RESUMEN
Hypozincemia, with hepatic zinc accumulation at the expense of other organs, occurs in infection, inflammation, and aseptic lung injury. Mechanisms underlying zinc partitioning or its impact on extrahepatic organs are unclear. Here we show that the major zinc-binding protein, metallothionein (MT), is critical for zinc transmigration from lung to liver during hyperoxia and preservation of intrapulmonary zinc during hyperoxia is associated with an injury-resistant phenotype in MT-null mice. Particularly, lung-to-liver zinc ratios decreased in wild-type (WT) and increased significantly in MT-null mice breathing 95% oxygen for 72 h. Compared with female adult WT mice, MT-null mice were significantly protected against hyperoxic lung injury indicated by reduced inflammation and interstitial edema, fewer necrotic changes to distal airway epithelium, and sustained lung function at 72 h hyperoxia. Lungs of MT-null mice showed decreased levels of immunoreactive LC3, an autophagy marker, compared with WT mice. Analysis of superoxide dismutase (SOD) activity in the lungs revealed similar levels of manganese-SOD activity between strains under normoxia and hyperoxia. Lung extracellular SOD activity decreased significantly in both strains at 72 h of hyperoxia, although there was no difference between strains. Copper-zinc-SOD activity was ~4× higher under normoxic conditions in MT-null compared with WT mice but was not affected in either group by hyperoxia. Collectively the data suggest that genetic deletion of MT-I/II in mice is associated with compensatory increase in copper-zinc-SOD activity, prevention of hyperoxia-induced zinc transmigration from lung to liver, and hyperoxia-resistant phenotype strongly associated with differences in zinc homeostasis during hyperoxic acute lung injury.
Asunto(s)
Lesión Pulmonar Aguda/metabolismo , Hígado/metabolismo , Pulmón/metabolismo , Metalotioneína/metabolismo , Superóxido Dismutasa/metabolismo , Zinc/metabolismo , Animales , Femenino , Hiperoxia , Inflamación/inmunología , Metalotioneína/genética , Ratones , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/análisis , Mucosa Respiratoria/metabolismoRESUMEN
INTRODUCTION: Promising preclinical results have been obtained with blood purification therapies as adjuvant treatment for sepsis. However, the mechanisms by which these therapies exert beneficial effects remain unclear. Some investigators have suggested that removal of activated leukocytes from the circulation might help ameliorate remote organ injury. We designed an extracorporeal hemoadsorption device capable of capturing both cytokines and leukocytes in order to test the hypothesis that leukocyte capture would alter circulating cytokine profiles and influence immunological cell-cell interactions in whole blood taken from patients with sepsis. METHODS: We performed a series of ex vivo studies in 21 patients with septic shock and 12 healthy volunteers. Blood circulated for four hours in closed loops with four specially designed miniaturized extracorporeal blood purification devices including two different hemoadsorption devices and a hemofilter in order to characterize leukocyte capture and to assess the effects of leukocyte removal on inflammation and immune function. RESULTS: Hemoadsorption was selective for removal of activated neutrophils and monocytes. Capture of these cells led to local release of certain cytokines, especially IL-8, and resulted in complex cell-cell interactions involved in cell-mediated immunity. Inhibition of cell adherence reversed the cytokine release and the effects on lymphocyte function. CONCLUSIONS: Monocyte and neutrophil capture using a sorbent polymer results in upregulation of IL-8 and modulation of cell-mediated immunity. Further studies are needed to understand better these cellular interactions in order to help design better blood purification therapies.
Asunto(s)
Circulación Extracorporea/métodos , Inmunidad Celular/fisiología , Leucocitos/inmunología , Sepsis/inmunología , Sepsis/terapia , Adsorción/fisiología , Circulación Extracorporea/instrumentación , Humanos , Sepsis/sangreRESUMEN
The authors report a case of endobronchial migration of a POD packing coil following embolization of a pulmonary artery pseudoaneurysm in a patient with cavitary tuberculosis and its successful management by bronchoscopy-assisted removal of the coil. Coil migration may be secondary to continued cough and persistence of a bronchial to pulmonary artery fistula from tuberculous infection and inflammation.
RESUMEN
Type II endoleak is a common complication following endovascular aortic aneurysm repair and can lead to an increased risk of aneurysmal expansion and rupture. The most frequently employed strategies to treat Type II endoleak involves catheterization of the branch vessels responsible for the endoleak or accessing the aneurysm sac through a percutaneous approach. An endovascular transcaval approach for embolization of the aneurysmal sac provides an alternate strategy with comparable success rates. This technique is advantageous when the endoleak is predominantly on the right side of the aneurysm sac and/or when a direct access to the aneurysm sac through a percutaneous approach is not feasible.
RESUMEN
Thrombin activates endothelial cell signaling by cleaving the protease-activated receptor-1 (PAR1). However, the function of the apparently nonsignaling receptor PAR3 also expressed in endothelial cells is unknown. We demonstrate here the crucial role of PAR3 in potentiating the responsiveness of PAR1 to thrombin. We tested the hypothesis that PAR1/PAR3 heterodimerization and its effect in modifying G protein selectivity was responsible for PAR3 regulation of PAR1 sensitivity. Using bioluminescent resonance energy transfer-2, we showed that PAR1 had comparable dimerization affinity for PAR3 as for itself. We observed increased Galpha(13) coupling between the PAR1/3 heterodimer compared with the PAR1/1 homodimer. Moreover, knockdown of PAR3 moderated the PAR1-activated increase in endothelial permeability. These results demonstrate a role of PAR3 in allosterically regulating PAR1 signaling governing increased endothelial permeability. Because PAR3 is a critical determinant of PAR1 function, targeting of PAR3 may mitigate the effects of PAR1 in activating endothelial responses such as vascular inflammation.
Asunto(s)
Endotelio Vascular/metabolismo , Regulación de la Expresión Génica , Receptor PAR-1/metabolismo , Receptores de Trombina/fisiología , Línea Celular , ADN Complementario/metabolismo , Dimerización , Proteínas de Unión al GTP/química , Humanos , Microscopía Fluorescente/métodos , Modelos Biológicos , Permeabilidad , ARN Interferente Pequeño/metabolismo , Receptores de Trombina/metabolismo , Transducción de Señal , Trombina/metabolismoRESUMEN
Thrombin-mediated activation of platelets is critical for hemostasis, but the signaling pathways responsible for this process are not completely understood. In addition, signaling within this cascade can also lead to thrombosis. In this study, we have defined a new signaling pathway for the thrombin receptor protease activated receptor-1 (PAR1) in human platelets. We show that PAR1 couples to G(i/o) in human platelets and activates phosphoinositide-3 kinase (PI3K). PI3K activation regulates platelet integrin alphaIIbbeta3 activation and platelet aggregation and potentiates the PAR1-mediated increase in intraplatelet calcium concentration. PI3K inhibitors eliminated these effects downstream of PAR1, but they had no effect on PAR4 signaling. This study has identified an important role for the direct activation of G(i/o) by PAR1 in human platelets. Given the efficacy of clopidogrel, which blocks the G(i/o)-coupled P2Y purinoceptor 12, as an antiplatelet/antithrombotic drug, our data suggest that specifically blocking only PAR1-mediated G(i/o) signaling could also be an effective therapeutic approach with the possibility of less unwanted bleeding.
Asunto(s)
Plaquetas/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Activación Plaquetaria , Receptor PAR-1/metabolismo , Transducción de Señal , Coagulación Sanguínea , Plaquetas/enzimología , Calcio/metabolismo , Activación Enzimática , Humanos , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria , Receptores de Trombina/metabolismoRESUMEN
Regulation of platelet activation plays a central role in hemostasis and pathophysiological processes such as coronary artery disease. Thrombin is the most potent activator of platelets. Human platelets express two thrombin receptors, PAR1 and PAR4, both of which signal platelet activation. Evidence is lacking on the mechanism by which PAR1 and PAR4 may differentially signal platelet aggregation. Here we show that at the relatively high concentration of agonist most likely found at the site of a local thrombus, dual inhibition of the P2Y12 receptor and calcium mobilization result in a complete inhibition of PAR4-induced aggregation, while having no effect on either thrombin or PAR1-mediated platelet aggregation. Both PAR1- and PAR4mediated aggregation are independent of calcium mobilization. Furthermore, we show that P2Y12 receptor activation is not required for protease-activated receptor-mediated aggregation at higher agonist concentrations and is only partially required for Rap1 as well as GPIIbIIIa activation. P2Y12 receptor inhibitors clinically in use such as clopidogrel are postulated to decrease platelet aggregation through partial inhibition of PAR1 signaling. Our data, however, indicate that at high local concentrations of thrombin, it is the signaling through PAR4 rather than PAR1 that may be regulated through purinergic feedback. Thus, our data identify an intra-platelet mechanism that may function as a future site for therapeutic intervention.
Asunto(s)
Plaquetas/metabolismo , Señalización del Calcio/fisiología , Calcio/metabolismo , Agregación Plaquetaria/fisiología , Receptor PAR-1/metabolismo , Receptores Purinérgicos P2/metabolismo , Receptores de Trombina/metabolismo , Adenosina Difosfato/análogos & derivados , Adenosina Difosfato/metabolismo , Quelantes/metabolismo , Ácido Egtácico/análogos & derivados , Ácido Egtácico/metabolismo , Fibrinógeno/metabolismo , Humanos , Activación Plaquetaria/fisiología , Inhibidores de Agregación Plaquetaria/metabolismo , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Antagonistas del Receptor Purinérgico P2 , Receptor PAR-1/genética , Receptores Purinérgicos P2Y12 , Receptores de Trombina/genética , Trombina/metabolismoRESUMEN
Phosducin-like protein (PhLP) is a widely expressed binding partner of the G protein betagamma subunit dimer (Gbetagamma). However, its physiological role is poorly understood. To investigate PhLP function, its cellular expression was blocked using RNA interference, resulting in inhibition of Gbetagamma expression and G protein signaling. This inhibition was caused by an inability of nascent Gbetagamma to form dimers. Phosphorylation of PhLP at serines 18-20 by protein kinase CK2 was required for Gbetagamma formation, while a high-affinity interaction of PhLP with the cytosolic chaperonin complex appeared unnecessary. PhLP bound nascent Gbeta in the absence of Ggamma, and S18-20 phosphorylation was required for Ggamma to associate with the PhLP-Gbeta complex. Once Ggamma bound, PhLP was released. These results suggest a mechanism for Gbetagamma assembly in which PhLP stabilizes the nascent Gbeta polypeptide until Ggamma can associate, resulting in membrane binding of Gbetagamma and release of PhLP to catalyze another round of assembly.
Asunto(s)
Subunidades beta de la Proteína de Unión al GTP/metabolismo , Proteínas de Unión al GTP/metabolismo , Chaperonas Moleculares/fisiología , Proteínas del Tejido Nervioso/fisiología , Proteínas Portadoras , Quinasa de la Caseína II/metabolismo , Línea Celular , Dimerización , Subunidades beta de la Proteína de Unión al GTP/química , Proteínas de Unión al GTP/química , Células HeLa , Humanos , Riñón , Modelos Biológicos , Chaperonas Moleculares/química , Proteínas del Tejido Nervioso/química , Fosforilación , Fosfoserina/metabolismo , Unión Proteica , Desnaturalización Proteica , Mapeo de Interacción de Proteínas , Procesamiento Proteico-Postraduccional , ARN Interferente Pequeño/farmacología , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/fisiologíaRESUMEN
Thrombin activates protease-activated receptor-1 (PAR-1) by cleavage of the amino terminus to unmask a tethered ligand. Although peptide analogs can activate PAR-1, we show that the functional responses mediated via PAR-1 differ between the agonists. Thrombin caused endothelial monolayer permeability and mobilized intracellular calcium with EC(50) values of 0.1 and 1.7 nm, respectively. The opposite order of activation was observed for agonist peptide (SFLLRN-CONH(2) or TFLLRNKPDK) activation. The addition of inactivated thrombin did not affect agonist peptide signaling, suggesting that the differences in activation mechanisms are intramolecular in origin. Although activation of PAR-1 or PAR-2 by agonist peptides induced calcium mobilization, only PAR-1 activation affected barrier function. Induced barrier permeability is likely to be Galpha(12/13)-mediated as chelation of Galpha(q)-mediated intracellular calcium with BAPTA-AM, pertussis toxin inhibition of Galpha(i/o), or GM6001 inhibition of matrix metalloproteinase had no effect, whereas Y-27632 inhibition of the Galpha(12/13)-mediated Rho kinase abrogated the response. Similarly, calcium mobilization is Galpha(q)-mediated and independent of Galpha(i/o) and Galpha(12/13) because pertussis toxin Y-27632 and had no effect, whereas U-73122 inhibition of phospholipase C-beta blocked the response. It is therefore likely that changes in permeability reflect Galpha(12/13) activation, and changes in calcium reflect Galpha(q) activation, implying that the pharmacological differences between agonists are likely caused by the ability of the receptor to activate Galpha(12/13) or Galpha(q). This functional selectivity was characterized quantitatively by a mathematical model describing each step leading to Rho activation and/or calcium mobilization. This model provides an estimate that peptide activation alters receptor/G protein binding to favor Galpha(q) activation over Galpha(12/13) by approximately 800-fold.
Asunto(s)
Ácido Egtácico/análogos & derivados , Proteínas de Unión al GTP/metabolismo , Péptidos/química , Receptor PAR-1/química , Trombina/química , Actinas/química , Adenosina Difosfato/química , Amidas/farmacología , Calcio/química , Calcio/metabolismo , Células Cultivadas , Quelantes/farmacología , Dipéptidos/farmacología , Relación Dosis-Respuesta a Droga , Ácido Egtácico/farmacología , Impedancia Eléctrica , Endotelio Vascular/citología , Inhibidores Enzimáticos/farmacología , Subunidades alfa de la Proteína de Unión al GTP G12-G13/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Cinética , Ligandos , Inhibidores de la Metaloproteinasa de la Matriz , Microcirculación , Modelos Biológicos , Modelos Teóricos , Toxina del Pertussis/farmacología , Inhibidores de Proteasas/farmacología , Unión Proteica , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Piridinas/farmacología , Receptor PAR-1/fisiología , Transducción de Señal , Factores de Tiempo , Quinasas Asociadas a rhoRESUMEN
Thrombospondin-1 (THBS1) is a large extracellular matrix glycoprotein that affects vasculature systems such as platelet activation, angiogenesis, and wound healing. Increases in THBS1 expression have been liked to disease states including tumor progression, atherosclerosis, and arthritis. The present study focuses on the effects of thrombin activation of the G-protein-coupled, protease-activated receptor-1 (PAR-1) on THBS1 gene expression in the microvascular endothelium. Thrombin-induced changes in gene expression were characterized by microarray analysis of approximately 11,000 different human genes in human microvascular endothelial cells (HMEC-1). Thrombin induced the expression of a set of at least 65 genes including THBS1. Changes in THBS1 mRNA correlated with an increase in the extracellular THBS1 protein concentration. The PAR-1-specific agonist peptide (TFLLRNK-PDK) mimicked thrombin stimulation of THBS1 expression, suggesting that thrombin signaling is through PAR-1. Further studies showed THBS1 expression was sensitive to pertussis toxin and protein kinase C inhibition indicating G(i/o)- and G(q)-mediated pathways. THBS1 up-regulation was also confirmed in human umbilical vein endothelial cells stimulated with thrombin. Analysis of the promoter region of THBS1 and other genes of similar expression profile identified from the microarray predicted an EBOX/EGRF transcription model. Expression of members of each family, MYC and EGR1, respectively, correlated with THBS1 expression. These results suggest thrombin formed at sites of vascular injury increases THBS1 expression into the extracellular matrix via activation of a PAR-1, G(i/o), G(q), EBOX/EGRF-signaling cascade, elucidating regulatory points that may play a role in increased THBS1 expression in disease states.
Asunto(s)
Endotelio Vascular/citología , Regulación de la Expresión Génica , Microcirculación/metabolismo , Trombina/química , Trombospondina 1/biosíntesis , Adenosina Difosfato/química , Algoritmos , Amidas/farmacología , Apoptosis , Células Cultivadas , Análisis por Conglomerados , Medios de Cultivo , Cartilla de ADN/química , ADN Complementario/metabolismo , Relación Dosis-Respuesta a Droga , Impedancia Eléctrica , Endotelio Vascular/metabolismo , Ensayo de Inmunoadsorción Enzimática , Matriz Extracelular/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Humanos , Indoles/farmacología , Maleimidas/farmacología , Modelos Biológicos , Hibridación de Ácido Nucleico , Análisis de Secuencia por Matrices de Oligonucleótidos , Péptidos/química , Toxina del Pertussis/farmacología , Regiones Promotoras Genéticas , Unión Proteica , Piridinas/farmacología , ARN/metabolismo , Receptor PAR-1/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Trombina/metabolismo , Factores de Tiempo , Venas Umbilicales/citología , Regulación hacia ArribaRESUMEN
Phosducin and phosducin-like protein (PhLP) bind G protein betagamma subunits and regulate their activity. This report describes a previously uncharacterized binding partner unique to PhLP that was discovered by coimmunoprecipitation coupled with mass spectrometric identification. Chaperonin containing tailless complex polypeptide 1 (CCT), a cytosolic chaperone responsible for the folding of many cellular proteins, binds PhLP with a stoichiometry of one PhLP per CCT complex. Unlike protein-folding substrates of CCT, which interact only in their nonnative conformations, PhLP binds in its native state. Native PhLP competes directly for binding of protein substrates of CCT and thereby inhibits CCT activity. Overexpression of PhLP inhibited the ability of CCT to fold newly synthesized beta-actin by 80%. These results suggest that the interaction between PhLP and CCT may be a means to regulate CCT-dependent protein folding or alternatively, to control the availability of PhLP to modulate G protein signaling.
Asunto(s)
Proteínas Portadoras/metabolismo , Chaperoninas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Actinas/metabolismo , Animales , Unión Competitiva , Células CHO , Proteínas Portadoras/aislamiento & purificación , Clonación Molecular , Cricetinae , Citosol/metabolismo , ADN Complementario , Escherichia coli/genética , Cinética , Espectrometría de Masas , Chaperonas Moleculares , Proteínas del Tejido Nervioso/aislamiento & purificación , Pliegue de Proteína , Ratas , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , TransfecciónRESUMEN
Phosducin-like protein (PhLP) is a broadly expressed member of the phosducin (Pd) family of G protein betagamma subunit (Gbetagamma)-binding proteins. Though PhLP has been shown to bind Gbetagamma in vitro, little is known about its physiological function. In the present study, the effect of PhLP on angiotensin II (Ang II) signaling was measured in Chinese hamster ovary cells expressing the type 1 Ang II receptor and various amounts of PhLP. Up to 3.6-fold overexpression of PhLP had no effect on Ang II-stimulated inositol trisphosphate (IP(3)) formation, whereas further increases caused an abrupt decrease in IP(3) production with half-maximal inhibition occurring at 6-fold PhLP overexpression. This threshold level for inhibition corresponds to the cellular concentration of cytosolic chaperonin complex, a recently described binding partner that preferentially binds PhLP over Gbetagamma. Results of pertussis toxin sensitivity, GTPgammaS binding, and immunoprecipitation experiments suggest that PhLP inhibits phospholipase Cbeta activation by dual mechanisms: (i) steric blockage of Gbetagamma activation of PLCbeta and (ii) interference with Gbetagamma-dependent cycling of G(q)alpha by the receptor. These results suggest that G protein signaling may be regulated through controlling the cellular concentration of free PhLP by inducing its expression or by regulating its binding to the chaperonin.
Asunto(s)
Angiotensina II/fisiología , Proteínas Portadoras/fisiología , Proteínas de Unión al GTP/metabolismo , Proteínas del Tejido Nervioso/fisiología , Transducción de Señal/fisiología , Angiotensina II/metabolismo , Animales , Células CHO , Cricetinae , Activación Enzimática , Inositol 1,4,5-Trifosfato/biosíntesis , Isoenzimas/metabolismo , Chaperonas Moleculares , Fosfolipasa C beta , Pruebas de Precipitina , Ratas , Receptores de Angiotensina/metabolismo , Fosfolipasas de Tipo C/metabolismoRESUMEN
The three-dimensional structure of the complex formed between the cytosolic chaperonin CCT (chaperonin containing TCP-1) and phosducin (Pdc)-like protein (PhLP), a regulator of CCT activity, has been solved by cryoelectron microscopy. Binding of PhLP to CCT occurs through only one of the chaperonin rings, and the protein does not occupy the central folding cavity but rather sits above it through interactions with two regions on opposite sides of the ring. This causes the apical domains of the CCT subunits to close in, thus excluding access to the folding cavity. The atomic model of PhLP generated from several atomic structures of the homologous Pdc fits very well with the mass of the complex attributable to PhLP and predicts the involvement of several sequences of PhLP in CCT binding. Binding experiments performed with PhLP/Pdc chimeric proteins, taking advantage of the fact that Pdc does not interact with CCT, confirm that both the N- and C-terminal domains of PhLP are involved in CCT binding and that several regions suggested by the docking experiment are indeed critical in the interaction with the cytosolic chaperonin.