 sodium channel reported in our earlier publication and prolongs AP duration both in ventricular myocytes and with computational simulation of human AP presented in this report.
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BACKGROUND: Guidance for periprocedural anticoagulant management is mainly based on limited data from Phase III or observational studies and expert opinion. HYPOTHESIS: EMIT-AF/VTE was designed to document the risks of bleeding and thromboembolic events in more than 1000 patients on edoxaban undergoing diagnostic and therapeutic procedures in clinical practice. METHODS: Routine care in a multinational multicenter, prospective observational study. Participants were adult patients with atrial fibrillation and/or venous thromboembolism treated with edoxaban for stroke prevention or for secondary prevention in venous thromboembolic disease, undergoing a wide range of diagnostic and therapeutic procedures. Edoxaban therapy was interrupted periprocedurally at the treating physician's discretion. Patients were evaluated from 5 days pre- until 30 days postprocedure. Primary outcome was the incidence of International Society on Thrombosis and Haemostasis defined major bleeding; secondary outcomes included incidence of clinically relevant non-major bleeding, acute coronary syndrome, and acute thromboembolic events. RESULTS: Outcomes and management are reported for the first procedures in 1155 unselected patients. Five cases of major bleeding (0.4%) and eight of clinically relevant non-major bleeding (0.7%) were documented, five (38%) of which occurred outside the period of likely edoxaban effect (last edoxaban dose ≥3 days prior to bleeding). Five (0.4%) deaths from any cause, seven acute thromboembolic events (0.6%) including two cardiac deaths (0.2%) in six patients, and one acute coronary event (0.1%) occurred. CONCLUSIONS: The periprocedural bleeding and acute thromboembolic event risks for patients treated with edoxaban were low. This can help inform both clinical routine and guidelines for the periprocedural management of edoxaban.
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Anticoagulantes/uso terapéutico , Fibrilación Atrial/tratamiento farmacológico , Fibrilación Atrial/cirugía , Inhibidores del Factor Xa/uso terapéutico , Piridinas/uso terapéutico , Tiazoles/uso terapéutico , Adulto , Ablación por Catéter , Femenino , Hemorragia/inducido químicamente , Humanos , Masculino , Persona de Mediana Edad , Periodo Perioperatorio , Estudios Prospectivos , Tromboembolia/inducido químicamente , Resultado del TratamientoRESUMEN
PURPOSE: The Tufts Center for the Study of Drug Development (CSDD) and the Drug Information Association (DIA) in collaboration with 8 pharmaceutical and biotechnology companies conducted a study examining the adoption and effect of artificial intelligence (AI), such as machine learning, on drug development. The study was conducted to clarify and understand AI adoption across the industry and to gather detailed insights into the spectrum of activities included in the definition of AI. The study investigated and identified analytical platforms and innovations across pharmaceutical and biotechnology companies currently being used or planned for in the future. METHODS: A 2-part method was used that comprised in-depth interviews with AI industry experts and a global survey conducted across pharmaceutical and biotechnology organizations. Eleven in-depth interviews focused on use and implementation of AI across drug development. The survey assessed use of AI and included perceptions about current and future use. The survey also examined technology definitions, assessment of organizational and personal AI expertise, and use of partnerships. A total of 402 responses, including data from 217 unique organizations, were analyzed. FINDINGS: Although 7 in 10 respondents reported using AI in some capacity, a wide range of use was reported by AI type. Patient selection and recruitment for clinical studies was the most commonly reported AI activity, with 34 respondents currently using AI for this activity. In addition, identification of medicinal products data gathering was the top activity being piloted or in the planning stages, reported by 49 respondents. The study also revealed that the most significant challenges to AI implementation included staff skills (55%), data structure (52%), and budgets (49%). Nearly 60% of respondents noted planned increases in staff within 1-2 years to support AI use or implementation. IMPLICATIONS: Despite the challenges to AI implementation, the survey revealed that most organizations use AI in some capacity and that it is important to the success of an organization's workforce. Many organizations reported expectations for increasing staff as implementation of AI expands. Further research should examine the changing development landscape as the role of AI evolves.
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Inteligencia Artificial , Desarrollo de Medicamentos , Biotecnología , Industria Farmacéutica , Humanos , Encuestas y CuestionariosRESUMEN
[This corrects the article DOI: 10.3389/fphar.2019.00070.].
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Xin Su Ning (XSN) is a China patented and certified herbal medicine used to treat premature ventricular contractions (PVCs) since 2005. A recent completed clinical trial of 861 patients showed that XSN had similar PVC inhibition rate to the class I antiarrhythmic drug mexiletine, at 65.85% for XSN and 63.10% for mexiletine. We have previously reported that XSN prolongs action potential duration (APD) and suppresses action potential amplitude (APA) of the cardiac ventricular myocytes. In this report we aim to reveal the effect of XSN on the ionic channels that govern APD and APA, which would help to explain the cellular electrophysiological mechanism of XSN. Our main findings are: (1) On ECG recorded in isolated rat, in the presence of XSN the amplitude of R wave was significantly decreased and the amplitude of T wave was increased significantly; (2) XSN blocked hNaV1.5 channel stably transfected cell line in a dose-dependent manner with an IC50 of 0.18 ± 0.02 g/L; and (3) XSN suppresses hERG channels in a dose-dependent manner with an IC50 of 0.34 ± 0.01 g/L. In conclusion, the clinical antiarrhythmic efficacy of XSN is based on its class I and Class III antiarrhythmic properties by suppression hNaV1.5 channel and hERG channels, which are directly responsible for XSN's effect on APA suppression and APD prolongation.
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A 57-year-old African American woman was being treated at our clinic for neurogenic urinary incontinence (UI). The UI, which occurred day and night, began 2 years earlier following a laminectomy of vertebrae C3 to C6 with spinal fusion of C3 to C7 for cervical spinal stenosis. The UI persisted despite physical therapy and trials of oxybutynin and imipramine. Since the surgery, the patient had also been experiencing chronic (debilitating) neuropathic pain in both legs, and the sensation of incomplete bladder emptying. She denied bowel incontinence or saddle anesthesia. Her prescription medications included hydrocodone-acetaminophen 7.5/325 mg every 6 hours as needed for pain and lisinopril 20 mg/d for essential hypertension. The patient's body mass index was 23.3.
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Acetaminofén/uso terapéutico , Analgésicos Opioides/uso terapéutico , Hidrocodona/uso terapéutico , Neuralgia/tratamiento farmacológico , Neuralgia/etiología , Vértebras Cervicales/cirugía , Combinación de Medicamentos , Femenino , Humanos , Laminectomía/efectos adversos , Persona de Mediana Edad , Resultado del TratamientoRESUMEN
BACKGROUND: Stent-based therapy in the superficial femoral and popliteal arteries in patients with peripheral artery disease is compromised by restenosis and risk of stent fracture or distortion. A novel self-expanding nitinol stent was developed that incorporates an interwoven-wire design (Supera stent, IDEV Technologies, Inc, Webster, TX) to confer greater radial strength, flexibility, and fracture resistance. METHODS AND RESULTS: This prospective, multicenter, investigational device exemption, single-arm trial enrolled 264 patients with symptomatic peripheral artery disease undergoing percutaneous treatment of de novo or restenotic lesions of the superficial femoral or proximal popliteal (femoropopliteal) artery. Freedom from death, target lesion revascularization, or any amputation of the index limb at 30 days (+ 7 days) postprocedure was achieved in 99.2% (258/260) of patients (P < 0.001). Primary patency at 12 months (360 ± 30 days) was achieved in 78.9% (180/228) of the population (P < 0.001). Primary patency by Kaplan-Meier analysis at 12 months (360 days) was 86.3%. No stent fracture was observed by independent core laboratory analysis in the 243 stents (228 patients) evaluated at 12 months. Clinical assessment at 12 months demonstrated improvement by at least 1 Rutherford-Becker category in 88.7% of patients. CONCLUSIONS: The SUPERB Trial, an investigational device exemption study using an interwoven nitinol wire stent in the femoropopliteal artery, achieved the efficacy and safety performance goals predesignated by the Food and Drug Administration. On the basis of the high primary patency rate, absence of stent fracture, and significant improvements in functional and quality-of-life measures, the Supera stent provides safe and effective treatment of femoropopliteal lesions in symptomatic patients with peripheral artery disease. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT00933270.
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Aleaciones , Procedimientos Endovasculares/instrumentación , Arteria Femoral/fisiopatología , Enfermedad Arterial Periférica/terapia , Arteria Poplítea/fisiopatología , Stents , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Enfermedad Arterial Periférica/fisiopatología , Estudios Prospectivos , Calidad de Vida , Resultado del Tratamiento , Grado de Desobstrucción VascularRESUMEN
Turnover of the cartilage extracellular matrix depends exclusively on chondrocytes and varies in response to load and osmolarity fluctuations. Obesity can affect chondrocyte physiology; adipokines, insulin and proinflammatory cytokines levels are all altered in the obese and are related to matrix turnover impairments and thus to osteoarthritis. TRPV4, a mechanosensitive cation channel, is responsible for reacting to hypotonic variations. In this study, the presence and activity of TRPV4 channels in bovine chondrocytes were evaluated using the whole-cell patch-clamp technique and fluorescence measurements to perform characterisations of these channels and to determine intracellular calcium responses. The expression of TRPV4 was determined by RT-PCR. The TRPV4 regulation by hypotonic shock, insulin and adipokines were analysed. Hypoosmolarity induced a Gd(3+)-, ruthenium red-, and HC-067047-sensitive current, predominantly inward, an intracellular Ca(2+) concentration increase and a membrane depolarisation. The current had a reversal potential of +28±4mV and exhibited preferential permeability to Ca(2+); 4αPDD, a specific TRPV4 agonist, evoked the same response. TNFα, IL-1ß, insulin, and, to a lesser degree, leptin and resistin attenuated the TRPV4-mediated effects; in contrast, adiponectin did not affect them. These results confirm the function of TRPV4 in bovine articular chondrocytes and its regulation by obesity-associated mediators.
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Cartílago Articular/metabolismo , Condrocitos/metabolismo , Insulina/farmacología , Interleucina-1beta/farmacología , Canales Catiónicos TRPV/efectos de los fármacos , Canales Catiónicos TRPV/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Animales , Calcio/metabolismo , Cartílago Articular/citología , Cartílago Articular/efectos de los fármacos , Bovinos , Células Cultivadas , Condrocitos/citología , Condrocitos/efectos de los fármacos , Gadolinio/farmacología , Insulina/metabolismo , Interleucina-1beta/metabolismo , Leptina/metabolismo , Leptina/farmacología , Morfolinas/farmacología , Obesidad/metabolismo , Presión Osmótica/efectos de los fármacos , Técnicas de Placa-Clamp , Pirroles/farmacología , Resistina/metabolismo , Resistina/farmacología , Rojo de Rutenio/farmacología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Chronic wounds present a significant societal burden in their cost of care, and they reduce patient quality of life. Key components of wound care include such measures as debridement, irrigation, and wound cleaning. Appropriate care removes necrotic tissue and reduces wound bioburden to enhance wound healing. Physical cleaning with debridement and irrigation is of documented efficacy. Wounds may be washed with water, saline, or Ringer's solution or cleaned with active ingredients, such as hydrogen peroxide, sodium hypochlorite, acetic acid, alcohol, ionized silver preparations, chlorhexidine, polyhexanide/betaine solution, or povidone-iodine--the majority of which are locally toxic and of limited or no proven efficacy in enhancing wound healing. Although the consensus opinion is that these topical cleaning agents should not be routinely used, recent clinical evidence suggests that polyhexanide/betaine may be nontoxic and effective in enhancing wound healing. Further well-designed studies are needed.
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Antiinfecciosos Locales/uso terapéutico , Cicatrización de Heridas/efectos de los fármacos , Heridas y Lesiones/tratamiento farmacológico , Antiinfecciosos Locales/farmacología , Enfermedad Crónica , Humanos , Heridas y Lesiones/enfermeríaRESUMEN
Hypoxia and acidosis are recognized features of inflammatory arthroses. This study describes the effects of IGF-1 and TGF-ß(1) on pH regulatory mechanisms in articular cartilage under hypoxic conditions. Acid efflux, reactive oxygen species (ROS), and mitochondrial membrane potential were measured in equine articular chondrocytes isolated in the presence of serum (10% fetal calf serum), IGF-1 (1, 10, 50, 100 ng/ml) or TGF-ß(1) (0.1, 1, 10 ng/ml) and then exposed to a short-term (3 h) hypoxic insult (1% O(2)). Serum and 100 ng/ml IGF-1 but not TGF-ß(1) attenuated hypoxic regulation of pH homeostasis. IGF-1 appeared to act through mitochondrial membrane potential stabilization and maintenance of intracellular ROS levels in very low levels of oxygen. Using protein phosphorylation inhibitors PD98059 (25 µM) and wortmannin (200 nM) and Western blotting, ERK1/2 and PI-3 kinase pathways are important for the effect of IGF-1 downstream to ROS generation in normoxia but only PI-3 kinase is implicated in hypoxia. These results show that oxygen and growth factors interact to regulate pH recovery in articular chondrocytes by modulating intracellular oxygen metabolites.
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Equilibrio Ácido-Base/efectos de los fármacos , Cartílago Articular/efectos de los fármacos , Condrocitos/efectos de los fármacos , Hipoxia/metabolismo , Factor I del Crecimiento Similar a la Insulina/farmacología , Factor de Crecimiento Transformador beta/farmacología , Androstadienos/farmacología , Animales , Flavonoides/farmacología , Homeostasis/efectos de los fármacos , Caballos , Concentración de Iones de Hidrógeno , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Oxígeno/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , WortmaninaRESUMEN
The unintended intravenous infusion of small volumes of air is common in clinical practice. International Electrotechnical Commission guidelines for infusion pumps permit infusion of up to 1 mL in 15 minutes and discount bubbles smaller than 50 µL. A review of the literature, however, suggests that these limits may be too generous. Neonates and patients with right-to-left cardiac shunts (eg, patent foramen ovale [PFO]) are at risk from lower volumes. Because PFO is prevalent in 20% to 27% of healthy adults and generally asymptomatic, all patients are at risk from small air bubbles, although clinically significant air embolism from intravenous infusion is rare. Attention to good clinical practice and use of an inline air filter should be considered to reduce any risk.
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Accidentes , Embolia Aérea/etiología , Adulto , Embolia Aérea/fisiopatología , HumanosRESUMEN
Polyhexanide and betaine topical solution is used in the management of infected wounds as a cleaning agent. An in vitro study was conducted to examine the antimicrobial effects of a solution containing 0.1% of the antimicrobial agent polyhexanide and 0.1% of the surfactant betaine. Three batches of each product were tested, and culture results of 13 microorganisms were evaluated after 7, 14, and 28 days using USP <51> methodology. Growth reduction was identical at each day following exposure to the solution in all micro-organisms except Aspergillus brasiliensis. A range of 5.3-log to 5.8-log reduction was seen for the following micro-organisms: Staphylococcus epidermidis, Pseudomonas aeruginosa, Serratia marcescens, Candida albicans, S. aureus, vancomycin-resistant Enterococcus faecalis, Proteus mirabilis, Escherichia coli, methicillin-resistant S. aureus, Acinetobacter baumannii, Enterobacter cloacae, and E. faecalis. For A. brasiliensis, reductions were 2.1-log, 2.3-log and 2.8-log at 7, 14, and 28 days, respectively. The results of this study indicate a 4+ log inhibition of activity in 12 of 13 micro-organisms exposed to the solution. Research to elucidate the potential clinical effects of these observations is needed.
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Betaína/farmacología , Biguanidas/farmacología , Heridas y Lesiones/tratamiento farmacológico , Recuento de Colonia Microbiana , Humanos , Técnicas In Vitro , Heridas y Lesiones/microbiologíaRESUMEN
AIM: Our objective was to identify the distribution of the endangered golden-cheeked warbler (Setophaga chrysoparia) in fragmented oak-juniper woodlands by applying a geoadditive semiparametric occupancy model to better assist decision-makers in identifying suitable habitat across the species breeding range on which conservation or mitigation activities can be focused and thus prioritize management and conservation planning. LOCATION: Texas, USA. METHODS: We used repeated double-observer detection/non-detection surveys of randomly selected (n = 287) patches of potential habitat to evaluate warbler patch-scale presence across the species breeding range. We used a geoadditive semiparametric occupancy model with remotely sensed habitat metrics (patch size and landscape composition) to predict patch-scale occupancy of golden-cheeked warblers in the fragmented oak-juniper woodlands of central Texas, USA. RESULTS: Our spatially explicit model indicated that golden-cheeked warbler patch occupancy declined from south to north within the breeding range concomitant with reductions in the availability of large habitat patches. We found that 59% of woodland patches, primarily in the northern and central portions of the warbler's range, were predicted to have occupancy probabilities ≤0.10 with only 3% of patches predicted to have occupancy probabilities >0.90. Our model exhibited high prediction accuracy (area under curve = 0.91) when validated using independently collected warbler occurrence data. MAIN CONCLUSIONS: We have identified a distinct spatial occurrence gradient for golden-cheeked warblers as well as a relationship between two measurable landscape characteristics. Because habitat-occupancy relationships were key drivers of our model, our results can be used to identify potential areas where conservation actions supporting habitat mitigation can occur and identify areas where conservation of future potential habitat is possible. Additionally, our results can be used to focus resources on maintenance and creation of patches that are more likely to harbour viable local warbler populations.
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K(+)-Cl(-) cotransporters (KCCs) play a fundamental role in epithelial cell function, both in the context of ionic homeostasis and also in cell morphology, cell division and locomotion. Unlike other ubiquitously expressed KCC isoforms, expression of KCC2 is widely considered to be restricted to neurons, where it is responsible for maintaining a low intracellular chloride concentration to drive hyperpolarising postsynaptic responses to the inhibitory neurotransmitters GABA and glycine. Here we report a novel finding that KCC2 is widely expressed in several human cancer cell lines including the cervical cancer cell line (SiHa). Membrane biotinylation assays and immunostaining showed that endogenous KCC2 is located on the cell membrane of SiHa cells. To elucidate the role of KCC2 in cervical tumuorigenesis, SiHa cells with stable overexpression or knockdown of KCC2 were employed. Overexpression of KCC2 had no significant effect on cell proliferation but dramatically suppressed cell spreading and stress fibre organization, while knockdown of KCC2 showed opposite effects. In addition, insulin-like growth factor 1 (IGF-1)-induced cell migration and invasiveness were significantly increased by overexpression of KCC2. KCC2-induced cell migration and invasion were not dependent on KCC2 transport function since overexpression of an activity-deficient mutant KCC2 still increased IGF-1-induced cell migration and invasion. Moreover, overexpression of KCC2 significantly diminished the number of focal adhesions, while knockdown of KCC2 increased their number. Taken together, our data establish that KCC2 expression and function are not restricted to neurons and that KCC2 serves to increase cervical tumourigenesis via an ion transport-independent mechanism.
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Movimiento Celular , Simportadores/metabolismo , Neoplasias del Cuello Uterino/metabolismo , Neoplasias del Cuello Uterino/patología , Línea Celular Tumoral , Proliferación Celular , Femenino , Células HeLa , Humanos , Factor I del Crecimiento Similar a la Insulina/farmacología , Transporte Iónico , Invasividad Neoplásica , Simportadores/genética , Transfección , Cotransportadores de K ClRESUMEN
BACKGROUND: Critical thinking is an important characteristic to develop in respiratory care students. METHODS: We used the short-form Watson-Glaser Critical Thinking Appraisal instrument to measure critical-thinking ability in 55 senior respiratory care students in a baccalaureate respiratory care program. We calculated the Pearson correlation coefficient to assess the relationships between critical-thinking score, age, and student performance on the clinical-simulation component of the national respiratory care boards examination. We used chi-square analysis to assess the association between critical-thinking score and educational background. RESULTS: There was no significant relationship between critical-thinking score and age, or between critical-thinking score and student performance on the clinical-simulation component. There was a significant (P = .04) positive association between a strong science-course background and critical-thinking score, which might be useful in predicting a student's ability to perform in areas where critical thinking is of paramount importance, such as clinical competencies, and to guide candidate-selection for respiratory care programs.
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Aptitud/fisiología , Competencia Clínica , Educación de Pregrado en Medicina , Terapia Respiratoria/educación , Estudiantes de Medicina/psicología , Pensamiento/fisiología , Adulto , Certificación , Escolaridad , Femenino , Humanos , Masculino , Adulto JovenRESUMEN
The effect of 5-aminoimidazole-4-carboxamide-ribonucleoside (AICAR) activation of the AMP-activated protein kinase (AMPK) on the transport of the model radiolabeled dipeptide [(3)H]-D-Phe-L-Gln was investigated in the human epithelial colon cancer cell line Caco-2. Uptake and transepithelial fluxes of [(3)H]-D-Phe-L-Gln were carried out in differentiated Caco-2 cell monolayers, and hPepT1 and glucose transporter 2 (GLUT2) protein levels were quantified by immunogold electron microscopy. AICAR treatment of Caco-2 cells significantly inhibited apical [(3)H]-D-Phe-L-Gln uptake, matched by a decrease in brush-border membrane hPepT1 protein but with a concomitant increase in the facilitated glucose transporter GLUT2. A restructuring of the apical brush-border membrane was seen by electron microscopy. The hPepT1-mediated transepithelial (A-to-B) peptide flux across the Caco-2 monolayers showed no significant alteration in AICAR-treated cells. The electrical resistance in the AICAR-treated monolayers was significantly higher compared with control cells. Inhibition of the sodium/hydrogen exchanger 3 (NHE3) had an additive effect to AICAR, suggesting that the AMPK effect is not via NHE3. Fluorescence measurement of intracellular pH showed no reduction in the proton gradient driving PepT1-mediated apical uptake. The reduction in apical hPepT1 protein and dipeptide uptake after AICAR treatment in Caco-2 cells demonstrates a regulatory effect of AMPK on hPepT1, along with an influence on both the microvilli and tight junction structures. The absence of an associated reduction in transepithelial peptide movement implies an additional stimulatory effect of AICAR on the basolateral peptide transport system in these cells. These results provide a link between the hPepT1 transporter and the metabolic state of this model enterocyte.
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Proteínas Quinasas Activadas por AMP/metabolismo , Dipéptidos/metabolismo , Células Epiteliales/enzimología , Mucosa Intestinal/enzimología , Simportadores/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacología , Transporte Biológico , Células CACO-2 , Polaridad Celular , Forma de la Célula , Relación Dosis-Respuesta a Droga , Impedancia Eléctrica , Activación Enzimática , Activadores de Enzimas/farmacología , Células Epiteliales/efectos de los fármacos , Fluorometría , Transportador de Glucosa de Tipo 2/metabolismo , Guanidinas/farmacología , Humanos , Concentración de Iones de Hidrógeno , Mucosa Intestinal/efectos de los fármacos , Cinética , Metacrilatos/farmacología , Microscopía Electrónica de Transmisión , Transportador de Péptidos 1 , Ribonucleótidos/farmacología , Intercambiador 3 de Sodio-Hidrógeno , Intercambiadores de Sodio-Hidrógeno/antagonistas & inhibidores , Intercambiadores de Sodio-Hidrógeno/metabolismoRESUMEN
The KCl cotransporter (KCC) is a major determinant of osmotic homeostasis and plays an emerging role in tumor biology. This study stresses the important role of KCC4 in tumor malignant behavior. Real-time reverse transcription-PCR on samples collected by laser microdissection and immunofluorescent stainings with different KCC isoform antibodies indicate that KCC4 is abundant in metastatic cervical and ovarian cancer tissues. Insulin-like growth factor I (IGF-I) and epidermal growth factor (EGF) stimulate KCC4 recruitment from a presumably inactive cytoplasmic pool of endoplasmic reticulum and Golgi to plasma membrane along actin cytoskeleton that is significantly inhibited by LY294002 and wortmannin. Throughout the trafficking process, KCC4 is incorporated into lipid rafts that function as a platform for the association between KCC4 and myosin Va, an actin-dependent motor protein. KCC4 and ezrin, a membrane cytoskeleton linker, colocalize at lamellipodia of migratory cancer cells. Interference with KCC activity by either an inhibitor or a dominant-negative loss-of-function mutant profoundly suppressed the IGF-I-induced membrane trafficking of KCC4 and the structural interaction between KCC4 and ezrin near the cell surface. Endogenous cancer cell invasiveness was significantly attenuated by small interfering RNA targeting KCC4, and the residual invasiveness was much less sensitive to IGF-I or EGF stimulation. In the metastatic cancer tissues, KCC4 colocalizes with IGF-I or EGF, indicating a likely in vivo stimulation of KCC4 function by growth factors. Thus, blockade of KCC4 trafficking and surface expression may provide a potential target for the prevention of IGF-I- or EGF-dependent cancer spread.
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Membrana Celular/metabolismo , Proteínas Motoras Moleculares/metabolismo , Invasividad Neoplásica , Neoplasias Ováricas/patología , Simportadores/metabolismo , Neoplasias del Cuello Uterino/patología , Adulto , Anciano , Línea Celular Tumoral , Factor de Crecimiento Epidérmico/metabolismo , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Factor I del Crecimiento Similar a la Insulina/metabolismo , Rayos Láser , Microdisección , Persona de Mediana Edad , Neoplasias Ováricas/metabolismo , Transporte de Proteínas , ARN Interferente Pequeño , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Neoplasias del Cuello Uterino/metabolismoRESUMEN
Articular chondrocytes experience low oxygen (O(2)) levels compared with many other tissues, and values fall further in disease states. Chondrocyte intracellular pH (pH(i)) is a powerful modulator of matrix synthesis and is principally regulated by Na(+)-H(+) exchange (NHE). In equine chondrocytes, NHE is inhibited when cells are incubated for 3 h at low O(2), leading to intracellular acidosis. O(2)-dependent changes in reactive oxygen species (ROS) levels appear to underlie this effect. The present study examines whether hypoxia can influence chondrocyte NHE activity and pH(i) over shorter timescales using the pH-sensitive fluoroprobe BCECF in cells isolated not only from equine cartilage but also from bovine tissue. O(2) levels in initially oxygenated solutions gassed with N(2) fell to approximately 1% within 2 h. A progressive fall in pH(i) and acid extrusion capacity was observed, with statistically significant effects (P < 0.05) apparent within 3 h. For equine and bovine cell populations subjected to step change in O(2) by resuspension in hypoxic (1%) solutions, a decline in acid extrusion and pH(i) was observed within 10 min and continued throughout the recording period. This effect represented inhibition of the NHE-mediated fraction of acid extrusion. Cells subjected to hypoxic solutions supplemented with CoCl(2) (100 microM) or antimycin A (100 microM) to raise levels of ROS did not acidify. The conserved nature and rapidity of the response to hypoxia has considerable implications for chondrocyte homeostasis and potentially for the maintenance of cartilage integrity.