Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 13 de 13
Filtrar
1.
Am J Physiol Regul Integr Comp Physiol ; 298(3): R713-9, 2010 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-20053959

RESUMEN

Hypertensive disorders of pregnancy are characterized by systemic and placental inflammation; however, treatment for these conditions has remained elusive. We tested whether administration of the anti-inflammatory cytokine interleukin-10 (IL-10) during pregnancy would attenuate the hypertension, endothelial dysfunction, proteinuria, and inflammation seen in pregnant DOCA/saline-treated (PDS) rats. Normal pregnant (NP) rats and PDS were given daily intraperitoneal injections of recombinant IL-10 from gestational day 13 until death on day 20. Systolic blood pressure, aortic endothelium-dependent relaxation responses, and urinary protein excretion were measured on days 13 and 20 of gestation. Fetal number and development, plasma endothelin-1 levels, serum and placental levels of IFNgamma and IL-10, and aortic and placental levels of platelet endothelial cell adhesion molecule (PECAM) were assessed on gestational day 20. Systolic blood pressure, aortic endothelial dysfunction, and urinary protein excretion were significantly increased at gestational day 13 in PDS rats. However, all of these were restored to NP levels following IL-10 treatment in PDS rats. IL-10 treatment also significantly increased the number of pups per litter in PDS rats and did not further affect fetal development. The beneficial effects of IL-10 in PDS rats were likely mediated by the decreased plasma levels of endothelin-1, decreased levels of circulating and placental IFNgamma, as well as decreased aortic and placental expression of PECAM. These data demonstrate that exogenous IL-10 can normalize blood pressure and endothelial function in pregnancy-induced hypertensive rats and may be beneficial in women with hypertensive disorders of pregnancy.


Asunto(s)
Presión Sanguínea/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Hipertensión Inducida en el Embarazo/tratamiento farmacológico , Inflamación/tratamiento farmacológico , Interleucina-10/farmacología , Animales , Aorta/metabolismo , Presión Sanguínea/inmunología , Anomalías Congénitas , Modelos Animales de Enfermedad , Endotelina-1/sangre , Endotelio Vascular/inmunología , Endotelio Vascular/fisiopatología , Femenino , Hipertensión Inducida en el Embarazo/inmunología , Hipertensión Inducida en el Embarazo/fisiopatología , Inflamación/inmunología , Inflamación/fisiopatología , Interferón gamma/sangre , Interleucina-10/sangre , Tamaño de la Camada/efectos de los fármacos , Masculino , Placenta/metabolismo , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Preeclampsia/tratamiento farmacológico , Preeclampsia/inmunología , Preeclampsia/fisiopatología , Embarazo , Proteinuria/tratamiento farmacológico , Proteinuria/inmunología , Proteinuria/fisiopatología , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/farmacología
2.
J Surg Res ; 152(1): 76-83, 2009 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-18621396

RESUMEN

BACKGROUND: Heart disease is one of the leading causes of death in the United States, killing nearly one million people every year. Inflammatory mediators or cytokines are released following myocardial infarction and ischemia/reperfusion injury. These cytokines, of which interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha (TNF-alpha) are among the most important, propagate the activation of a multitude of signaling pathways, such as the protein kinase C (PKC) and myosin light chain kinase (MLCK) pathways, which lead to deleterious changes in the structure and function of the coronary microvascular endothelium. METHODS: The effects of cytokines on rat heart microvascular endothelial cell monolayer integrity, PKC activity, and adherens junction protein alteration were examined. Further, an in vivo rat coronary ischemia/reperfusion injury model was used to determine vascular leakage and TNF-alpha release. RESULTS: Administration of the above mentioned cytokines to cell monolayers resulted in significant increases in PKC activation, gap formation, and hyperpermeability across the monolayer and beta-catenin phosphorylation/reorganization. Inhibition of conventional PKC and MLCK attenuated permeability increases. Ischemia/reperfusion injury to the left ventricle resulted in TNF-alpha release as well as conventional PKC- and MLCK-dependent protein extravasation from the circulation to the heart tissue. CONCLUSION: These results identify the conventional PKC and MLCK pathways as important factors in coronary endothelial dysfunction elicited by IR injury and cytokine release. Further examination of these molecular signaling cascades has the potential of identifying targets for therapeutic intervention following ischemic events in the heart.


Asunto(s)
Citocinas/metabolismo , Células Endoteliales/enzimología , Insuficiencia Cardíaca/enzimología , Daño por Reperfusión Miocárdica/enzimología , Quinasa de Cadena Ligera de Miosina/metabolismo , Proteína Quinasa C/metabolismo , Animales , Permeabilidad de la Membrana Celular , Células Cultivadas , Ligadura , Masculino , Fosforilación , Ratas , Ratas Sprague-Dawley , beta Catenina/metabolismo
3.
Circ Res ; 90(11): 1214-21, 2002 Jun 14.
Artículo en Inglés | MEDLINE | ID: mdl-12065325

RESUMEN

Neutrophil-induced coronary microvascular leakage represents an important pathophysiological consequence of ischemic and inflammatory heart diseases. The precise mechanism by which neutrophils regulate endothelial barrier function remains to be established. The aim of this study was to examine the microvascular endothelial response to neutrophil activation with a focus on myosin light chain kinase (MLCK)-mediated myosin light chain (MLC) phosphorylation, a regulatory process that controls cell contraction. The apparent permeability coefficient of albumin (Pa) was measured in intact isolated porcine coronary venules. Incubation of the vessels with C5a-activated neutrophils induced a time- and concentration-dependent increase in Pa. The hyperpermeability response was significantly attenuated during inhibition of endothelial MLC phosphorylation with the selective MLCK inhibitor ML-7 and transfection of a specific MLCK-inhibiting peptide. In contrast, transfection of constitutively active MLCK elevated Pa, which was abolished by ML-7. In addition to the vessel study, albumin transendothelial flux was measured in cultured bovine coronary venular endothelial monolayers, which displayed a hyperpermeability response to neutrophils and MLCK in a pattern similar to that in venules. Importantly, neutrophil stimulation caused MLC phosphorylation in endothelial cells in a time course closely correlated with that of the hyperpermeability response. Consistently, the MLCK inhibitors abolished neutrophil-induced MLC phosphorylation. Furthermore, immunohistochemical observation of neutrophil-stimulated endothelial cells revealed an increased staining for phosphorylated MLC in association with contractile stress fiber formation and intercellular gap development. Taken together, the results suggest that endothelial MLCK activation and MLC phosphorylation play an important role in mediating endothelial barrier dysfunction during neutrophil activation.


Asunto(s)
Permeabilidad Capilar/fisiología , Vasos Coronarios/fisiología , Cadenas Ligeras de Miosina/metabolismo , Neutrófilos/fisiología , Animales , Azepinas/farmacología , Permeabilidad Capilar/efectos de los fármacos , Bovinos , Células Cultivadas , Complemento C5a/farmacología , Relación Dosis-Respuesta a Droga , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Inhibidores Enzimáticos/farmacología , Humanos , Quinasa de Cadena Ligera de Miosina/antagonistas & inhibidores , Quinasa de Cadena Ligera de Miosina/genética , Quinasa de Cadena Ligera de Miosina/metabolismo , Naftalenos/farmacología , Neutrófilos/citología , Neutrófilos/efectos de los fármacos , Fosforilación/efectos de los fármacos , Porcinos , Factores de Tiempo , Transfección
4.
Trends Pharmacol Sci ; 25(2): 64-6, 2004 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-15106625

RESUMEN

Inflammatory lung diseases result in high rates of morbidity and mortality. Central to the pathogenesis of these diseases is disruption of endothelial barrier function. Activation of myosin light chain kinase (MLCK) is a key regulatory step in the modulation of endothelial permeability. Recent studies show that mice with selective knockout of the endothelial MLCK are less susceptible to endotoxin-induced acute lung injury and that a new small-molecule inhibitor of MLCK also protects against lung injury.


Asunto(s)
Quinasa de Cadena Ligera de Miosina/antagonistas & inhibidores , Síndrome de Dificultad Respiratoria/etiología , Animales , Humanos , Isoenzimas , Ratones , Quinasa de Cadena Ligera de Miosina/genética , Quinasa de Cadena Ligera de Miosina/fisiología , Fosforilación , Síndrome de Dificultad Respiratoria/prevención & control
5.
Shock ; 37(3): 306-11, 2012 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-22089197

RESUMEN

ß-Catenin, a key regulator of barrier integrity, is an important component of the adherens junctional complex. Although the roles of ß-catenin in maintaining the adherens junctions and Wnt signaling are known, the dynamics of ß-catenin following insult and its potential role in vascular recovery/repair remain unclear. Our objective was to define ß-catenin's dynamics following disruption of the adherens junctional complex and subsequent recovery. Rat lung microvascular endothelial cells were treated with active caspase 3 enzyme, by protein transference method, as an inducer of junctional damage and permeability. The disruption and subsequent recovery of ß-catenin to the adherens junctions were studied via immunofluorescence. Rat lung microvascular endothelial cell monolayers were used to measure hyperpermeability. To understand the role of ß-catenin on nuclear translocation/transcriptional regulation in relationship to the recovery of the adherens junctions, Tcf-mediated transcriptional activity was determined. Active caspase 3 induced a loss of ß-catenin at the adherens junctions at 1 and 2 h followed by its recovery at 3 h. Transference of Bak peptide, an inducer of endogenous caspase 3 activation, induced hyperpermeability at 1 h followed by a significant decrease at 2 h. Inhibition of GSK-3ß and the transfection of ß-catenin vector increased Tcf-mediated transcription significantly (P < 0.05). The dissociated adherens junctional protein ß-catenin translocates into the cytoplasm, resulting in microvascular hyperpermeability followed by a time-dependent recovery and relocation to the cell membrane. Our data suggest a recycling pathway for ß-catenin to the cell junction.


Asunto(s)
Uniones Adherentes/efectos de los fármacos , Caspasa 3/metabolismo , Células Endoteliales/fisiología , Permeabilidad/efectos de los fármacos , beta Catenina/metabolismo , Uniones Adherentes/fisiología , Animales , Células Cultivadas , Endotelio Vascular/citología , Activación Enzimática , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3 beta , Pulmón/citología , Transporte de Proteínas , Ratas , Transfección , Proteína Destructora del Antagonista Homólogo bcl-2/farmacología
6.
Am J Hypertens ; 22(12): 1314-9, 2009 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-19779466

RESUMEN

BACKGROUND: Preeclampsia (PE), a pregnancy-specific hypertensive syndrome, is one of the leading causes of premature births as well as fetal and maternal death. There is strong evidence that maternal immune system activation, of which Toll-like receptors (TLRs) play a major role, contributes to the development of PE. Viral infections, sensed by TLR3, are associated with hypertensive disorders of pregnancy. We tested the hypothesis that TLR3 activation during pregnancy would cause hypertension, endothelial dysfunction, proteinuria, and intrauterine growth restriction in normal pregnant rats. METHODS: We treated pregnant and nonpregnant rats with the viral mimetic polyinosinic:polycytidylic acid (poly I:C) or vehicle every other day beginning at day 10 of gestation and measured systolic blood pressure, aortic vasodilation, urinary protein concentration, fetal growth, and serum and placental cytokine levels. RESULTS: Pregnant rats treated with poly I:C displayed significantly elevated systolic blood pressures compared to pregnant rats and nonpregnant rats treated with poly I:C on day 18 of gestation. Poly I:C-treated pregnant rats also exhibited significantly decreased aortic vasodilation, significantly increased urinary protein concentrations, and had more malformed pups/litter. Additionally, poly I:C-treated rats exhibited a significant increase in placental TLR3 expression and proinflammatory/anti-inflammatory cytokine ratio compared to vehicle-treated rats. Poly I:C treatment of nonpregnant control rats had no effect on systolic blood pressure, aortic vasodilation, or urinary protein concentrations. CONCLUSION: These findings demonstrate that sustained maternal immune system activation via TLR3 during pregnancy causes PE-like symptoms in rats and suggest that viral infection during pregnancy may contribute to the development of PE.


Asunto(s)
Preeclampsia/etiología , Receptor Toll-Like 3/fisiología , Animales , Femenino , Poli I-C/farmacología , Embarazo , Ratas , Receptor Toll-Like 3/efectos de los fármacos
7.
Am J Hypertens ; 22(10): 1107-14, 2009 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-19617880

RESUMEN

BACKGROUND: Hypertensive disorders of pregnancy, including preeclampsia (PE), affect approximately 7-10% of pregnancies in the US. Clinical and experimental studies strongly suggest that the maternal immune system plays a role in the development of these disorders; however, few therapeutic options exist aside from delivery. METHODS: Using a deoxycorticosterone acetate (DOCA)/salt-low renin rat model, which exhibits hypertension, proteinuria, endothelial dysfunction, and intrauterine growth restriction (IUGR), we measured serum cytokine levels as an indication of immune system activation. In addition, we suppressed the immune system with either azathioprine (Aza) or mycophenolate mofetil (MMF) during the second half of pregnancy to determine whether the these symptoms could be ameliorated. RESULTS: Our results demonstrate that serum T helper-1 (Th1)-type inflammatory cytokines interleukin (IL)-2, IL-12, interferon-gamma (IFNgamma), and RANTES were significantly elevated in hypertensive pregnant rats while the Th2-type cytokine IL-4 was elevated in normal pregnant animals. Either Aza or MMF significantly attenuated the hypertension, proteinuria, and endothelial dysfunction as well as the increased proinflammatory Th1 cytokine profile in pregnant rats treated with DOCA/salt, and had no effect on these parameters in normal pregnant rats. CONCLUSION: These data strongly suggest that maternal immune system activation plays a role in the development of pregnancy-induced hypertension (PIH).


Asunto(s)
Presión Sanguínea/efectos de los fármacos , Endotelio Vascular/fisiopatología , Hipertensión Inducida en el Embarazo/tratamiento farmacológico , Inmunosupresores/uso terapéutico , Animales , Azatioprina/farmacología , Azatioprina/uso terapéutico , Citocinas/antagonistas & inhibidores , Citocinas/sangre , Desoxicorticosterona , Modelos Animales de Enfermedad , Endotelio Vascular/metabolismo , Femenino , Retardo del Crecimiento Fetal/tratamiento farmacológico , Hipertensión Inducida en el Embarazo/inducido químicamente , Hipertensión Inducida en el Embarazo/fisiopatología , Terapia de Inmunosupresión/métodos , Inmunosupresores/farmacología , Ácido Micofenólico/análogos & derivados , Ácido Micofenólico/farmacología , Ácido Micofenólico/uso terapéutico , Embarazo , Proteinuria/tratamiento farmacológico , Ratas , Ratas Sprague-Dawley
8.
Am J Physiol Heart Circ Physiol ; 294(5): H2285-95, 2008 May.
Artículo en Inglés | MEDLINE | ID: mdl-18344375

RESUMEN

Studies from our laboratory demonstrated the involvement of intrinsic apoptotic signaling in hyperpermeability following hemorrhagic shock (HS). Angiopoietin 1 (Ang-1), a potent inhibitor of hyperpermeability, was recently shown to inhibit apoptosis. The purpose of our study was to determine the effectiveness of Ang-1 in attenuating HS-induced hyperpermeability and its relationship to apoptotic signaling. HS was induced in rats by withdrawing blood to reduce the mean arterial pressure to 40 mmHg for 1 h, followed by reperfusion. Mesenteric postcapillary venules were examined for changes in hyperpermeability by intravital microscopy. Mitochondrial release of second mitochondrial derived activator of caspases (smac) and cytochrome c were determined by Western blot and ELISA, respectively. Caspase-3 activity was determined by fluorometric assay. Parallel studies were performed in rat lung microvascular endothelial cell (RLMEC) monolayers, utilizing HS serum and the proapoptotic Bcl-2 homologous antagonist/killer [BAK (BH3)] peptide as inducers of hyperpermeability. In rats, Ang-1 (200 ng/ml) attenuated HS-induced hyperpermeability versus the HS group (P < 0.05). Ang-1 prevented HS-induced collapse of mitochondrial transmembrane potential (DeltaPsi(m)), smac and cytochrome c release, and caspase-3 activity (P < 0.05). In RLMEC monolayers, HS serum and BAK (BH3) peptide both induced hyperpermeability that was inhibited by Ang-1 (P < 0.05). Ang-1 attenuated HS and BAK (BH3) peptide-induced collapse of DeltaPsi(m), smac release, cytochrome c release, activation of caspase-3, and vascular hyperpermeability. In vivo, BAK (BH3) induced vascular hyperpermeability that was attenuated by Ang-1 (P < 0.05). These findings suggest that Ang-1's role in maintaining microvascular endothelial barrier integrity involves the intrinsic apoptotic signaling cascade.


Asunto(s)
Angiopoyetina 1/metabolismo , Apoptosis , Permeabilidad Capilar , Mesenterio/irrigación sanguínea , Choque Hemorrágico/metabolismo , Transducción de Señal , Uniones Adherentes/metabolismo , Uniones Adherentes/patología , Animales , Proteínas Reguladoras de la Apoptosis , Western Blotting , Proteínas Portadoras/metabolismo , Caspasa 3/metabolismo , Células Cultivadas , Citocromos c/metabolismo , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Células Endoteliales/patología , Activación Enzimática , Ensayo de Inmunoadsorción Enzimática , Masculino , Potencial de la Membrana Mitocondrial , Microscopía Confocal , Microscopía por Video , Mitocondrias/enzimología , Mitocondrias/patología , Proteínas Mitocondriales/metabolismo , Fragmentos de Péptidos/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Ratas , Ratas Sprague-Dawley , Choque Hemorrágico/patología , Factores de Tiempo , Vénulas/metabolismo , Vénulas/patología
9.
Am J Physiol Heart Circ Physiol ; 292(6): H3179-89, 2007 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-17307990

RESUMEN

Hemorrhagic shock (HS) disrupts the endothelial cell barrier, resulting in microvascular hyperpermeability. Recent studies have also demonstrated that activation of the apoptotic signaling cascade is involved in endothelial dysfunction, which may result in hyperpermeability. Here we report involvement of the mitochondrial "intrinsic" pathway in microvascular hyperpermeability following HS in rats. HS resulted in the activation of the mitochondrial intrinsic pathway, as is evident from an increase in the proapoptotic Bcl-2 family member BAK, release of mitochondrial cytochrome c into the cytoplasm, and activation of caspase-3. This, along with the in vivo transfection of the proapoptotic peptide BAK (BH3), resulted in hyperpermeability (as visualized by intravital microscopy), release of mitochondrial cytochrome c into the cytoplasm, and activation of caspase-3. Conversely, transfection of the BAK (BH3) mutant had no effect on hyperpermeability. Together, these results demonstrate involvement of the mitochondrial intrinsic apoptotic pathway in HS-induced hyperpermeability and that the attenuation of this pathway may provide an alternative strategy in preserving vascular barrier integrity.


Asunto(s)
Apoptosis , Permeabilidad Capilar , Endotelio Vascular/fisiopatología , Mesenterio/irrigación sanguínea , Choque Hemorrágico/fisiopatología , Transducción de Señal , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismo , Animales , Caspasa 3/metabolismo , Inhibidores de Caspasas , Inhibidores de Cisteína Proteinasa/farmacología , Citocromos c/metabolismo , Modelos Animales de Enfermedad , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Activación Enzimática , Masculino , Potencial de la Membrana Mitocondrial , Microcirculación/metabolismo , Microcirculación/patología , Microcirculación/fisiopatología , Microscopía por Video , Mitocondrias/metabolismo , Mitocondrias/patología , Oligopéptidos/farmacología , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Ratas , Ratas Sprague-Dawley , Choque Hemorrágico/metabolismo , Choque Hemorrágico/patología , Transfección , Proteína Destructora del Antagonista Homólogo bcl-2/genética , Factor de von Willebrand/metabolismo
10.
Am J Physiol Lung Cell Mol Physiol ; 289(2): L217-23, 2005 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-15821015

RESUMEN

Rat lung microvascular endothelial cell monolayers were exposed to donor plasma from burned rats (25% total body surface area) at 1:3 dilution for 30 min. Immunofluorescence analysis revealed that concomitant with gap formation alterations were seen in the adherens junction (AJ) proteins beta-catenin and vascular endothelial-cadherin. Both of these components were shown to exist in a smooth, uniform arrangement at the cell periphery in untreated cells. However, upon exposure to burn plasma, this uniformity was lost, and the AJ proteins showed a disrupted, zipper-like pattern at the cells' edge. In addition, these proteins were absent from areas of gap formation between the cells, and an increase in punctate staining throughout the cells suggests they were internalized in response to burn plasma. Measurements of both transendothelial electrical resistance and FITC-albumin flux across the cell monolayer were used to assess barrier integrity. Our study found that exposure to burn plasma rapidly caused the electrical resistance across confluent monolayers to decrease and albumin flux to increase, phenomena associated with barrier dysfunction. Furthermore, all the above responses to burn plasma were attenuated when cells were pretreated with the PKC inhibitor bisindolylmaleimide, suggesting that PKC is required for burn-induced pulmonary endothelial dysfunction.


Asunto(s)
Uniones Adherentes/metabolismo , Quemaduras/sangre , Endotelio Vascular/patología , Pulmón/irrigación sanguínea , Proteína Quinasa C/metabolismo , Animales , Western Blotting , Cadherinas/metabolismo , Células Cultivadas , Proteínas del Citoesqueleto/metabolismo , Impedancia Eléctrica , Endotelio Vascular/metabolismo , Indoles/farmacología , Maleimidas/farmacología , Fosforilación , Proteína Quinasa C/antagonistas & inhibidores , Ratas , Ratas Sprague-Dawley , Transactivadores/metabolismo , beta Catenina
11.
Am J Physiol Lung Cell Mol Physiol ; 286(4): L841-7, 2004 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-14672924

RESUMEN

Major cutaneous burns result in not only localized tissue damage but broad systemic inflammation causing organ system damage distal to the burn site. It is well recognized that many problems result from the release of inflammatory mediators that target vascular endothelial cells, causing organ dysfunction. The pulmonary microvessels are particularly susceptible to functional abnormalities as a direct consequence of exposure to burn-induced inflammatory mediators. Traditional therapeutic intervention is quite often ineffective in treating burn patients suffering from systemic problems. A possible explanation for this ineffectiveness may be that because so many mediators are released, supposedly activating numerous signaling cascades that interact with each other, targeting of upstream factors in these cascades on an individual basis becomes futile. Therefore, if an end-point effector responsible for endothelial dysfunction following burn injury could be identified, it may present a target for intervention. In this study, we identified phosphorylation of myosin light chain (MLC) as a required element of burn plasma-induced hyperpermeability across rat lung microvascular endothelial cell monolayers. In addition, pharmacological inhibition of myosin light chain kinase (MLCK) and Rho kinase as well as transfection of MLCK-inhibiting peptide blocked actin stress fiber formation and MLC phosphorylation in response to burn plasma. The results suggest that blocking MLC phosphorylation may provide therapeutic intervention in burn patients with the goal of alleviating systemic inflammation-induced endothelial dysfunction.


Asunto(s)
Quemaduras/metabolismo , Endotelio Vascular/metabolismo , Pulmón/irrigación sanguínea , Cadenas Ligeras de Miosina/metabolismo , Actinas/metabolismo , Animales , Proteínas Sanguíneas/metabolismo , Permeabilidad Capilar , Células Cultivadas , Endotelio Vascular/citología , Péptidos y Proteínas de Señalización Intracelular , Quinasa de Cadena Ligera de Miosina/antagonistas & inhibidores , Quinasa de Cadena Ligera de Miosina/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Ratas Sprague-Dawley , Transducción de Señal/fisiología , Quinasas Asociadas a rho
12.
Am J Physiol Cell Physiol ; 286(1): C105-11, 2004 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-13679307

RESUMEN

The involvement of PKC, the isoforms of which are categorized into three subtypes: conventional (alpha, betaI, betaII, and gamma), novel [delta, epsilon, eta, and mu (also known as PKD), theta], and atypical (zeta and iota/lambda), in the regulation of endothelial monolayer integrity is well documented. However, isoform activity varies among different cell types. Our goal was to reveal isoform-specific PKC activity in the microvascular endothelium in response to phorbol 12-myristate 13-acetate (PMA) and diacylglycerol (DAG). Isoform activity was demonstrated by cytosol-to-membrane translocation after PMA treatment and phosphorylation of the myristoylated alanine-rich C kinase substrate (MARCKS) protein after PMA and DAG treatment. Specific isoforms were inhibited by using both antisense oligonucleotides and pharmacological agents. The data showed partial cytosol-to-membrane translocation of isoforms alpha, betaI, and epsilon and complete translocation of PKCdelta and PKD in response to PMA. Furthermore, antisense treatment and pharmacological studies indicated that the novel isoform PKCdelta and PKD are both required for PMA- and DAG-induced MARCKS phosphorylation and hyperpermeability in pulmonary microvascular endothelial cells, whereas isoforms alpha, betaI, and epsilon were dispensable with regard to these same phenomena.


Asunto(s)
Permeabilidad Capilar/fisiología , Endotelio Vascular/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana , Proteína Quinasa C/fisiología , Circulación Pulmonar , Animales , Transporte Biológico/efectos de los fármacos , Permeabilidad Capilar/efectos de los fármacos , Células Cultivadas , Diglicéridos/farmacología , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Glucosidasas , Microcirculación , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada , Oligonucleótidos Antisentido/farmacología , Fosfoproteínas/metabolismo , Fosforilación/efectos de los fármacos , Proteína Quinasa C-delta , Ratas , Acetato de Tetradecanoilforbol/farmacología
13.
Am J Physiol Cell Physiol ; 283(6): C1745-51, 2002 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-12388068

RESUMEN

The hyperpermeability response of microvessels in inflammation involves complex signaling reactions and structural modifications in the endothelium. Our goal was to determine the role of Src-family kinases (Src) in neutrophil-mediated venular hyperpermeability and possible interactions between Src and endothelial barrier components. We found that inhibition of Src abolished the increases in albumin permeability caused by C5a-activated neutrophils in intact, perfused coronary venules, as well as in cultured endothelial monolayers. Activated neutrophils increased Src phosphorylation at Tyr416, which is located in the catalytic domain, and decreased phosphorylation at Tyr527 near the carboxyl terminus, events consistent with reports that phosphorylating and transforming activities of Src are upregulated by Tyr416 phosphorylation and negatively regulated by Tyr527 phosphorylation. Furthermore, neutrophil stimulation resulted in association of Src with the endothelial junction protein beta-catenin and beta-catenin tyrosine phosphorylation. These phenomena were abolished by blockage of Src activity. Taken together, our studies link for the first time neutrophil-induced hyperpermeability to a pathway involving Src kinase activation, Src/beta-catenin association, and beta-catenin tyrosine phosphorylation in the microvascular endothelium.


Asunto(s)
Permeabilidad Capilar/fisiología , Proteínas del Citoesqueleto/metabolismo , Neutrófilos/fisiología , Transactivadores/metabolismo , Familia-src Quinasas/fisiología , Animales , Células Cultivadas , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Activación Enzimática/fisiología , Humanos , Fosforilación , Porcinos , Tirosina/metabolismo , beta Catenina
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA