RESUMEN
The aim of this study was to determine the association of benign recurrent vertigo (BRV) and migraine, using standardized questionnaire-based interview of 208 patients with BRV recruited through a University Neurotology clinic. Of 208 patients with BRV, 180 (87%) met the International Classification of Headache Disorders 2004 criteria for migraine: 112 migraine with aura (62%) and 68 without aura (38%). Twenty-eight (13%) did not meet criteria for migraine. Among patients with migraine, 70% experienced headache, one or more auras, photophobia, or auditory symptoms with some or all of their vertigo attacks, meeting the criteria for definite migrainous vertigo. Thirty per cent never experienced migraine symptoms concurrent with vertigo attacks. These met criteria for probable migrainous vertigo. Among patients without migraine, 21% experienced either photophobia or auditory symptoms with some or all of their vertigo attacks; 79% experienced only isolated vertigo. The age of onset and duration of vertigo attacks did not differ significantly between patients with (34 +/- 1.2 years) and patients without migraine (31 +/- 3.0 years). In patients with migraine, the age of onset of migraine headache preceded the onset of vertigo attacks by an average of 14 years and aura preceded vertigo by 8 years. The most frequent duration of vertigo attacks was between 1 h and 1 day. Benign recurrent vertigo is highly associated with migraine, but a high proportion of patients with BRV and migraine never have migraine symptoms during their vertigo attacks. Other features such as age of onset and duration of vertigo are similar between patients with or without migraine.
Asunto(s)
Trastornos Migrañosos/complicaciones , Vértigo/complicaciones , Adulto , Edad de Inicio , Femenino , Humanos , Masculino , Trastornos Migrañosos/epidemiología , Prevalencia , Encuestas y Cuestionarios , Vértigo/epidemiologíaRESUMEN
A new inhibitory peptide binding exosite on the protease domain of coagulation Factor VIIa (FVIIa) has been identified. A novel series of peptide inhibitors of FVIIa, termed the "A-series" peptides, identified from peptide phage libraries and exemplified by peptide A-183 [Dennis, M. S., Roberge, M., Quan, C., and Lazarus, R. A. (2001) Biochemistry 40, 9513-9521], specifically bind at a site that is distinct from both the active site and the exosite of another recently described peptide inhibitor of FVIIa, E-76 [Dennis, M. S., Eigenbrot, C., Skelton, N. J., Ultsch, M. H., Santell, L., Dwyer, M. A., O'Connell, M. P., and Lazarus, R. A. (2000) Nature 404, 465-4701. Peptide A-183 prolonged TF-dependent clotting in human, but not rabbit plasma. Thus, a panel of human FVIIa mutants, containing 70 of the 76 rabbit sequence differences in the protease domain, localized the binding site to residues in the 60s loop and the C-terminus. The location of the exosite was refined by a series of FVIIa alanine mutants, which showed that proximal residues Trp 61 and Leu 251 were critical for binding. Kinetic and equilibrium binding constants for zymogen FVII, FVIIa and TF x FVIIa were determined using immobilized N-terminal biotinylated A-183 by surface plasmon resonance. No peptide binding to nine other human serine proteases was observed. Key residues on the peptide were determined from binding to FVIIa and inhibition of FX activation using a series of alanine mutants of A-183 fused to the Z domain of protein A. Analysis of the mutagenesis data is presented in the context of a crystal structure of A-183 in complex with a version of zymogen FVII [Eigenbrot, C., Kirchhofer, D., Dennis, M. S., Santell, L., Lazarus, R. A., Stamos, J., and Ultsch, M. H. (2001) Structure 9, 627-636]. The shape and proximity of this exosite to the active site may lend itself towards the design of new anticoagulants that inhibit FVIIa.
Asunto(s)
Coagulación Sanguínea/efectos de los fármacos , Factor VIIa/antagonistas & inhibidores , Péptidos/farmacología , Inhibidores de Proteasas/farmacología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Relación Dosis-Respuesta a Droga , Factor VIIa/química , Factor VIIa/metabolismo , Humanos , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Biblioteca de Péptidos , Péptidos/química , Péptidos/metabolismo , Inhibidores de Proteasas/química , Inhibidores de Proteasas/metabolismo , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Conejos , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Relación Estructura-ActividadRESUMEN
BACKGROUND: Coagulation factor VIIa (FVIIa) contains a Trypsin-like serine protease domain and initiates the cascade of proteolytic events leading to Thrombin activation and blood clot formation. Vascular injury allows formation of the complex between circulating FVIIa and its cell surface bound obligate cofactor, Tissue Factor (TF). Circulating FVIIa is nominally activated but retains zymogen-like character and requires TF in order to complete the zymogen-to-enzyme transition. The manner in which TF exerts this effect is unclear. The structure of TF/FVIIa is known. Knowledge of the zymogen structure is helpful for understanding the activation transition in this system. RESULTS: The 2 A resolution crystal structure of a zymogen form of FVII comprising the EGF2 and protease domains is revealed in a complex with the exosite binding inhibitory peptide A-183 and a vacant active site. The activation domain, which includes the N terminus, differs in ways beyond those that are expected for zymogens in the Trypsin family. There are large differences in the TF binding region. An unprecedented 3 residue shift in registration between beta strands B2 and A2 in the C-terminal beta barrel and hydrogen bonds involving Glu154 provide new insight into conformational changes accompanying zymogen activation, TF binding, and enzymatic competence. CONCLUSIONS: TF-mediated allosteric control of the activity of FVIIa can be rationalized. The reregistering beta strand connects the TF binding region and the N-terminal region. The zymogen registration allows H bonds that prevent the N terminus from attaining a key salt bridge with the active site. TF binding may influence an equilibrium by selecting the enzymatically competent registration.
Asunto(s)
Factor VII/química , Factor VII/metabolismo , Ácido Aspártico/química , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Precursores Enzimáticos/química , Factor VII/genética , Ácido Glutámico/química , Isoleucina/química , Modelos Moleculares , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tromboplastina/metabolismoRESUMEN
Potent anticoagulants have been derived by targeting the tissue factor-factor VIIa complex with naive peptide libraries displayed on M13 phage. The peptides specifically block the activation of factor X with a median inhibitory concentration of 1 nM and selectively inhibit tissue-factor-dependent clotting. The peptides do not bind to the active site of factor VIIa; rather, they work by binding to an exosite on the factor VIIa protease domain, and non-competitively inhibit activation of factor X and amidolytic activity. One such peptide (E-76) has a well defined structure in solution determined by NMR spectroscopy that is similar to the X-ray crystal structure when complexed with factor VIIa. These structural and functional studies indicate an allosteric 'switch' mechanism of inhibition involving an activation loop of factor VIIa and represent a new framework for developing inhibitors of serine proteases.
Asunto(s)
Anticoagulantes/farmacología , Factor VIIa/antagonistas & inhibidores , Oligopéptidos/farmacología , Péptidos/farmacología , Inhibidores de Serina Proteinasa/farmacología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Secuencia de Consenso , Cristalografía por Rayos X , Precursores Enzimáticos/metabolismo , Factor VIIa/química , Factor X/antagonistas & inhibidores , Factor X/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Oligopéptidos/aislamiento & purificación , Biblioteca de Péptidos , Péptidos/aislamiento & purificación , Conformación Proteica , Conejos , Inhibidores de Serina Proteinasa/análisis , Tromboplastina/metabolismoRESUMEN
The incorporation of sialic acid into therapeutic recombinant glycoprotein expressed in Chinese hamster ovary (CHO) cells during growth in large bioreactors (10 l) has been monitored under high productivity conditions induced by the presence of sodium butyrate. Samples of the bioreactor culture (approximately 4 x 10(6) cells) were labeled with 3H-N-acetylmannosamine, a metabolic precursor of sialic acid. After 24 h, the recombinant glycoprotein, an immunoadhesion chimeric molecule, was purified and the amount of sialic acid incorporated was determined as radioactive counts. The labeling profile of the protein over the course of the culture was compared with the sialic acid content of the molecule as determined by direct chemical analysis. Early in the culture, the two methods of analysis gave a similar sialylation profile. However, after sodium butyrate was included in the culture, the metabolically incorporated sialic acid rapidly and dramatically decreased to near undetectable levels. In contrast, sialic acid content of the protein, as determined by chemical analysis, decreased only moderately and gradually over the culture period, from a maximum of 6.1 to about 5. 0 mol sialic acid/mole of protein after 10 days in culture. These results suggest that butyrate may enhance reutilization of existing glycoproteins in the culture, generating sialic acid for biosynthesis through lysosomal degradation and thereby bypassing de novo biosynthesis.
Asunto(s)
Ácido N-Acetilneuramínico/metabolismo , Proteínas Recombinantes/metabolismo , Animales , Células CHO , Técnicas de Cultivo de Célula , Cromatografía de Afinidad , Cromatografía Líquida de Alta Presión , Cricetinae , Hexosaminas/metabolismoRESUMEN
Plasma levels of tissue plasminogen activator (tPA) increase rapidly in response to specific vasoactive agents, trauma, and neural stimulation. This response has been attributed to acute release of tPA from stored pools within the vascular endothelium and from catecholamine storage vesicles of chromaffin cells. We have tested directly whether tPA can be sorted into the regulated secretory pathway using the murine pituitary-derived neuroendocrine cell line AtT-20 transfected with tPA cDNA. Clones of AtT-20 cells expressing tPA were isolated, and targeting of tPA into the regulated secretory pathway was demonstrated by (1) stimulation of tPA secretion with 8-bromo-cAMP, the secretagogue which promotes the release of dense granule contents; (2) colocalization with ACTH, an endogenous protein that is stored in dense core granules; and (3) retention of newly synthesized tPA in the cell for prolonged periods of time. Laser scanning confocal microscopy analysis of cells immunostained with antibodies to tPA and ACTH showed colocalization at the tips of the neuritic processes under the cytoplasmic membrane, a region where dense granules are known to migrate after maturation. Treatment of the cells with 5 mM 8-bromo-cAMP for 30 min resulted in a 2.41+/-0.36-fold increase in tPA secretion. Both the magnitude of the stimulatory effect and the fraction of the intracellular tPA released were the same regardless of the tPA expression level in the various clones. Pulse-chase experiments showed that a portion of newly synthesized tPA is retained in the cell for at least 4 h and is released into the culture medium in response to 8-bromo-cAMP. These studies indicate that tPA, under the appropriate conditions, can be targeted into the regulated secretory pathway and can be stored for later release by cellular stimuli.
Asunto(s)
Adenohipófisis/metabolismo , Señales de Clasificación de Proteína/fisiología , Activador de Tejido Plasminógeno/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Hormona Adrenocorticotrópica/metabolismo , Animales , Células CHO , Línea Celular , Células Clonales , Cricetinae , Espacio Extracelular/metabolismo , Inmunohistoquímica , Líquido Intracelular/metabolismo , Ratones , Factores de Tiempo , Activador de Tejido Plasminógeno/genética , TransfecciónRESUMEN
Plasma tissue plasminogen activator (tPA) has long been considered to be the product of the endothelial cells that line the various parts of the vascular system regardless of vessel size or location. To determine whether this was truly the case in vivo, the distribution of tPA in the endothelium of the mouse lung and other tissues was evaluated. Immunohistochemical analysis of normal lung tissue showed positive staining limited to the endothelial cells of the bronchial arteries regardless of size with few cells of the pulmonary circulation associated with tPA. The pulmonary vessels that did contain endothelial cell-derived tPA were consistently between 7 and 30 microns in diameter. No capillary or large vessel pulmonary endothelium ever stained positive. These results were also observed in primate lung tissue where the bronchial endothelium of all vessels, even down to capillary size, contained tPA while none of the pulmonary endothelium did. Prolonged exposure of mice to hyperoxic conditions promotes acute lung injury and associated inflammation. Using this model, the effect of inflammation on endothelial cell tPA expression was evaluated. A 4.5-fold increase in the number of pulmonary vessels staining positive for tPA was observed after 66 hours with all of these vessels having a diameter between 7 and 30 microns. Again, none of the endothelium of large arteries or veins nor the capillaries had tPA. Whole tissue tPA mRNA increased dramatically with hyperoxia and in situ hybridization analysis showed tPA mRNA in the endothelium of the same types of vessels as antigen. The tPA localized to both the bronchial and pulmonary endothelium was active with neither tPA-PAI-1 complexes nor urokinase found in perfused lung tissue. These results indicate that endothelial cell tPA expression, either constitutive or induced by a pathologic event, is a function of a highly select group of endothelial cells which are defined by their association with vessels of discrete size and/or anatomic location. Thus, the widely held concept that the steady state level of plasma tPA is maintained through its constitutive production by all endothelial cells of the vascular system is invalid. Also suggested is the possibility that endothelial cell tPA might play a broader role than simply maintaining vessel patency as a component of the fibrinolytic pathway and contribute to complex dynamic processes such as inflammation.
Asunto(s)
Activadores Plasminogénicos/metabolismo , Animales , Antígenos/metabolismo , Endotelio , Pulmón/metabolismo , Pulmón/patología , Ratones , Ratones Endogámicos C57BL , Oxígeno/metabolismo , Activadores Plasminogénicos/genética , Primates/metabolismo , ARN Mensajero/análisis , Conejos , RatasRESUMEN
Placement of endothelial cells under hypoosmotic or hyperosmotic conditions results in the reduction and increase, respectively, in the phosphorylation of a M(r) = 16,500 protein (P17). The changes were dose-dependent with a 3.3 +/- 0.3-fold increase occurring at 485 mosm/kg H2O and negligible phosphorylation observed at 202 mosm/kg H2O. The phosphorylation and dephosphorylation were rapid and prolonged; modified phosphorylation levels were maintained as long as the anisotonic conditions were present. However, return to isotonic medium reversed the phosphorylation back to normal within 1 h. Cellular fractionation studies showed that P17 was associated only with the nuclear compartment under isotonic, hypertonic, or hypotonic conditions. Two forms of P17 with pI values of 9.2 and 9.6 were resolved by isoelectric focusing; both forms showed enhanced phosphorylation by hyperosmotic treatment. Phosphorylation occurred on serines exclusively. These studies demonstrate that a nuclear protein with characteristics similar to histones is affected by cell shrinkage or swelling through changes in its phosphorylation state.
Asunto(s)
Histonas/metabolismo , Células Cultivadas , Cromatografía en Capa Delgada , Electroforesis en Gel de Poliacrilamida , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Humanos , Cinética , Concentración Osmolar , FosforilaciónRESUMEN
Shear, stretch, and the generation of oxygen radicals stimulate increases in tissue plasminogen activator (t-PA) mRNA levels and antigen production, suggesting that environmental stress may regulate t-PA gene expression. We have examined whether t-PA production is also responsive to a hyperosmotic environment. Endothelial and HeLa cells were treated with hyperosmotic medium, and t-PA mRNA and antigen secretion were measured. Endothelial cells incubated in hyperosmotic medium showed a dose-dependent decrease in cell volume and a 1.9 +/- 0.3- and 3.7 +/- 0.9-fold increase in t-PA secretion at 425 and 485 mosmol/kgH2O, respectively. HeLa cells showed a 3.3 +/- 0.6- and 5.1 +/- 1.2-fold increase at the same osmolalities. Increased secretion began between 8 and 16 h and continued through 24 h. Cultures returned to isosmotic medium after 8 h of treatment continued to release 98.1 +/- 7% of the maximum levels of t-PA for the next 16 h, despite the reversal of other responses to hyperosmotic environment. t-PA mRNA levels also increased between 8 and 16 h to five times control levels but returned to baseline by 24 h. No change in intracellular Ca2+ concentration, inositol 1,4,5-trisphosphate, or diacylglycerol content was detected, suggesting that a different intracellular signal pathway may be involved in the response to hyperosmolar stimulus. Thus environmental stress may be a general stimulatory signal through which t-PA production can be induced.
Asunto(s)
Ósmosis , Proteína Quinasa C/fisiología , Estrés Fisiológico/metabolismo , Activador de Tejido Plasminógeno/metabolismo , Secuencia de Bases , Calcio/metabolismo , Medios de Cultivo , Diglicéridos/metabolismo , Epoprostenol/metabolismo , Células HeLa , Humanos , Fosfatos de Inositol/metabolismo , Soluciones Isotónicas , Manitol/farmacología , Datos de Secuencia Molecular , Concentración Osmolar , ARN Mensajero/metabolismo , Activador de Tejido Plasminógeno/genéticaRESUMEN
The expression of certain proteolytic enzymes involved in cell migration (collagenase, urokinase) can be enhanced by the disruption of cellular cytoskeletal organization, suggesting an association between cell shape and gene expression. We have examined the effect of cytoskeleton-disrupting agents on the production and secretion of another proteolytic enzyme, tissue plasminogen activator (tPA), and its inhibitor, plasminogen activator inhibitor-1 (PAI-1), in human endothelial cells. Addition of 1 x 10(-6) M colchicine, 5 x 10(-6) M cytochalasin B, 10(-6) M nocodazole, or 10(-6) M tubulazole had no effect on the constitutive rate of release of tPA. However, the three microtubule-disrupting agents--colchicine, nocodazole, and tubulazole--depressed the stimulation of tPA secretion by phorbol myristate acetate (PMA) by 50- to 65%. Disruption of microfilament structure by cytochalasin B had no effect. In contrast, microtubule disruption in the absence or presence of PMA stimulated PAI-1 secretion by 2.5 and 2 times, respectively. The depression of tPA secretion was not due to inhibition of the secretory function since tPA did not accumulate intracellularly during colchicine treatment. Nor did colchicine affect the PMA activation of protein kinase C-alpha, upon which stimulation of tPA is dependent; neither translocation of the kinase nor phosphorylation of the protein kinase C substrate protein, P80, was inhibited. Measurement of tPA mRNA levels demonstrated that the increase which precedes PMA-enhanced tPA secretion was also inhibited by colchicine by 50%. However, tPA gene transcriptional activity was only reduced 13%, suggesting that a post-transcriptional event was affected by microtubule disruption. PAI-1 mRNA levels and transcription rates were elevated 3.5 times. This study suggests that the changes that occur in endothelial cells during PMA-induced signal transmission leading to enhanced tPA mRNA levels and tPA antigen production can be partly blocked by agents that disrupt microtubule organization.
Asunto(s)
Expresión Génica/efectos de los fármacos , Microtúbulos/efectos de los fármacos , Inactivadores Plasminogénicos/metabolismo , Activador de Tejido Plasminógeno/metabolismo , Transcripción Genética/efectos de los fármacos , Células Cultivadas , Colchicina/farmacología , Dioxolanos/farmacología , Endotelio/efectos de los fármacos , Humanos , Nocodazol/farmacología , Ésteres del Forbol/farmacologíaRESUMEN
Trans retinoic acid (t-RA) stimulated the production of tissue plasminogen activator (tPA) in HeLa-S3 and human umbilical vein endothelial cells (huvecs) in a dose-dependent manner with maximal release (four to five times control) at 40 nmol/L and 40 mumol/L, respectively. In endothelial cells, the stimulation of tPA production by phorbol 12-myristate 13-acetate (PMA) was potentiated 1.9-fold by 10 mumol/L t-RA, or 1.8 times the additive effect. In HeLa cells, total tPA secretion with 10 nmol/L PMA was increased from 43 ng/mL to 96 ng/mL by 40 nmol/L t-RA, which was two times the additive effect. Higher concentrations of t-RA (400 nmol/L) depressed tPA secretion by itself and also suppressed PMA-induced tPA production by 50%. Histamine and thrombin also synergized with t-RA. t-RA (40 nmol/L) and 10 micrograms/mL histamine or 10 U/mL thrombin combined to induce tPA production 3.4 and 1.3 times the additive effect in HeLa cells. Cyclic adenosine monophosphate (cAMP) levels were not significantly affected by 10 nmol/L to 10 mumol/L t-RA. Nor did 10 nmol/L PMA and 40 nmol/L t-RA together affect cAMP levels, suggesting that t-RA-mediated potentiation of PMA-induced tPA production occurred via a mechanism that was independent of cAMP levels. Downregulation of protein kinase C (PKC) by pretreatment of huvecs with 100 nmol/L PMA completely blocked a secondary response to PMA, but did not have a significant effect on t-RA induction. Pretreatment with 10 mumol/L t-RA, on the other hand, did not significantly affect a secondary stimulus by 100 nmol/L PMA, but completely suppressed a secondary stimulation by 10 mumol/L t-RA alone. These studies suggest that the mechanism mediating t-RA stimulation of tPA production interacts with the PKC pathway, resulting in synergism.
Asunto(s)
Endotelio Vascular/metabolismo , Proteína Quinasa C/metabolismo , Activador de Tejido Plasminógeno/biosíntesis , Tretinoina/farmacología , Células Cultivadas , AMP Cíclico/metabolismo , Sinergismo Farmacológico , Endotelio Vascular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Células HeLa/metabolismo , Histamina/farmacología , Humanos , Cinética , Acetato de Tetradecanoilforbol/farmacología , Trombina/farmacología , Activador de Tejido Plasminógeno/metabolismo , Venas UmbilicalesRESUMEN
Several agonists of endothelial cell function (thrombin, histamine, dioctanoylglycerol, phorbol 12-myristate 13-acetate, interleukin-1) have previously been shown to enhance the level of phosphorylation of an undefined 29,000-M(r) protein (P29). Comparison of this protein with other phosphoproteins suggested that it may be related to the mammalian heat-shock protein HSP27. Immunoprecipitation and immunoblot analysis with antibodies specific for human HSP27 demonstrated that P29 was immunochemically identical with HSP27. Further characterization of agonist-induced phosphorylation of HSP27 indicated that phosphorylation occurred exclusively on serine residues, and phosphopeptide analysis of tryptic- and chymotryptic-cleavage products demonstrated that the phosphopeptides generated were identical for each agonist and okadaic acid. Down-regulation of protein kinase C-alpha by prolonged treatment with phorbol esters eliminated the ability of phorbol 12-myristate 13-acetate, dioctanoylglycerol, thrombin and histamine to phosphorylate HSP27 above background levels and deceased interleukin-1-stimulated HSP27 phosphorylation by 60%. These data suggest that the various agonists employed stimulate HSP27 phosphorylation through similar mechanisms and that protein kinase C is probably involved.
Asunto(s)
Endotelio Vascular/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteína Quinasa C/metabolismo , Autorradiografía , Western Blotting , Células Cultivadas , Electroforesis en Gel Bidimensional , Electroforesis en Gel de Poliacrilamida , Endotelio Vascular/efectos de los fármacos , Proteínas de Choque Térmico/aislamiento & purificación , Humanos , Mapeo Peptídico , Fosfatos/metabolismo , Fosfopéptidos/análisis , Fosfoproteínas/aislamiento & purificación , Fosfoproteínas/metabolismo , Radioisótopos de Fósforo , Fosforilación , Acetato de Tetradecanoilforbol/farmacología , Tripsina , Venas UmbilicalesRESUMEN
IL-1 treatment of human endothelial cells leads to the rapid phosphorylation of a Mr = 29,000 (P29) set of proteins to 18 times that of control cultures. Approximately 80% of the phosphorylated P29 (pP29) disappeared within 60 min although the remaining component was stable and remained for at least another 2 h. IL-1R antagonist protein blocked phosphorylation completely. Secondary treatment of IL-1 failed to increase the level of pP29 above that remaining after 1 h although other unrelated agonists that stimulated pP29 generation could. Removal of the cytokine and incubation of the cells in agonist-free medium for 2 h resulted in the total loss of the remaining pP29. Readdition of IL-1 2 h after washout restimulated P29 phosphorylation but only back to the lower level. Maximum rephosphorylation could not be attained until 16 h after IL-1 removal. Protein kinase inhibitors 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine and staurosporine, the calcium chelators bis(2-amino-5-methylphenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester and EGTA, and the calmodulin inhibitor N-(6-aminohexyl)-1-naphthalene-sulfonamide had no effect on IL-I-induced phosphorylation. However, when cultures were treated with the protein phosphatase inhibitor okadaic acid alone, the level of pP29 increased after 1 h and the presence of okadaic acid during prolonged IL-1 treatment blocked the decline in pP29. The protein synthesis inhibitors puromycin, emetine, and cycloheximide also blocked the decline in pP29 during IL-1 treatment. These data suggest that IL-1-stimulated P29 phosphorylation is made up of two components, one susceptible to prolonged down-regulation even in the absence of the cytokine and one refractory to desensitization but that remains active only in the presence of IL-1. IL-1-induced changes in pP29 levels may be dependent on the relative activities of protein kinase and protein phosphatase activities.
Asunto(s)
Endotelio Vascular/metabolismo , Interleucina-1/farmacología , Fosfoproteínas/metabolismo , Células Cultivadas , Regulación hacia Abajo , Éteres Cíclicos/farmacología , Humanos , Peso Molecular , Ácido Ocadaico , FosforilaciónRESUMEN
Treatment of human endothelial cells with thrombin, histamine, or dioctanoylglycerol (DiC8), a synthetic diacylglycerol, resulted in the rapid and transient phosphorylation of a Mr = 29,000 protein (P29) in a dose-dependent manner. Various tumor promoters also promoted P29 phosphorylation while the adenylate cyclase activator, forskolin, did not. The level of phosphorylation with all three agonists was similar (2.5-4 fold), and analysis of P29 by two-dimensional gel electrophoresis revealed identical patterns in each case. Receptor specificity was demonstrated for the histamine-stimulated changes; pyrilamine (10(-6) M; H1) but not cimetidine (10(-4); H2) blocked the response. The thrombin effect was active site-dependent. Phosphorylation induced by thrombin and histamine occurred within 1 min, peaked between 5 and 10 min, and returned to control levels by 1 h. DiC8-induced phosphorylation occurred more slowly but was also reduced by 1 h while phorbol ester treatment prolonged phosphorylation for at least 4 h. Treatment of these cells with thrombin or histamine for 1 h desensitized P29 to further phosphorylation by the homologous agonist although secondary phosphorylation could occur with heterologous compounds. However, if the primary agonist was removed following the onset of a desensitized state, secondary phosphorylation of P29 could be stimulated by the same compound. These same results were observed with two other phosphoproteins Mr = 18,000 (P18) and 80,000 (P80) which became more highly phosphorylated in response to thrombin treatment and with histamine/thrombin-stimulated prostaglandin I2 production. In contrast, homologous down-regulation of P29 phosphorylation was not observed with DiC8-treated cells, and the decline in phosphorylated P29 was associated with the loss of functional DiC8. The protein kinase inhibitors staurosporine and H-7 blocked P18 and P80 phosphorylation by thrombin but had no effect on P29 phosphorylation by histamine, thrombin, or DiC8 suggesting distinct pathways leading to the phosphorylation of these different proteins. These data suggest that multiple and independent thrombin/histamine-induced events are susceptible to receptor occupancy-dependent homologous down-regulation.
Asunto(s)
Endotelio Vascular/metabolismo , Histamina/farmacología , Fosfoproteínas/metabolismo , Transducción de Señal , Trombina/farmacología , 6-Cetoprostaglandina F1 alfa/metabolismo , Calcio/metabolismo , Células Cultivadas , Diglicéridos/farmacología , Electroforesis en Gel Bidimensional , Electroforesis en Gel de Poliacrilamida , Endotelio Vascular/efectos de los fármacos , Humanos , Cinética , Ésteres del Forbol/farmacología , Fosfoproteínas/aislamiento & purificación , FosforilaciónRESUMEN
Tumor-promoting phorbol esters stimulate tissue plasminogen activator (tPA) release from human endothelial cells, and simultaneous elevation of cyclic AMP potentiates this response 5-fold (Santell, L., and Levin, E. G. (1988) J. Biol. Chem. 263, 16802-16808). A similar effect on tPA mRNA was observed, with phorbol myristate acetate inducing a 3.5-fold increase in steady state tPA mRNA levels and forskolin enhancing that increase to 25-fold. Peak levels occurred at 8 h after agonist addition and returned to baseline levels by 16 h. As was found with tPA antigen secretion, delayed addition of forskolin reduced the level of potentiation, and, at 6 h after phorbol 12-myristate 13-acetate (PMA), forskolin was no longer effective. The protein synthesis inhibitor cycloheximide did not inhibit the rise in tPA mRNA levels in response to PMA/forskolin nor the decline in mRNA levels between 8 and 12 h. However, peak levels (8 h) were approximately 1.5-fold higher than in cultures not treated with cycloheximide. The effect of two inhibitors of protein kinases, H-7 and staurosporine, on PMA-induced tPA antigen secretion and tPA mRNA levels were examined. H-7 and staurosporine inhibited PMA, and PMA/forskolin induced tPA secretion in a dose-dependent manner. This effect was time-dependent; the inhibitory effect was reduced with delayed H-7 addition, and, by 6 h after PMA treatment, no inhibition was observed. H-7 and staurosporine also inhibited the PMA/forskolin-induced increase in tPA mRNA levels and were less effective the later they were added. The same time-dependent effect on the potentiation of PMA-induced tPA mRNA levels by forskolin was observed. Again, delayed addition reduced the effect, and, by 6 h, potentiation was absent. The results of this study indicate that changes in mRNA levels in response to PMA and PMA/forskolin precede and determine those that occur to tPA antigen secretion. In addition, the maximal response is dependent upon the prolonged activation of an H-7- and cAMP-sensitive pathway.
Asunto(s)
Endotelio Vascular/metabolismo , Proteína Quinasa C/metabolismo , ARN Mensajero/genética , Sulfonamidas , Activador de Tejido Plasminógeno/biosíntesis , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina , Alcaloides/farmacología , Northern Blotting , Bloqueadores de los Canales de Calcio/farmacología , Células Cultivadas , Clonación Molecular , Colforsina/farmacología , Cicloheximida/farmacología , ADN/genética , ADN/aislamiento & purificación , Endotelio Vascular/efectos de los fármacos , Humanos , Isoquinolinas/farmacología , Cinética , Piperazinas/farmacología , Estaurosporina , Acetato de Tetradecanoilforbol/farmacología , Activador de Tejido Plasminógeno/genética , Activador de Tejido Plasminógeno/metabolismoRESUMEN
We have examined the effect of phorbol esters and cAMP elevating compounds on tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) secretion. Phorbol esters induce a time- and dose-dependent increase in tPA release from endothelial cells, while forskolin, isobutylmethylxanthine, dibutyryl cAMP, and 8-bromo-cAMP had no significant stimulatory effect on tPA secretion. However, elevation of cAMP simultaneously with phorbol ester treatment potentiated the phorbol ester-induced release of tPA 6 times from 22.2 ng/ml with phorbol myristate acetate (PMA) alone to 122.1 ng/ml (PMA and forskolin). Potentiation was dose-dependent (half-maximal potentiation = 4 microM forskolin), and tPA release was enhanced at all stimulatory concentrations of PMA with no change in the PMA concentrations causing half-maximal or maximum tPA release. The kinetics of release was also similar in PMA versus PMA-forskolin-treated cells. A 4-h delay was observed, enhanced release was transient, and was followed by the onset of a refractory period. In contrast, elevation of cAMP reduced constitutive secretion of PAI-1 by 30-40% and prevented the increase in PAI-1 secretion stimulated by PMA. Elevated cAMP also decreased the rate of PAI-1 deposition into the endothelial substratum. These studies indicate that activation of a cAMP-dependent pathway(s) in coordination with phorbol ester-induced responses plays a central role in modifying the tPA and PAI-1 secretion from endothelial cells, leading to a profibrinolytic state in the endothelial environment.
Asunto(s)
AMP Cíclico/farmacología , Endotelio Vascular/efectos de los fármacos , Glicoproteínas/metabolismo , Ésteres del Forbol/farmacología , Activador de Tejido Plasminógeno/metabolismo , 1-Metil-3-Isobutilxantina/farmacología , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Bucladesina/farmacología , Células Cultivadas , Colforsina/farmacología , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Endotelio Vascular/metabolismo , Humanos , Inactivadores Plasminogénicos , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Tumor-promoting phorbol esters and histamine induce tissue plasminogen activator (tPA) release from human endothelial cells in a dose- and time-dependent manner. Phorbol myristate acetate (PMA) and phorbol dibutyrate (PDBu) increased tPA concentration in the culture medium by eight to 12 times after 24 h with half-maximal stimulation at 13 and 55 nM, respectively. Maximum release by histamine was only half that of the phorbol esters and required 18 microM for half-maximal response. Kinetics of enhanced release was similar with both types of agonists: a 4-h lag period followed by a period of rapid release (4 h in PMA-treated and 10 h in histamine-treated cultures) followed by a decline toward pretreatment rates. The PMA and histamine effects were additive while histamine and thrombin, which also stimulates tPA release in human endothelial cells, were no more effective together than they were alone. Exposure of the cells to PMA, PDBu, or phorbol 12,13-didecanoate caused a loss of responsiveness to second treatment of the homologous agent that was time- and dose-dependent, sustained, and specific to active tumor promoters (half-maximal desensitization = 52 nM PDBu). A partial desensitized state was also established by histamine which resulted in a 60% lower response to a second challenge dose. Histamine-induced desensitization did not interfere with the PMA response. However, PMA-induced desensitization caused a 75% loss of the histamine and a 67% loss of the thrombin effects. These studies indicate that tumor promoters are potent agonists of tPA release from human endothelial cells and establish a desensitized state to further stimulation. Treatment of these cells with histamine has similar effects which may be mediated at least in part by pathways common to phorbol ester stimulation.
Asunto(s)
Endotelio Vascular/metabolismo , Histamina/farmacología , Ésteres del Forbol/farmacología , Activador de Tejido Plasminógeno/metabolismo , Células Cultivadas , Cimetidina/farmacología , Difenhidramina/farmacología , Endotelio Vascular/efectos de los fármacos , Humanos , Cinética , Venas UmbilicalesRESUMEN
We have studied the distribution of the plasminogen activator inhibitor type 1 (PAI-1) in cultures of confluent human umbilical vein endothelial cells. Plasminogen activator inhibitor activity measured by the 125I-fibrin plate assay was detected in the cytosol (2.85 +/- 0.16 U), 100,000 g particulate fraction (1.26 +/- 0.30 U), and in the growth substratum (9.82 +/- 1.80 U). Characterization of the protein responsible for this activity by reverse fibrin autography, immunoprecipitation, and immunoblotting demonstrated that it had an Mr of 46,000 and was antigenically related to PAI-1. Only the active form of the inhibitor was found in all three fractions. Inhibitor in the cytosol and particulate fraction converted to the latent form during 37 degrees C incubation while the substratum inhibitor remained fully active. Extracellular PAI-1 was detected in the growth substratum before its appearance in conditioned medium and represented the major protein deposited beneath the cells. The inhibitor was only transiently localized in the substratum, disappearing within 6 h and concomitantly appearing in the culture medium. Incubation of isolated metabolically labeled substratum with tissue plasminogen activator (tPA) resulted in the appearance and release of an immunologically related inactive 44,000 Mr form as well as the tPA-PAI-1 complex (110,000 Mr). PAI-1 was also converted into its 44,000-Mr form and released by treatment of the substratum with human leukocyte elastase. The rapid deposition and predominance of PAI-1 in the underlying compartment of endothelial cells may explain how the basement membrane is protected from proteolytic degradation by plasmin-generating enzymes.
Asunto(s)
Endotelio Vascular/citología , Glicoproteínas/fisiología , Activadores Plasminogénicos/antagonistas & inhibidores , Inactivadores Plasminogénicos , Membrana Celular/fisiología , Células Cultivadas , Citosol/análisis , Femenino , Fibrinólisis , Glicoproteínas/aislamiento & purificación , Humanos , Peso Molecular , Venas UmbilicalesRESUMEN
The plasminogen activator inhibitor from human endothelial cells (PAI-1) exists in two forms in the culture medium: an active form that binds to and inactivates plasminogen activators and a latent form that in its native state has no anti-activator activity. Inhibitor activity associated with the latent form can be generated by treatment with protein denaturants and makes up more than 98% of the total inhibitor activity in conditioned medium. Plasminogen activator inhibitor activity is also found in cell cytosol. This inhibitor activity is stable to SDS-treatment but is not enhanced by it. We investigated the relationship between this active cell-associated inhibitor and the latent PAI-1 found in the conditioned medium. Both intracellular and extracellular inhibitors were immunoprecipitated by a monoclonal antibody produced against the latent inhibitor from HT1080 fibrosarcoma cells and electrophoresis on SDS gels of various acrylamide concentrations demonstrated that both forms had the same Mr. Incubation of cytosol inhibitor at 37 degrees C resulted in a decline in inhibitor activity with a half-life of approximately 4 hours, a rate of decline similar to that of the active PAI-1 in conditioned medium, with less than 10% of the original activity present after eight hours. This decline is accelerated at higher temperatures and is not affected by the presence of a variety of protease inhibitors. Approximately 90% of the activity can be regenerated after SDS treatment suggesting that the cell associated inhibitor, during incubation at 37 degrees C, converts to a form similar to that found in conditioned medium. Despite these similarities, the apparent Stoke's radii of the active intracellular inhibitor and the latent inhibitor in conditioned medium were significantly different with values of 2.77 nm and 2.40 nm for active and latent PAI-1, respectively. Incubation of the active form at 37 degrees C resulted in the shift of the Stoke's radius to that similar to the latent PAI-1 (2.45 nm). Thus, the active and latent PAI-1, while being immunologically similar and of the same apparent Mr, can be differentiated by their behavior on gel permeation columns. This suggests that the intracellular inhibitor is a precursor to the latent form.