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1.
J Cardiovasc Electrophysiol ; 21(11): 1276-83, 2010 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-20487124

RESUMEN

UNLABELLED: Effect of Stretch on Conduction and Cx43. INTRODUCTION: In disease states such as heart failure, myocardial infarction, and hypertrophy, changes in the expression and location of Connexin43 (Cx43) occur (Cx43 remodeling), and may predispose to arrhythmias. Stretch may be an important stimulus to Cx43 remodeling; however, it has only been investigated in neonatal cell cultures, which have different physiological properties than adult myocytes. We hypothesized that localized stretch in vivo causes Cx43 remodeling, with associated changes in conduction, mediated by the renin-angiotensin system (RAS). METHODS AND RESULTS: In an open-chest canine model, a device was used to stretch part of the right ventricle (RV) by 22% for 6 hours. Activation mapping using a 312-electrode array was performed before and after stretch. Regional stretch did not change longitudinal conduction velocity (post-stretch vs baseline: 51.5 ± 5.2 vs 55.3 ± 8.1 cm/s, P = 0.24, n = 11), but significantly reduced transverse conduction velocity (28.7 ± 2.5 vs 35.4 ± 5.4 cm/s, P < 0.01). It also reduced total Cx43 expression, by Western blotting, compared with nonstretched RV of the same animal (86.1 ± 32.2 vs 100 ± 19.4%, P < 0.02, n = 11). Cx43 labeling redistributed to the lateral cell borders. Stretch caused a small but significant increase in the proportion of the dephosphorylated form of Cx43 (stretch 9.95 ± 1.4% vs control 8.74 ± 1.2%, P < 0.05). Olmesartan, an angiotensin II blocker, prevented the stretch-induced changes in Cx43 levels, localization, and conduction. CONCLUSION: Myocardial stretch in vivo has opposite effects to that in neonatal myocytes in vitro. Stretch in vivo causes conduction changes associated with Cx43 remodeling that are likely caused by local stretch-induced activation of the RAS.


Asunto(s)
Conexina 43/metabolismo , Sistema de Conducción Cardíaco/fisiología , Contracción Miocárdica/fisiología , Conducción Nerviosa/fisiología , Sistema Renina-Angiotensina/fisiología , Función Ventricular Derecha/fisiología , Animales , Perros , Módulo de Elasticidad/fisiología , Regulación de la Expresión Génica/fisiología , Distribución Tisular
2.
Circ Res ; 92(4): 437-43, 2003 Mar 07.
Artículo en Inglés | MEDLINE | ID: mdl-12600896

RESUMEN

The epicardial border zone (EBZ) of canine infarcts has increased anisotropy because of transverse conduction slowing. It remains unknown whether changes in gap junctional conductance (Gj) accompany the increased anisotropy. Ventricular cell pairs were isolated from EBZ and normal hearts (NZ). Dual patch clamp was used to quantify Gj. At a transjunctional voltage (Vj) of +10 mV, side-to-side Gj of EBZ pairs (9.2+/-3.4 nS, n=16) was reduced compared with NZ side-to-side Gj (109.4+/-23.6 nS, n=14, P<0.001). Gj of end-to-end coupled cells was not reduced in EBZ. Steady-state Gj of both NZ and EBZ showed voltage dependence, described by a two-way Boltzmann function. Half-maximal activation voltage in EBZ was shifted to higher Vj in positive and negative directions. Immunoconfocal planimetry and quantification showed no change in connexin43 per unit cell volume or surface area in EBZ. Decreased side-to-side coupling occurs in EBZ myocytes, independent of reduced connexin43 expression, and is hypothesized to contribute to increased anisotropy and reentrant arrhythmias.


Asunto(s)
Conexina 43/fisiología , Uniones Comunicantes/fisiología , Infarto del Miocardio/fisiopatología , Pericardio/fisiopatología , Animales , Conexina 43/análisis , Perros , Inmunohistoquímica , Potenciales de la Membrana/fisiología , Infarto del Miocardio/patología , Miocitos Cardíacos/química , Miocitos Cardíacos/fisiología , Pericardio/química , Pericardio/patología , Cicatrización de Heridas
3.
BMC Cardiovasc Disord ; 6: 25, 2006 Jun 06.
Artículo en Inglés | MEDLINE | ID: mdl-16756651

RESUMEN

BACKGROUND: Organ transplantation is presently often the only available option to repair a damaged heart. As heart donors are scarce, engineering of cardiac grafts from autologous skeletal myoblasts is a promising novel therapeutic strategy. The functionality of skeletal muscle cells in the heart milieu is, however, limited because of their inability to integrate electrically and mechanically into the myocardium. Therefore, in pursuit of improved cardiac integration of skeletal muscle grafts we sought to modify primary skeletal myoblasts by overexpression of the main gap-junctional protein connexin 43 and to study electrical coupling of connexin 43 overexpressing myoblasts to cardiac myocytes in vitro. METHODS: To create an efficient means for overexpression of connexin 43 in skeletal myoblasts we constructed a bicistronic retroviral vector MLV-CX43-EGFP expressing the human connexin 43 cDNA and the marker EGFP gene. This vector was employed to transduce primary rat skeletal myoblasts in optimised conditions involving a concomitant use of the retrovirus immobilising protein RetroNectin and the polycation transduction enhancer Transfectam. The EGFP-positive transduced cells were then enriched by flow cytometry. RESULTS: More than four-fold overexpression of connexin 43 in the transduced skeletal myoblasts, compared with non-transduced cells, was shown by Western blotting. Functionality of the overexpressed connexin 43 was demonstrated by microinjection of a fluorescent dye showing enhanced gap-junctional intercellular transfer in connexin 43 transduced myoblasts compared with transfer in non-transduced myoblasts. Rat cardiac myocytes were cultured in multielectrode array culture dishes together with connexin 43/EGFP transduced skeletal myoblasts, control non-transduced skeletal myoblasts or alone. Extracellular field action potential activation rates in the co-cultures of connexin 43 transduced skeletal myoblasts with cardiac myocytes were significantly higher than in the co-cultures of non-transduced skeletal myoblasts with cardiac myocytes and similar to the rates in pure cultures of cardiac myocytes. CONCLUSION: The observed elevated field action potential activation rate in the co-cultures of cardiac myocytes with connexin 43 transduced skeletal myoblasts indicates enhanced cell-to-cell electrical coupling due to overexpression of connexin 43 in skeletal myoblasts. This study suggests that retroviral connexin 43 transduction can be employed to augment engineering of the electrocompetent cardiac grafts from patients' own skeletal myoblasts.


Asunto(s)
Conexina 43/genética , Mioblastos Esqueléticos/fisiología , Miocitos Cardíacos/fisiología , Transfección/métodos , Animales , Línea Celular , Conexina 43/metabolismo , Electromiografía , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Masculino , Plásmidos/genética , Ratas , Ratas Wistar , Retroviridae/genética
4.
JACC Clin Electrophysiol ; 2(5): 574-582, 2016 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-27807593

RESUMEN

OBJECTIVES: The purpose of this study was to investigate the effects of enhancing gap junction (GJ) coupling during acute myocardial infarction (MI) on the healed infarct scar morphology and late post-MI arrhythmia susceptibility. BACKGROUND: Increased heterogeneity of myocardial scarring after MI is associated with greater arrhythmia susceptibility. We hypothesized that short-term enhancement of GJ coupling during acute MI can produce more homogeneous infarct scars, reducing late susceptibility to post-MI arrhythmias. METHODS: Following arrhythmic characterization of a rat 4-week post-MI model (n = 24), another 27 Sprague-Dawley rats were randomized to receive rotigaptide to enhance GJ coupling (n = 13) or to saline control (n = 14) by osmotic minipump immediately prior to and for the first 7 days following surgically induced MI. At 4 weeks post-MI, hearts were explanted for ex vivo programmed electrical stimulation (PES) and optical mapping. Heterogeneity of infarct border zone (IBZ) scarring was quantified by histomorphometry. RESULTS: Despite no detectable differences in infarct size at 4 weeks post-MI, rotigaptide-treated hearts had reduced arrhythmia susceptibility during PES (inducibility score for rotigaptide: 2.4 ± 0.8; for control: 5.0 ± 0.6; p = 0.02) and less heterogeneous IBZ scarring (dispersion of IBZ complexity score: rotigaptide: 1.1 ± 0.1; control: 1.4 ± 0.1; p = 0.04), associated with an improvement in IBZ conduction velocity (rotigaptide: 43.1 ± 3.4 cm/s; control: 34.8 ± 2.0 cm/s; p = 0.04). CONCLUSIONS: Enhancement of GJ coupling for only 7 days at the time of acute MI produced more homogeneous IBZ scarring and reduced arrhythmia susceptibility at 4 weeks post-MI. Short-term GJ modulation at the time of MI may represent a novel treatment strategy to modify the healed infarct scar morphology and reduce late post-MI arrhythmic risk.

5.
J Am Coll Cardiol ; 39(1): 116-23, 2002 Jan 02.
Artículo en Inglés | MEDLINE | ID: mdl-11755296

RESUMEN

OBJECTIVES: The aim of this study was to determine the relationship between immunolocalized gap-junctional proteins and human atrial conduction. BACKGROUND: As a determinant of intercellular conductance, gap-junctional coupling is considered to influence myocardial conduction velocity. This study tested the hypothesis that the quantity of immunodetectable atrial gap-junctional proteins, connexin40 (Cx40) and connexin43 (Cx43), are related to atrial conduction velocity in humans. METHODS: Epicardial mapping was performed on 16 patients undergoing cardiac surgery using an array of 56 unipolar electrodes. The conduction velocity was measured over the right atrial free wall during sinus rhythm and at a paced cycle length 500 ms. A biopsy from this region was excised for quantitative confocal immunodetection of Cx40 and Cx43. RESULTS: There was no correlation between conduction velocity and Cx43 signal or total connexin signal (Cx40 + Cx43). Connexin40 signal was inversely correlated with conduction velocity (p = 0.036). However, the relative quantity of connexin immunolabeling (expressed as Cx40/[Cx40+Cx43] or the inverse equivalent Cx43/[Cx40+Cx43]) was strongly associated with conduction velocity during sinus rhythm, such that, as the proportion of Cx40 signal increased (and that for Cx43 decreased), the conduction velocity decreased (p < 0.005, r = -0.66). Furthermore, with paced atrial activation at 500 ms cycle length, the relative quantity of connexin labeling (Cx40/[Cx40+Cx43]) correlated with the rate-related change in atrial conduction velocity (p < 0.02, r = 0.59). CONCLUSIONS: In human right atrium, conduction velocity is inversely related to immunodetectable Cx40 levels. The relative level of connexins 40 and 43 signal is strongly associated with atrial conduction properties, suggesting that interactions between the two connexins may result in novel coupling properties.


Asunto(s)
Función Atrial , Conexina 43/metabolismo , Conexinas/metabolismo , Sistema de Conducción Cardíaco/fisiología , Miocardio/metabolismo , Adulto , Anciano , Humanos , Inmunohistoquímica , Microscopía Confocal , Persona de Mediana Edad , Proteína alfa-5 de Unión Comunicante
6.
Heart Rhythm ; 12(2): 397-408, 2015 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-25444850

RESUMEN

BACKGROUND: Electrogram fractionation and atrial fibrosis are both thought to be pathophysiological hallmarks of evolving persistence of atrial fibrillation (AF), but recent studies in humans have shown that they do not colocalize. The interrelationship and relative roles of fractionation and fibrotic change in AF persistence therefore remain unclear. OBJECTIVE: The aim of the study was to examine the hypothesis that electrogram fractionation with increasing persistence of AF results from localized conduction slowing or block due to changes in atrial connexin distribution in the absence of fibrotic change. METHODS: Of 12 goats, atrial burst pacemakers maintained AF in 9 goats for up to 3 consecutive 4-week periods. After each 4-week period, 3 goats underwent epicardial mapping studies of the right atrium and examination of the atrial myocardium for immunodetection of connexins 43 and 40 (Cx43 and Cx40) and quantification of connective tissue. RESULTS: Despite refractoriness returning to normal in between each 4-week period of AF, there was a cumulative increase in the prevalence of fractionated atrial electrograms during both atrial pacing (control and 1, 2, and 3 months period of AF 0.3%, 1.3% ± 1.5%, 10.6% ± 2%, and 17% ± 5%, respectively; analysis of variance, P < .05) and AF (0.3% ± 0.1%, 2.3% ± 1.2%, 14% ± 2%, and 23% ± 3%; P < .05) caused by colocalized areas of conduction block during both pacing (local conduction velocity <10 cm/s: 0.1% ± 0.1%, 0.3% ± 0.6%, 6.5% ± 3%, and 6.9% ± 4%; P < .05) and AF (1.5% ± 0.5%, 2.7% ± 1.1%, 10.1% ± 1.2%, and 13.6% ± 0.4%; P < .05), associated with an increase in the heterogeneity of Cx40 and lateralization of Cx43 (lateralization scores: 1.75 ± 0.89, 1.44 ± 0.31, 2.85 ± 0.96, and 2.94 ± 0.31; P < .02), but not associated with change in connective tissue content or net conduction velocity. CONCLUSION: Electrogram fractionation with increasing persistence of AF results from slow localized conduction or block associated with changes in atrial connexin distribution in the absence of fibrotic change.


Asunto(s)
Fibrilación Atrial/fisiopatología , Conexinas/metabolismo , Técnicas Electrofisiológicas Cardíacas , Atrios Cardíacos/fisiopatología , Bloqueo Cardíaco/fisiopatología , Sistema de Conducción Cardíaco/fisiopatología , Animales , Fibrilación Atrial/diagnóstico , Fibrilación Atrial/metabolismo , Modelos Animales de Enfermedad , Femenino , Fibrosis , Cabras , Atrios Cardíacos/metabolismo , Bloqueo Cardíaco/diagnóstico , Bloqueo Cardíaco/metabolismo , Sistema de Conducción Cardíaco/metabolismo , Imagen por Resonancia Cinemagnética , Pronóstico
7.
Circ Arrhythm Electrophysiol ; 7(6): 1198-204, 2014 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-25313260

RESUMEN

BACKGROUND: We tested the hypothesis that alterations to action potential conduction velocity (CV) and conduction anisotropy in left ventricular hypertrophy are associated with topographical changes to gap-junction coupling and intracellular conductance by measuring these variables in the same preparations. METHODS AND RESULTS: Left ventricular papillary muscles were excised from aortic-banded or sham-operated guinea-pig hearts. With intracellular stimulating and recording microelectrodes, CV was measured in 3 dimensions with simultaneous conductance mapping with subthreshold stimuli and correlated with quantitative histomorphometry of myocardial architecture and connexin 43 distribution. In hypertrophied myocardium, CV in the longitudinal axis was smaller and transverse velocity was greater compared with control; associated with similar differences of intracellular conductance, consistent with more cell contacts per cell (5.7 ± 0.2 versus 8.1 ± 0.5; control versus hypertrophy), and more intercalated disks mediating side-to-side coupling (8.2 ± 0.2 versus 10.2 ± 0.4 per cell). Intercalated disk morphology and connexin 43 immunolabelling were not different in hypertrophy. Hypertrophied preparations showed local submillimeter (≈250 µm) regions with slow conduction and low intracellular conductance, which, although not affecting CV on the millimeter scale, were consistent with discontinuities from increased microscopical connective tissue content. CONCLUSIONS: With myocardial hypertrophy, altered longitudinal and transverse CV, and greater nonuniformity of CV anisotropy correspond to changes of intracellular conductance. These are associated with alteration of myocardial architecture, specifically the topography of cell-cell coupling and gap-junction connectivity.


Asunto(s)
Potenciales de Acción , Acoplamiento Excitación-Contracción , Hipertrofia Ventricular Izquierda/fisiopatología , Contracción Miocárdica , Músculos Papilares/fisiopatología , Animales , Conexina 43/metabolismo , Conexinas/metabolismo , Modelos Animales de Enfermedad , Técnicas Electrofisiológicas Cardíacas , Cobayas , Hipertrofia Ventricular Izquierda/metabolismo , Hipertrofia Ventricular Izquierda/patología , Masculino , Músculos Papilares/metabolismo , Músculos Papilares/patología , Factores de Tiempo , Proteína alfa-5 de Unión Comunicante
8.
Circ Arrhythm Electrophysiol ; 7(2): 321-9, 2014 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-24610741

RESUMEN

BACKGROUND: The relative roles of the gap-junctional proteins connexin40 (Cx40) and connexin43 (Cx43) in determining human atrial myocardial resistivity is unknown. In addressing the hypothesis that changing relative expression of Cx40 and Cx43 underlies an increase in human atrial myocardial resistivity with age, this relationship was investigated by direct ex vivo measurement of gap-junctional resistivity and quantitative connexin immunoblotting and immunohistochemistry. METHODS AND RESULTS: Oil-gap impedance measurements were performed to determine resistivity of the intracellular pathway (Ri), which correlated with total Cx40 quantification by Western blotting (rs=0.64, P<0.01, n=20). Specific gap-junctional resistivity (Rj) correlated not only with Western immunoquantification of Cx40 (rs=0.63, P=0.01, n=20), but also more specifically, with the Cx40 fraction localized to the intercalated disks on immunohistochemical quantification (rs=0.66, P=0.02, n=12). Although Cx43 expression showed no correlation with resistivity values, the proportional expression of the 2 connexins, (Cx40/[Cx40+Cx43]) correlated with Ri and Rj (rs=0.58, P<0.01 for Ri and rs=0.51, P=0.02 for Rj). Advancing age was associated with a rise in Ri (rs=0.77, P<0.0001), Rj (rs=0.65, P<0.001, n=23), Cx40 quantity (rs=0.54, P=0.01, n=20), and Cx40 gap-junction protein per unit area of en face disk (rs=0.61, P=0.02, n=12). CONCLUSIONS: Cx40 is associated with human right atrial gap-junctional resistivity such that increased total, gap-junctional, and proportional Cx40 expression increases gap-junctional resistivity. Accordingly, advancing age is associated with an increase in Cx40 expression and a corresponding increase in gap-junctional resistivity. These findings are the first to demonstrate this relationship and a mechanistic explanation for changing atrial conduction and age-related arrhythmic tendency.


Asunto(s)
Fibrilación Atrial/metabolismo , Conexinas/biosíntesis , Atrios Cardíacos/metabolismo , Sistema de Conducción Cardíaco/metabolismo , Miocardio/metabolismo , Anciano , Anciano de 80 o más Años , Fibrilación Atrial/patología , Western Blotting , Femenino , Uniones Comunicantes/metabolismo , Atrios Cardíacos/patología , Humanos , Inmunohistoquímica , Masculino , Microscopía Confocal , Persona de Mediana Edad , Miocardio/patología , Proteína alfa-5 de Unión Comunicante
9.
Optometry ; 83(4): 125-6, 2012 Apr 30.
Artículo en Inglés | MEDLINE | ID: mdl-23231435

RESUMEN

BACKGROUND: Neovascularization can occur in various ocular structures including the retina, iris, anterior chamber angle and cornea; however, it rarely occurs in the crystalline lens. Neovascularization results secondary to hypoxic conditions within the eye. A natural balance of angiogenic and antiangiogenic factors are critical for the eye to prevent the formation of neovascularization. Various factors can upset this natural balance, resulting in angiogenesis. Due to the lack of an intrinsic blood supply, intralenticular neovascularization is rare. CASE REPORT: A 61-year-old black male presented with a previous diagnosis of proliferative diabetic retinopathy (PDR). His ocular history included cataract extraction in the left eye (OS) and tractional retinal detachment in both eyes that was surgically repaired OS. His entering visual acuities were NLP OD and 20/400 OS. Upon biomicroscopic evaluation OD, there was evidence of a hypermature cataract with iris neovascularization and neovascularization coursing over and within the anterior capsule of the lens. CONCLUSIONS: Intralenticular neovascularization is a rarely reported ocular complication. We present a patient with uncontrolled diabetes resulting in proliferative diabetic retinopathy, extreme ocular ischemia, and consequent intralenticular neovascularization.


Asunto(s)
Catarata/etiología , Retinopatía Diabética/complicaciones , Cristalino/irrigación sanguínea , Neovascularización Patológica/etiología , Catarata/patología , Catarata/fisiopatología , Retinopatía Diabética/patología , Diagnóstico Diferencial , Humanos , Masculino , Persona de Mediana Edad , Neovascularización Patológica/patología , Neovascularización Patológica/fisiopatología , Agudeza Visual
10.
Eur Urol ; 56(2): 346-53, 2009 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-18602741

RESUMEN

BACKGROUND: During bladder filling, the trigone contracts help keep the ureteral orifices open and the bladder neck shut. The trigone generates spontaneous activity as well as responding to neuromuscular transmitters, but the relationship between these phenomena are unclear. OBJECTIVES: To characterise the cellular mechanisms that regulate and modify spontaneous activity in trigone smooth muscle. DESIGN, SETTING, AND PARTICIPANTS: Muscle strips from the superficial trigone of male guinea-pigs were used for tension experiments and immunofluorescent studies. MEASUREMENTS: In isolated trigonal cells, intracellular Ca(2+) was measured by epifluorescence microscopy using the fluorescent Ca(2+) indicator Fura-2. RESULTS AND LIMITATIONS: Spontaneous intracellular Ca(2+) transients and contractions were observed in trigonal single cells and strips and were significantly higher compared to the bladder dome. Ca-free superfusate and verapamil terminated spontaneity. T-type Ca(2+) channel block with NiCl(2) depressed slightly Ca(2+) transients but not spontaneous contractions. Neither the BK(Ca) channel blocker iberiotoxin nor the SK(Ca) channel blocker apamin had any effect on single cell activity. By contrast, the Cl(-) channel blocker niflumic acid attenuated significantly both Ca(2+) transients and muscle contractions. Agonist stimulation (carbachol, phenylephrine) up-regulated activity. Gap junction labelling (Cx43) was approximately 5 times denser in the trigone than in detrusor smooth muscle. The gap junction blocker 18-beta-glycyrrhetinic acid modulated spontaneous contractions in the trigone but not in the bladder dome. CONCLUSIONS: Trigone myocytes employ membrane L-type-Ca(2+) channels and Cl(-) channels to generate spontaneous activity. Intercellular electrical coupling ensures its propagation and, thus, sustains contraction of the whole trigone.


Asunto(s)
Músculo Liso/fisiología , Vejiga Urinaria/fisiología , Animales , Fenómenos Electrofisiológicos , Cobayas , Técnicas In Vitro , Masculino , Contracción Muscular
11.
Eur Urol ; 55(6): 1440-8, 2009 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-19054608

RESUMEN

BACKGROUND: An increasing body of evidence suggests a possible role of suburothelial myofibroblasts (MFs) in bladder mechanosensation and in the pathophysiology of detrusor overactivity (DO). OBJECTIVE: To determine whether markers of MFs, including gap junction protein connexin43 (Cx43) and c-kit have altered immunohistochemical expression in the suburothelium of patients with neurogenic DO (NDO) or idiopathic DO (IDO) and whether this is affected by successful treatment of DO with botulinum neurotoxin type A (BoNTA). DESIGN, SETTING, AND PARTICIPANTS: Patients with NDO (n=10) or IDO (n=11) were treated in a single-centre, open-label study of intradetrusor BoNTA injections. Control tissue was obtained from 10 patients undergoing pelvic-floor repair procedures who had no overactive bladder (OAB) symptoms. This study is registered with ClinicalTrials.gov, number NCT00662064. INTERVENTIONS: Bladder biopsies performed with flexible cystoscopes were obtained from control subjects and from NDO and IDO patients before BoNTA treatment and at 4 wk and 16 wk after treatment. They were studied with quantitative immunofluorescence using antibodies to connexin 43 (Cx43), vimentin, and c-kit. MEASUREMENTS: Differences in Cx43, vimentin, and c-kit immunoreactivity between control subjects and NDO or IDO patients (primary outcomes). Changes in NDO or IDO, Cx43 immunoreactivity, and c-kit immunoreactivity after BoNTA treatment (secondary outcomes). RESULTS AND LIMITATIONS: Cx43 immunoreactivity was increased in both IDO and NDO patients compared to controls, but remained unchanged after BoNTA treatment. C-kit immunoreactivity was similar in NDO/IDO patients and controls and remained unchanged after BoNTA treatment. CONCLUSIONS: Increased gap junction formation in the suburothelium has been demonstrated in biopsies from humans with DO. It is hypothesised that this change could have a significant role in the pathogenesis of the detrusor abnormality. Successful treatment of NDO or IDO does not appear to be associated with changes in the expression of Cx43 or c-kit on suburothelial MFs.


Asunto(s)
Toxinas Botulínicas Tipo A/administración & dosificación , Conexina 43/metabolismo , Fármacos Neuromusculares/administración & dosificación , Vejiga Urinaria Hiperactiva/tratamiento farmacológico , Vejiga Urinaria Hiperactiva/patología , Adulto , Anciano , Biomarcadores/metabolismo , Biopsia con Aguja , Conexina 43/efectos de los fármacos , Cistoscopía , Femenino , Estudios de Seguimiento , Humanos , Inmunohistoquímica , Inyecciones Intramusculares , Persona de Mediana Edad , Valores de Referencia , Medición de Riesgo , Índice de Severidad de la Enfermedad , Resultado del Tratamiento , Vejiga Urinaria Hiperactiva/metabolismo , Urodinámica/efectos de los fármacos , Urotelio/efectos de los fármacos , Urotelio/metabolismo
12.
J Biol Chem ; 279(36): 37771-8, 2004 Sep 03.
Artículo en Inglés | MEDLINE | ID: mdl-15215250

RESUMEN

Aldosterone induces expression and activation of the GTP-dependent signaling switch K-Ras. This small monomeric G protein is both necessary and sufficient for activation of the epithelial Na(+) channel (ENaC). The mechanism by which K-Ras enhances ENaC activity, however, is uncertain. We demonstrate here that K-Ras activates human ENaC reconstituted in Chinese hamster ovary cells in a GTP-dependent manner. K-Ras influences ENaC activity most likely by affecting open probability. Inhibition of phosphoinositide 3-OH kinase (PI3K) abolished K-Ras actions on ENaC. In contrast, inhibition of other K-Ras effector cascades, including the MAPK and Ral/Rac/Rho cascades, did not affect K-Ras actions on ENaC. Activation of ENaC by K-Ras, moreover, was sensitive to co-expression of dominant negative p85(PI3K). The G12:C40 effector-specific double mutant of Ras, which preferentially activates PI3K, enhanced ENaC activity in a manner sensitive to inhibition of PI3K. Other effector-specific mutants preferentially activating MAPK and RalGDS signaling had no effect. Constitutively active PI3K activated ENaC independent of K-Ras with the effects of PI3K and K-Ras on ENaC not being additive. We conclude that K-Ras activates ENaC via the PI3K cascade.


Asunto(s)
Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/fisiología , Transducción de Señal , Canales de Sodio/metabolismo , Animales , Células CHO , Cricetinae , Canales Epiteliales de Sodio , Guanosina Trifosfato/metabolismo , Microscopía Fluorescente
13.
J Biol Chem ; 279(26): 27729-34, 2004 Jun 25.
Artículo en Inglés | MEDLINE | ID: mdl-15096495

RESUMEN

Activity of the epithelial Na(+) channel (ENaC) is rate-limiting for Na(+) (re)absorption across electrically tight epithelia. ENaC is a heteromeric channel comprised of three subunits, alpha, beta, and gamma, with each subunit contributing to the functional channel pore. The subunit stoichiometry of ENaC remains uncertain with electrophysiology and biochemical experiments supporting both a tetramer with a 2alpha:1beta:1gamma stoichiometry and a higher ordered channel with a 3alpha:3beta:3gamma stoichiometry. Here we used an independent biophysical approach based upon fluorescence resonance energy transfer (FRET) between differentially fluorophore-tagged ENaC subunits to determine the subunit composition of mouse ENaC functionally reconstituted in Chinese hamster ovary and COS-7 cells. We found that when all three subunits were co-expressed, ENaC contained at least two of each type of subunit. Findings showing that ENaC subunits interact with similar subunits in immunoprecipitation studies are consistent with these FRET results. Upon native polyacrylamide gel electrophoresis, moreover, oligomerized ENaC runs predominantly as a single species with a molecular mass of >600 kDa. Because single ENaC subunits have a molecular mass of approximately 90 kDa, these results also agree with the FRET results. The current results as a whole, thus, are most consistent with a higher ordered channel possibly with a 3alpha:3beta:3gamma stoichiometry.


Asunto(s)
Epitelio/metabolismo , Canales de Sodio/química , Canales de Sodio/metabolismo , Animales , Células CHO , Células COS , Chlorocebus aethiops , Cricetinae , Cricetulus , Transferencia Resonante de Energía de Fluorescencia , Expresión Génica , Ratones , Microscopía Fluorescente , Modelos Moleculares , Peso Molecular , Subunidades de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Canales de Sodio/genética
14.
J Biol Chem ; 277(37): 34480-8, 2002 Sep 13.
Artículo en Inglés | MEDLINE | ID: mdl-12093811

RESUMEN

The activity of membrane proteins is controlled, in part, by protein-protein interactions localized to the plasma membrane. In the current study, domains within the epithelial Na(+) channel (ENaC) reactive at the plasma membrane were identified using a novel yeast one-hybrid screen. The cytosolic N terminus of alphaENaC and the cytosolic C termini of alpha-, beta-, and gammaENaC contained domains reactive at the plasma membrane. Fluorescent micrographs of epithelial cells overexpressing fusion proteins of enhanced green fluorescent protein and mENaC cytosolic domains were consistent with those in yeast. A novel membrane reactive domain within the cytosolic C terminus of gamma-mENaC was localized to the 17 amino acids between residues Thr(584)-Pro(600). Two overlapping internalization signals within the C terminus of gamma-mENaC, a WW-binding domain (PY motif) and a tyrosine-based endocytic signal, were additive with respect to decreasing complementation and expression levels of hybrid proteins. Decreases in expression levels of hybrid proteins containing the PY and endocytic motif were reversed with latrunculin A, an inhibitor of endosomal movement. Decreases in complementation and expression levels of hybrid proteins mediated by the combined PY and overlapping endocytic motif proceeded in the absence of established ubiquitination sites within ENaC. In addition, the endocytic motif was active in the absence of the PY motif, demonstrating that these two domains, while possibly interacting, also have discrete functions. The novel domains within the cytosolic N terminus of alphaENaC and the C termini of alpha-, beta-, and gammaENaC identified here are likely to be involved in protein-protein and/or protein-lipid interactions localized to the plasma membrane. We hypothesize that these newly identified domains play a role in modulating ENaC activity.


Asunto(s)
Canales de Sodio/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Células CHO , Membrana Celular/química , Cricetinae , Citoplasma/química , Endocitosis , Canales Epiteliales de Sodio , Datos de Secuencia Molecular , Subunidades de Proteína , Canales de Sodio/fisiología
15.
J Biol Chem ; 278(42): 41367-79, 2003 Oct 17.
Artículo en Inglés | MEDLINE | ID: mdl-12897071

RESUMEN

We used a yeast one-hybrid complementation screen to identify regions within the cytosolic tails of the mouse alpha, beta, and gamma epithelial Na+ channel (ENaC) important to protein-protein and/or protein-lipid interactions at the plasma membrane. The cytosolic COOH terminus of alphaENaC contained a strongly interactive domain just distal to the second transmembrane region (TM2) between Met610 and Val632. Likewise, gammaENaC contained such a domain just distal to TM2 spanning Gln573-Pro600. Interactive domains were also localized within Met1-Gln54 and the last 17 residues of alpha- and betaENaC, respectively. Confocal images of Chinese hamster ovary cells transfected with enhanced green fluorescent fusion proteins of the cytosolic tails of mENaC subunits were consistent with results in yeast. Fusion proteins of the NH2 terminus of alphaENaC and the COOH termini of all three subunits co-localized with a plasma membrane marker. The functional importance of the membrane interactive domain in the COOH terminus of gammaENaC was established with whole-cell patch clamp experiments of wild type (alpha, beta, and gamma) and mutant (alpha, beta, and gammadeltaQ573-P600) mENaC reconstituted in Chinese hamster ovary cells. Mutant channels had about 13% of the activity of wild type channels with 0.33 +/- 0.14 versus 2.5 +/- 0.80 nA of amiloridesensitive inward current at -80 mV. Single channel analysis of recombinant channels demonstrated that mutant channels had a decrease in Po with 0.16 +/- 0.03 versus 0.67 +/- 0.07 for wild type. Mutant gammaENaC associated normally with the other two subunits in co-immunoprecipitation studies and localized to the plasma membrane in membrane labeling experiments and when visualized with evanescent-field fluorescence microscopy. Similar to deletion of Gln573-Pro600, deletion of Gln573-Arg583 but not Thr584-Pro600 decreased ENaC activity. The current results demonstrate that residues within Gln573-Arg583 of gammaENaC are necessary for normal channel gating.


Asunto(s)
Canales de Sodio/química , Canales de Sodio/fisiología , Secuencias de Aminoácidos , Animales , Biotinilación , Western Blotting , Células CHO , Membrana Celular/metabolismo , Cricetinae , Citosol/metabolismo , Electrofisiología , Canales Epiteliales de Sodio , Eliminación de Gen , Ratones , Microscopía Confocal , Microscopía Fluorescente , Mutación , Técnicas de Placa-Clamp , Plásmidos/metabolismo , Pruebas de Precipitina , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/metabolismo , Canales de Sodio/metabolismo , Relación Estructura-Actividad , Temperatura , Transfección , Técnicas del Sistema de Dos Híbridos
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