RESUMEN
Perihepatic lymph node enlargement (PLNE) which has been shown to be negatively associated with hepatocellular carcinoma (HCC) occurrence is frequently observed in chronic liver disease; however, changes in the state of perihepatic lymph nodes after eradication of hepatitis C virus (HCV) have not been investigated yet. We aimed to evaluate this issue. We enrolled 472 patients with chronic HCV infection who achieved viral eradication with direct-acting antivirals (DAA). We investigated whether the status of perihepatic lymph nodes changed before and after HCV eradication (primary endpoint). We also evaluated the association between PLNE and clinical findings such as liver fibrosis or hepatocellular injury before HCV eradication (secondary endpoint). Perihepatic lymph node enlargement was detected in 164 of 472 (34.7%) patients before DAA treatment. Surprisingly, disappearance of PLNE was observed in 23.8% (39 patients) of all PLNE-positive patients after eradication of HCV. Disappearance of PLNE was not associated with baseline clinical parameters or changing rates of clinical findings before and after DAA treatment. At baseline, presence of PLNE was significantly associated with a lower serum HCV-RNA level (P = .03), a higher serum AST level (P = .004) and a higher ALT level (P < .001) after adjustment for sex and age. In conclusion, PLNEs became undetectable after DAA treatment in 23.8% of PLNE-positive patients. Further study with a longer follow-up period is needed to clarify the clinical importance of this phenomenon especially in relationship with the risk of HCC development.
Asunto(s)
Antivirales/uso terapéutico , Hepatitis C Crónica/tratamiento farmacológico , Hepatitis C Crónica/patología , Ganglios Linfáticos/patología , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Resultado del TratamientoRESUMEN
BACKGROUND AND OBJECTIVES: The effect of leukoreduction and storage periods on the accumulation of bioactive lysophospholipids and substances in human autologous blood (AB units) has not been fully investigated. MATERIALS AND METHODS: The accumulation of bioactive lysophospholipids such as sphingosine 1-phosphate (S1P) and lysophosphatidylserine (LysoPS) in AB units during the storage was investigated. The time-dependent changes and the effect of the filtration in pre-storage leuckoreduction (LR) and unmodified samples derived from 46 AB units were analysed. Additionally, the changes of lysophospholipids and platelet releasate, namely ß-thromboglobulin (ß-TG), induced by exposure of whole blood (WB) or platelet-rich plasma (PRP) to the filter material were analysed. RESULTS: LysoPS, but not S1P levels, time-dependently and significantly increased in both unmodified and LR samples. LysoPS significantly decreased in LR compared with unmodified samples, whereas S1P increased in LR compared with unmodified samples. In addition, exposure of WB and/or PRP to the filter material in vitro resulted in increased levels of S1P, LysoPS and ß-TG. CONCLUSIONS: LR effectively reduced the accumulation of LysoPS in AB units. On the other hand, it increased concentrations of S1P due to platelet activation by exposure to the filter material. These suggest that increases of S1P levels in LR and LysoPS in the unmodified samples were mainly caused by the leukocytes and/or platelets and that LR was effective in inhibiting the accumulation of LysoPS.
Asunto(s)
Conservación de la Sangre , Transfusión de Sangre Autóloga , Procedimientos de Reducción del Leucocitos , Lisofosfolípidos/sangre , Esfingosina/análogos & derivados , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Esfingosina/sangreRESUMEN
Activation of the PDGF receptor on human arterial smooth muscle cells (SMC) induces migration and proliferation via separable signal transduction pathways. Sphingosine-1-phosphate (Sph-1-P) can be formed following PDGF receptor activation and therefore may be implicated in PDGF-receptor signal transduction. Here we show that Sph-1-P does not significantly affect PDGF-induced DNA synthesis, proliferation, or activation of mitogenic signal transduction pathways, such as the mitogen-activated protein (MAP) kinase cascade and PI 3-kinase, in human arterial SMC. On the other hand, Sph-1-P strongly mimics PDGF receptor-induced chemotactic signal transduction favoring actin filament disassembly. Although Sph-1-P mimics PDGF, exogenously added Sph-1-P induces more prolonged and quantitatively greater PIP2 hydrolysis compared to PDGF-BB, a markedly stronger calcium mobilization and a subsequent increase in cyclic AMP levels and activation of cAMP-dependent protein kinase. This excessive and prolonged signaling favors actin filament disassembly by Sph-1-P, and results in inhibition of actin nucleation, actin filament assembly and formation of focal adhesion sites. Sph-1-P-induced interference with the dynamics of PDGF-stimulated actin filament disassembly and assembly results in a marked inhibition of cell spreading, of extension of the leading lamellae toward PDGF, and of chemotaxis toward PDGF. The results suggest that spatial and temporal changes in phosphatidylinositol turnover, calcium mobilization and actin filament disassembly may be critical to PDGF-induced chemotaxis and suggest a possible role for endogenous Sph-1-P in the regulation of PDGF receptor chemotactic signal transduction.
Asunto(s)
Quimiotaxis/efectos de los fármacos , Lisofosfolípidos , Músculo Liso Vascular/fisiología , Factor de Crecimiento Derivado de Plaquetas/antagonistas & inhibidores , Receptores del Factor de Crecimiento Derivado de Plaquetas/fisiología , Esfingosina/análogos & derivados , Citoesqueleto de Actina/ultraestructura , Actinas/metabolismo , Calcio/metabolismo , Calcio/fisiología , Adhesión Celular , División Celular/efectos de los fármacos , Membrana Celular/fisiología , Movimiento Celular/efectos de los fármacos , Células Cultivadas , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Humanos , Técnicas In Vitro , Fosfatidilinositoles/metabolismo , Transducción de Señal , Esfingosina/farmacologíaRESUMEN
Cancer-specific gene promoter methylation has been described in many types of cancers, and various semi-quantified results have shown their usefulness. Here, we show a more sensitive and specific second-generation system for profiling the DNA methylation status. This method is based on bisulfite reaction of DNA and real-time PCR using two TaqMan MGB probes labeled with different fluorescence, followed by clustering analysis. Primers were designed with CpG-less sequences, and TaqMan MGB probes were designed to contain three or four CpG sites and to be shorter than conventional TaqMan probes. We have added new criteria for primer and probe design for further specificity. We confirmed the reliability of this system and applied it to analysis of lung cancers. Using 10 promoters, 90 primary lung cancers were clustered into six groups consisting of cases having similar smoking status and pathological findings. EGFR mutation and p16 promoter DNA methylation were exclusive, as previously reported; however, DNA methylation in other genes was unrelated to EGFR mutation. This system was also useful to distinguish double primary lung cancers from a single cancer with intrapulmonary metastasis. As above, our system has widespread availability in clinical use and biological research.
Asunto(s)
Metilación de ADN , Neoplasias Pulmonares/genética , Regiones Promotoras Genéticas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Análisis por Conglomerados , Islas de CpG , Sondas de ADN , Genes erbB-1 , Humanos , Técnicas de Sonda Molecular , MutaciónRESUMEN
BACKGROUND: Sphingosine 1-phosphate (S1P), known to play important roles in vascular biology, is a bioactive lysophospholipid mediator that maintains endothelial integrity via its cell-surface receptors (S1Ps). In this in vitro study, we aimed to examine the role of S1P in monocyte-endothelium adhesion, which is an important event in the pathophysiology of atherosclerosis. METHODS AND RESULTS: S1P pretreatment of human umbilical vein endothelial cells (ECs), but not U937 cells, effectively suppressed U937-EC adhesion independently from the expression of adhesion molecules, namely ICAM-1, VCAM-1, and E-selectin. This S1P-induced suppressive effect was inhibited by the blockage of S1P(1) and S1P(3) receptors and the specific inhibitors of G(i) protein, Src family proteins, phosphatidylinositol 3-kinase, and Rac1, indicating involvement of these key downstream pathways. Moreover, the RGD peptide and antibodies, which neutralize adhesion via alpha(5)beta(1) and alpha(v)beta(3), effectively inhibited U937-EC adhesion with a degree similar to S1P pretreatment. Both an adhesion assay and flow-cytometric analysis demonstrated that U937 cells adhered through integrins alpha(5)beta(1) and alpha(v)beta(3) expressed on the apical surface of monolayer ECs, and S1P shifted the localization of these integrins from the apical surface to the basal surface. CONCLUSIONS: From the present results, we propose that S1P may contribute to the maintenance of vascular integrity and the regulation of atherogenesis through the rearrangement of endothelial integrins.
Asunto(s)
Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Integrina alfa5beta1/metabolismo , Integrina alfaVbeta3/metabolismo , Lisofosfolípidos/farmacología , Monocitos/citología , Monocitos/efectos de los fármacos , Esfingosina/análogos & derivados , Adulto , Adhesión Celular/efectos de los fármacos , Adhesión Celular/fisiología , Células Cultivadas , Células Endoteliales/metabolismo , Humanos , Técnicas In Vitro , Células Jurkat , Monocitos/metabolismo , Óxido Nítrico Sintasa de Tipo III/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Esfingosina/farmacología , Células U937RESUMEN
The inhibitory effects of ticlopidine on platelet function were evaluated in 18 healthy male volunteers using three different platelet function tests. Three methods include the recently developed small collagen-beads method, which evaluates the platelet response in whole blood samples under shear stress conditions, the conventional platelet aggregometry and a cone-plate viscometer which measures shear-induced platelet aggregation (SIPA). The latter two methods use platelet-rich plasma as measurement samples instead of whole blood. SIPA was significantly inhibited by the oral intake of ticlopidine. The conventional platelet aggregometry detected significant inhibition of ticlopidine on ADP-induced platelet aggregation. In contrast, ticlopidine moderately inhibited platelet aggregation induced by low concentrations of collagen, but not by high concentrations of collagen. With the collagen-bead column method, ticlopidine significantly inhibited platelet retention rates when the retention rates exceeded 30% prior to ticlopidine uptake. On the other hand, there was no significant inhibition when the original retention rates prior to ticlopidine uptake were below 30%. The three methods all proved useful to evaluate the effect of ticlopidine on platelet function. However, taking into consideration easy procedures, lower costs and use of whole blood samples under shear stress conditions, we suggest the collagen-bead column can serve as an appropriate method for monitoring ticlopidine therapy.
Asunto(s)
Plaquetas/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/farmacología , Agregación Plaquetaria/efectos de los fármacos , Ticlopidina/farmacología , Plaquetas/fisiología , Humanos , Masculino , Inhibidores de Agregación Plaquetaria/administración & dosificación , Pruebas de Función Plaquetaria/métodos , Ticlopidina/administración & dosificaciónRESUMEN
BACKGROUND: Radiofrequency ablation (RFA) is a minimally invasive treatment for hepatocellular carcinoma (HCC). There is increasing evidence of an association between increasing hospital volume and lower postoperative mortality for many surgical procedures, but this is difficult to establish with minimally invasive treatments, where postoperative mortality is low. The aim of this study was to investigate the relationship between hospital volume and in-hospital mortality following RFA using a Japanese nationwide database. METHODS: Data from the Diagnostic Procedure Combination database were analysed from 1 July 2010 to 31 March 2012. Multivariable logistic regression was used to analyse the relationship between hospital volume and in-hospital mortality following RFA, with adjustment for patient background. RESULTS: Some 36 675 patients with HCC were identified in the database. The overall in-hospital mortality rate from RFA was 0·31 per cent. In-hospital mortality was significantly higher in low-volume than high-volume hospitals (odds ratio 2·57, 95 per cent c.i. 1·61 to 4·09; P < 0·001). Higher in-hospital mortality was significantly associated with older age and a higher Charlson Co-morbidity Index score. CONCLUSION: RFA for HCC was associated with acceptably low mortality in Japan, but in-hospital mortality following RFA was affected by hospital procedural volume.
RESUMEN
OBJECTIVES: Although the concept of aspirin resistance is extensively reported in medical literature, its precise mechanisms and clinical outcomes are largely unknown. In this study, we examined individual thromboxane biosynthesis and platelet aggregation in aspirin-treated patients, and whether the results of a platelet aggregation test influenced clinical outcomes. RESULTS: Subjects taking 81 mg of aspirin (n = 50) and controls (n = 38) were evaluated for platelet aggregation and platelet cyclooxygenase-1 (COX-1) activity by measuring collagen-induced thromboxane B2 production. For aggregometry, both light transmission (LT) and laser-light scattering methods were employed to quantitatively evaluate aggregate sizes and numbers. Aspirin treatment resulted in the inhibition of collagen-induced platelet aggregation, particularly the transition from small to large platelet aggregates. Although platelet COX-1 activity seemed to be uniformly inhibited in all patients, platelet aggregation studies showed great inter-individual differences; variation in platelet COX-1 activity only accounted for 6-20% of the individual aggregations. Factor analysis revealed the existence of a common factor (other than platelet COX-1) that explained 48.4% of the variations in platelet aggregation induced by collagen, adenosine diphosphate (ADP), and collagen-related peptide. We then prospectively enrolled 136 aspirin-treated patients in our study, and we found that being in the upper quartile level of LT, or with large aggregate formation induced by collagen, was an independent risk factor for developing cardiovascular events within 12 months [hazard ratio (HR) = 7.98, P = 0.008 for LT; HR = 7.76, P = 0.007 for large aggregates]. On the other hand, the existence of diabetes mellitus was an independent risk factor for overall outcomes (HR 1.30-11.9, P = 0.015-0.033). CONCLUSIONS: Aspirin resistance expressed as unsuppressed platelet COX-1 activity is a rare condition in an out-patient population. Other factor(s) affecting collagen-induced platelet aggregation may influence early outcomes in aspirin-treated patients.
Asunto(s)
Aspirina/farmacología , Plaquetas/efectos de los fármacos , Enfermedades Cardiovasculares/sangre , Inhibidores de la Ciclooxigenasa/farmacología , Resistencia a Medicamentos , Inhibidores de Agregación Plaquetaria/farmacología , Agregación Plaquetaria/efectos de los fármacos , Anciano , Aspirina/efectos adversos , Plaquetas/enzimología , Enfermedades Cardiovasculares/etiología , Enfermedades Cardiovasculares/orina , Colágeno/farmacología , Ciclooxigenasa 1/metabolismo , Inhibidores de la Ciclooxigenasa/efectos adversos , Femenino , Humanos , Masculino , Inhibidores de Agregación Plaquetaria/efectos adversos , Pruebas de Función Plaquetaria/métodos , Estudios Prospectivos , Factores de Riesgo , Transducción de Señal , Tromboxano B2/análogos & derivados , Tromboxano B2/metabolismo , Tromboxano B2/orina , Resultado del TratamientoRESUMEN
Blood platelets are very unique in that they store sphingosine 1-phosphate (Sph-1-P) abundantly (possibly due to the existence of highly active sphingosine kinase and a lack of Sph-1-P lyase) and release this bioactive lipid extracellularly upon stimulation. Vascular endothelial cells (ECs) and smooth muscle cells (SMCs) respond dramatically to this platelet-derived bioactive lipid mainly through a family of G protein-coupled Sph-1-P receptors named S1P1, 2, 3, 4, and 5, originally referred to as EDG-1, 5, 3, 6, and 8, respectively. In fact, the importance of Sph-1-P in platelet-vascular cell interactions has been revealed in a number of recent reports. Through interaction with ECs, Sph-1-P can mediate physiological wound healing processes such as vascular repair, although this important bioactive lipid can become atherogenic and thrombogenic, and cause or aggravate cardiovascular diseases especially under certain pathological conditions. On the other hand, Sph-1-P induces vasoconstriction through interaction with SMCs. It is likely that regulation of Sph-1-P biological activities is important for the therapeutical purpose to control vascular disorders. Particularly, the development of specific S1P receptor agonists or antagonists seems a reasonable strategy to selectively regulate the bioactivity of Sph-1-P, considering that a great diversity of Sph-1-P actions has been reported and that this diversity depends mainly on the S1P receptor subtype involved. In this review, I will summarize recent findings on possible roles of Sph-1-P in vascular biology and its therapeutical implications.
Asunto(s)
Vasos Sanguíneos/fisiología , Lisofosfolípidos/fisiología , Lisofosfolípidos/uso terapéutico , Esfingosina/análogos & derivados , Enfermedades Vasculares/tratamiento farmacológico , Animales , Plaquetas/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/fisiología , Humanos , Lisofosfolípidos/sangre , Lisofosfolípidos/deficiencia , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Neovascularización Patológica/tratamiento farmacológico , Esfingosina/sangre , Esfingosina/deficiencia , Esfingosina/fisiología , Esfingosina/uso terapéuticoRESUMEN
The present studies were undertaken to characterize the potential role of sphingosine in the regulation of apoptosis in HL-60 promyelocytic leukemia cells. A 6-h exposure of HL-60 cells to sphingosine or its methylated derivative, N,N-dimethylsphingosine, caused internucleosomal DNA fragmentation and stereotypical morphological changes characteristic of apoptosis (i.e., cell shrinkage, nuclear condensation, and the formation of apoptotic bodies), as well as that to pharmacological inhibitors of protein kinase C such as 1-(5-isoquinolinesulfonyl)-2-methylpiperazine and staurosporine. Apoptosis by sphingosine and N,N-dimethylsphingosine was measured using a flow cytometric method. The percentages of apoptotic cells in cultures treated with sphingosine (10 microM) and N,N-dimethylsphingosine (10 microM) for 6 h were 55.6 +/- 7.8% and 84.2 +/- 11.6%, respectively. HL-60 cells were induced to differentiate toward macrophages by treatment with 5 nM 4 beta-phorbol 12-myristate 13-acetate (PMA). Internucleosomal DNA fragmentation, which was a hallmark of apoptosis, was first detected after 10-h exposure to PMA and increased with longer treatment. Sphingosine concentrations in the cells increased concomitantly with the increasing proportion of apoptotic cells during cell differentiation. Sphingosine level in HL-60 cells differentiated by treatment with PMA for 48 h was about 3.3-fold greater than that in untreated cells. Differentiated HL-60 cells exhibited a markedly increased conversion of exogenously added [3H]ceramide to [3H]sphingosine, indicating elevation of ceramidase activity. Moreover, exposure to sphingosine resulted in down-regulation of c-myc mRNA. These observations suggest the possible role of sphingosine in induction of apoptotic DNA fragmentation during PMA-induced differentiation in myeloid leukemia cells. Sphingosine may function as an endogenous modulator mediating the apoptotic signal.
Asunto(s)
Apoptosis/efectos de los fármacos , ADN de Neoplasias/efectos de los fármacos , Esfingosina/farmacología , Acetato de Tetradecanoilforbol/farmacología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Línea Celular , Ceramidas/metabolismo , Daño del ADN , Citometría de Flujo/métodos , Expresión Génica/efectos de los fármacos , Genes myc , Humanos , Cinética , Leucemia Promielocítica Aguda , Fosfolípidos/análisis , Fosfolípidos/metabolismo , Proteínas Proto-Oncogénicas c-myc/biosíntesis , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Esfingosina/análogos & derivados , Factores de Tiempo , Tritio , Células Tumorales CultivadasRESUMEN
A megakaryoblastic cell line, termed T-33, was established from the peripheral blood of a patient with Philadelphia chromosome-positive chronic myelogenous leukemia in megakaryoblastic crisis. T-33 cells have been maintained in RPMI 1640 medium containing 10% fetal calf serum in a single cell suspension with a doubling time of 24-36 h for over 2 years. Giemsa-banded karyotypes were female hyperdiploid with a modal chromosomal number of 51, all cells including Philadelphia chromosome. The cells showed strong positivity for periodic acid-Schiff and alpha-naphthyl acetate esterase, and weak for alpha-naphthyl butyrate esterase, but were negative for myeloperoxidase. Flow cytometric analysis of cell surface markers showed the existence of HLA-DR, MY-7, MY-9, and a platelet antigen (Yukb), and no markers for T- or B-lymphocytes. Most of the cells fixed with acetone were positive for Factor VIII, platelet glycoprotein IIb-IIIa, IIIa (Yukb), and Ib, but negative for glycophorin A and hemoglobin. Ultrastructural platelet peroxidase was demonstrated in 2-3% of cells and the percentage of positive cells increased up to 20% after the treatment with 12-O-tetradecanoylphorbol-13-acetate. The cells contained small dense granules negative for platelet peroxidase, their number increasing threefold after 12-O-tetradecanoylphorbol-13-acetate treatment. Such treated cells frequently showed a complex of the demarcation membrane in the cytoplasm. T-33 responded thrombin to exhibit calcium influx. This cell line may be useful for the study of the early stage of megakaryocytic differentiation in human megakaryopoiesis.
Asunto(s)
Leucemia Megacarioblástica Aguda/patología , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Antígenos de Superficie/análisis , Diferenciación Celular , Femenino , Humanos , Cariotipificación , Leucemia Megacarioblástica Aguda/genética , Leucemia Megacarioblástica Aguda/inmunología , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/inmunología , Persona de Mediana Edad , Peroxidasas/análisis , Glicoproteínas de Membrana Plaquetaria/análisis , Trombina/farmacología , Células Tumorales CultivadasRESUMEN
Sodium fluoride (NaF) alone below the concentration of 10 mM had no effect on platelet intracellular Ca2+ ([Ca2+]i). When platelets were incubated with low concentrations of NaF (< 10 mM) prior to thrombin stimulation, the second phase of [Ca2+]i elevation which is attributable to Ca2+ influx was suppressed, while the initial rapid peak of [Ca2+]i which is attributable to internal Ca2+ release was unaffected. Ca2+ influx assessed by the addition of extracellular Ca2+ to cells preactivated by thrombin in the absence of extracellular Ca2+ was also inhibited by NaF in a dose-dependent manner. NaF was also effective in inhibiting thrombin- or U-46619-induced Mn2+ entry. This inhibitory effect of NaF on Ca2+ influx occurred after a lag of at least 30 s. However, Ca2+ influx induced by ionomycin-induced Ca2+ depletion or by thapsigargin, a Ca(2+)-ATPase inhibitor, was only partially suppressed by NaF treatment. It is suggested that Ca2+ entry induced by receptor-mediated activation is NaF-sensitive and that the depletion of Ca2+ storage sites by artificial procedures facilitates the opening of Ca2+ channels via NaF-insensitive pathways.
Asunto(s)
Plaquetas/efectos de los fármacos , Calcio/metabolismo , Activación Plaquetaria , Receptores de Superficie Celular/fisiología , Fluoruro de Sodio/farmacología , Plaquetas/metabolismo , Canales de Calcio/efectos de los fármacos , Proteínas Portadoras/efectos de los fármacos , Humanos , Manganeso/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Intercambiadores de Sodio-Hidrógeno , Trombina/farmacologíaRESUMEN
The potentiation by 1,2-dioctanoyl-sn-glycerol (DiC8) of ionomycin-induced platelet production of 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) was investigated in correlation with extracellular Ca2+ concentrations and increases in [Ca2+]i, as detected with aequorin and fura-2. Extracellular Ca2+ concentrations greatly influenced the production of arachidonic acid metabolites induced by DiC8 and ionomycin, while that induced by ionomycin alone was minimally affected by variation of the extracellular Ca2+ concentration. In the synergy between ionomycin and 20 microM DiC8, the optimal concentrations of ionomycin shifted from high to low with increasing concentrations of extracellular Ca2+, suggesting that there might be a range of optimal [Ca2+]i for the production of the arachidonic acid metabolites. This hypothesis was confirmed by simultaneous measurements of [Ca2+]i increases, and the production of the arachidonic acid metabolites. With the aequorin method, the optimal concentrations of [Ca2+]i fell to between 10 microM and 20 microM, and with the fura-2 method, it fell to between 800 nM and 1800 nM. Direct measurements of [14C]arachidonic acid release suggested that the DiC8-potentiated production of arachidonic acid metabolites induced by ionomycin was attributable to increased arachidonic acid release. Since ionomycin and DiC8 induced relatively low levels of phosphatidic acid production, an indicator of phospholipase C activation, it was suggested that the increased arachidonic acid release was largely dependent upon phospholipase A2. Synergy between DiC8 and ionomycin was also observed with aggregation and serotonin release. Aggregation was induced by lower concentrations of ionomycin, and appeared to be more dependent upon extracellular Ca2+, while serotonin release required higher concentrations of ionomycin, and variations in extracellular Ca2+ affected the response minimally. These findings suggest that the mechanisms underlying the synergy between protein kinase C activation and Ca2+ mobilization differ among the three functions evaluated in this study.
Asunto(s)
Ácidos Araquidónicos/sangre , Plaquetas/metabolismo , Calcio/sangre , Diglicéridos/farmacología , Glicéridos/farmacología , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico , Aequorina , Ácido Araquidónico , Benzofuranos , Plaquetas/efectos de los fármacos , Cationes Bivalentes , Sinergismo Farmacológico , Éteres/farmacología , Espacio Extracelular/metabolismo , Ácidos Grasos Insaturados/sangre , Colorantes Fluorescentes , Fura-2 , Humanos , Ácidos Hidroxieicosatetraenoicos/sangre , Ionomicina , Ácidos Fosfatidicos/sangre , Fosfolipasas A/sangre , Fosfolipasas A2 , Agregación Plaquetaria/efectos de los fármacos , Serotonina/sangreRESUMEN
To elucidate the mechanism involving synthesis of phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2), which is the main species of 3-phosphorylated phosphoinositides in activated blood platelets, we observed a correlation among protein-tyrosine phosphorylation, protein kinase C (PKC) activation, and PtdIns(3,4)P2 synthesis in these anucleate cells. Thrombin (1 U/ml) elicited marked protein-tyrosine phosphorylation, PKC activation, and PtdIns(3,4)P2 synthesis. In contrast, 1 microM 12-O-tetrade-canoylphorbol 13-acetate barely induced tyrosine phosphorylation and PtdIns(3,4)P2 synthesis although it strongly activated PKC. A variety of kinase inhibitors were tested for their ability to inhibit the thrombin effects. Both staurosporine and tyrphostin inhibited thrombin-stimulated tyrosine phosphorylation and PtdIns(3,4)P2 synthesis. H-7, which specifically, although weakly, inhibited PKC activation, had no effect on tyrosine phosphorylation and PtdIns(3,4)P2 production. Among the various kinase inhibitors tested, staurosporine was the most potent inhibitor of protein tyrosine phosphorylation and PtdIns(3,4)P2 synthesis, and there was a good correlation of the inhibition between these two parameters, although it also inhibited PKC activation. To examine the involvement of PtdIns 3-kinase, which is believed to play an important role in 3-phosphorylated phosphoinositide synthesis, we studied tyrosine phosphorylation and the association with tyrosine-phosphorylated proteins of the p85 alpha subunit of PtdIns 3-kinase in thrombin-stimulated platelets. We did not detect tyrosine-phosphorylated protein by Western blotting where p85 alpha was located. Similarly, when platelet lysates were precipitated with anti-p85 alpha antibodies and then blotted with anti-phosphotyrosine antibodies, tyrosine-phosphorylated p85 alpha was undetectable. Furthermore, when the cell lysates were precipitated with anti-phosphotyrosine antibodies, no p85 alpha was found in the immunoprecipitates. These results show that PtdIns(3,4)P2 synthesis in stimulated platelets is mediated by tyrosine phosphorylation, as it is in proliferating cells, but the p85 alpha subunit of PtdIns 3-kinase may not be a target for tyrosine kinases and that staurosporine, though non-specific, would be a useful tool for elucidating signal transduction involving D-3-phosphorylated phosphoinositide generation and protein-tyrosine phosphorylation in blood platelets.
Asunto(s)
Plaquetas/efectos de los fármacos , Fosfatos de Fosfatidilinositol/biosíntesis , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Trombina/farmacología , Tirfostinos , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina , Alcaloides/farmacología , Plaquetas/metabolismo , Catecoles/farmacología , Activación Enzimática/efectos de los fármacos , Humanos , Isoquinolinas/farmacología , Nitrilos/farmacología , Fosfatidilinositol 3-Quinasas , Fosforilación , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Piperazinas/farmacología , Proteína Quinasa C/metabolismo , Estaurosporina , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
The interaction between von Willebrand factor (vWF) and glycoprotein Ib (GPIb) induced by ristocetin or botrocetin resulted in associated platelet aggregation, protein tyrosine phosphorylation (PTP) of a 64 kDa protein, as detected by a monoclonal antibody against phosphotyrosine (PY-20), and intracellular Ca2+ elevation that is largely dependent upon Ca2+ influx in human platelets. It is of interest that 75-80, 97 and 125 kDa proteins which are strongly tyrosine-phosphorylated in platelet activation induced by thrombin and other agonists were not detected. Neither vWF nor a coaggregating agent (ristocetin or botrocetin) alone induced aggregation, [Ca2+]i elevation or the 64 kDa PTP. NMC-4, an antibody which inhibits both ristocetin- or botrocetin-induced vWF binding to GPIb, abolished the appearance of the 64 kDa PTP as well as other responses, suggesting that it is specifically induced by the GPIb-vWF interaction. Aspirin, or ONO-3708, a competitive inhibitor of thromboxane A2, did not modify the 64 kDa PTP, while [Ca2+]i elevation was moderately suppressed. Depletion of extracellular Ca2+ or RGD peptides suppressed neither the 64 kDa PTP nor aggregation. H-7, a protein kinase C inhibitor, did not inhibit the 64 kDa PTP, while staurosporine, a potent protein kinase inhibitor, inhibited the 64 kDa PTP and Ca2+ influx, but not aggregation, in a dose-dependent manner. It is suggested that the 64 kDa PTP is associated with platelet aggregation induced by the interaction between GPIb and vWF.
Asunto(s)
Plaquetas/metabolismo , Fosfoproteínas/sangre , Glicoproteínas de Membrana Plaquetaria/metabolismo , Tirosina/análogos & derivados , Factor de von Willebrand/metabolismo , Alcaloides/farmacología , Plaquetas/efectos de los fármacos , Calcio/sangre , Venenos de Crotálidos/farmacología , Epoprostenol/farmacología , Humanos , Fosforilación , Fosfotirosina , Agregación Plaquetaria/efectos de los fármacos , Ristocetina/farmacología , Estaurosporina , Tirosina/sangre , Factor de von Willebrand/farmacologíaRESUMEN
Tyrosine kinases, c-Src and Fyn, in their active form, have their C-terminal tyrosine residue dephosphorylated. In this study, we used clone 28, a monoclonal antibody (MoAb) that recognizes dephosphorylated C-terminal tyrosine of c-Src and Fyn, to investigate the mode of activation and mobilization of these kinases. Independently of integrin alphaIIbbeta3 signaling, the Fyn activity increased by 8.3-fold 5 s after stimulation with 20 microM TRAP (thrombin receptor agonist peptide), while that of c-Src increased only by 2.9-fold 15 s after stimulation. Both c-Src and Fyn translocated to the Triton-insoluble cytoskeletal fraction in an aggregation-dependent manner. Five minutes after TRAP-stimulation, 85% of Fyn translocated to the cytoskeleton, while only about 20% of c-Src was recovered in this fraction. The Triton-insoluble fraction was further fractionated by RIPA (radioimmunoprecipitation assay) buffer containing 0.1% SDS. While active c-Src was predominantly present in the Triton-insoluble/RIPA-insoluble fraction, clone 28-negative c-Src was present in the Triton-insoluble/RIPA-soluble fraction. On the other hand, Fyn was present only in the Triton-insoluble/RIPA-insoluble fraction. These findings suggest that the mode of activation and redistribution into the cytoskeleton differs between c-Src and Fyn, and that clone 28 provides a useful tool for investigating the activation and mobilization of Src family tyrosine kinases.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Plaquetas/enzimología , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Secuencia de Aminoácidos , Plaquetas/efectos de los fármacos , Fraccionamiento Celular , Humanos , Datos de Secuencia Molecular , Octoxinol , Fragmentos de Péptidos/farmacología , Fosforilación , Proteínas Tirosina Quinasas/química , Proteínas Tirosina Quinasas/inmunología , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/inmunología , Proteínas Proto-Oncogénicas c-fyn , Proteínas Proto-Oncogénicas pp60(c-src)/química , Proteínas Proto-Oncogénicas pp60(c-src)/inmunología , Ensayo de Radioinmunoprecipitación , Solubilidad , Fracciones Subcelulares/enzimología , Tirosina/metabolismoRESUMEN
Using glutathione S-transferase Syk fusion proteins, we evaluated the mode of platelet FcgammaRII tyrosine phosphorylation induced by FcgammaRII cross-linking or anti-CD9 monoclonal antibodies (mAb). The N-terminal SH2 domain of Syk (Syk-N-SH2), the C-terminal SH2 domain of Syk (Syk-C-SH2), and the domain having both the N- and C-terminal SH2 of Syk (Syk-NC-SH2) all bound to tyrosine-phosphorylated FcgammaRII with FcgammaRII cross-linking. In the case of anti-CD9 mAb-induced platelet activation, only Syk-C-SH2 and Syk-NC-SH2 bound to tyrosine-phosphorylated FcgammaRII. Since the SH2 domain is specific for a particular structure containing phosphotyrosine, these findings suggest that only one tyrosine residue in the immunoreceptor tyrosine-based activation motif (ITAM) is phosphorylated with anti-CD9 mAb, and that both are phosphorylated with FcgammaRII cross-linking. Synthetic peptides corresponding to the ITAM of human platelet FcgammaRII with the N-terminal tyrosine residue phosphorylated (N-P) or the C-terminal tyrosine residue phosphorylated (C-P), were used. N-P more potently dissociated Syk-C-SH2 from tyrosine-phosphorylated FcgammaRII than C-P, suggesting that the N-terminal tyrosine residue is phosphorylated upon anti-CD9 mAb-induced activation. Furthermore, these findings imply that Syk-N-SH2 binds to the phosphorylated C-terminal tyrosine residue of ITAM, and Syk-C-SH2 to the N-terminal tyrosine. Taken together, our findings suggest that FcgammaRII-dependent platelet activation without FcgammaRII dimerization, such as with anti-CD9 mAb, is distinct from that induced by FcgammaRII cross-linking.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Antígenos CD/inmunología , Plaquetas/inmunología , Receptores de IgG/inmunología , Secuencia de Aminoácidos , Antígenos CD/química , Unión Competitiva , Precursores Enzimáticos/química , Humanos , Péptidos y Proteínas de Señalización Intracelular , Datos de Secuencia Molecular , Fosforilación , Activación Plaquetaria , Proteínas Tirosina Quinasas/química , Receptores de IgG/química , Proteínas Recombinantes de Fusión/química , Quinasa Syk , Tirosina/química , Dominios Homologos srcRESUMEN
We analyzed the intracellular action of sphingosine 1-phosphate (Sph-1-P), formed from sphingosine (Sph) by sphingosine kinase (SPHK), in platelets. When sphingosine kinase activity was inhibited by N,N-dimethylsphingosine (DMS), Ca2+ mobilization induced by convulxin, an agonist of the collagen receptor glycoprotein VI (GPVI), was moderately but specifically abolished; that induced via G protein-coupled receptors was not affected. Under the same conditions, however, tyrosine phosphorylation of Syk and phospholipase Cgamma2, which is essential for the GPVI-mediated signaling, was not inhibited. Sphingosine kinase activity of the platelet membrane fraction increased specifically upon stimulation with convulxin or collagen. Our results suggest that intracellular sphingosine 1-phosphate is related to Ca2+ mobilization in GPVI-mediated signaling pathways.
Asunto(s)
Plaquetas/efectos de los fármacos , Señalización del Calcio/efectos de los fármacos , Calcio/metabolismo , Lisofosfolípidos/metabolismo , Glicoproteínas de Membrana Plaquetaria/metabolismo , Esfingosina/análogos & derivados , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacología , Plaquetas/metabolismo , Señalización del Calcio/fisiología , Colágeno/farmacología , Venenos de Crotálidos/farmacología , Humanos , Lectinas Tipo C , Lisofosfolípidos/sangre , Fosforilación/efectos de los fármacos , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Glicoproteínas de Membrana Plaquetaria/agonistas , Receptores de Trombina/química , Esfingosina/sangre , Esfingosina/metabolismo , Esfingosina/farmacología , Tirosina/efectos de los fármacos , Tirosina/metabolismoRESUMEN
OBJECTIVE: Sphingosine 1-phosphate is a naturally occurring biologically active lysophospholipid. Recent studies suggested that sphingosine 1-phosphate is released into the blood flow from activated platelets upon stimulation to exert multiple biological phenomenon. The purpose of this study was to assess the effects of sphingosine 1-phosphate on sinus automaticity, ventricular contraction and coronary blood flow. METHODS: The canine isolated, blood-perfused sinoatrial node and papillary muscle preparations were used. RESULTS: Sphingosine 1-phosphate increased the sinoatrial rate, while it decreased the coronary blood flow, which was followed by a weak negative inotropic effect. These positive chronotropic and coronary vasoconstrictor effects were not attenuated by the beta- and alpha-adrenoceptor antagonists atenolol and prazosin, respectively. Furthermore, sphingosine 1-phosphate did not affect the adenylate cyclase activity of the membrane preparations made from the canine right atrium and right ventricle, indicating the involvement of a novel signaling pathway in sphingosine 1-phosphate-induced cardiac effects. CONCLUSIONS: These results may provide a clue to better understanding the physiological as well as the pathophysiological regulation of sphingosine 1-phosphate in the heart.
Asunto(s)
Circulación Coronaria/efectos de los fármacos , Lisofosfolípidos , Sistemas de Mensajero Secundario , Esfingosina/análogos & derivados , Taquicardia Sinusal/inducido químicamente , Adenilil Ciclasas/metabolismo , Antagonistas Adrenérgicos alfa/farmacología , Antagonistas Adrenérgicos beta/farmacología , Análisis de Varianza , Animales , Atenolol/farmacología , Perros , Femenino , Masculino , Contracción Miocárdica/efectos de los fármacos , Miocardio/enzimología , Perfusión , Prazosina/farmacología , Esfingosina/farmacologíaRESUMEN
Diabetes mellitus (DM) is a major risk factor for stroke and it exacerbates tissue damage after ischemic insult. Diabetes is one of the important causes of the progression of white matter lesion, however, the pathological mechanisms remain unclear. The present study evaluated the influences of type 2 DM on ischemic subcortical white matter injury and the recruitment of oligodendrocyte progenitor cells (OPCs) under chronic cerebral hypoperfusion using type 2 diabetic (db/db) mice. After bilateral common carotid artery stenosis (BCAS), the rarefaction in the white matter was more severe in db/db mice than in db/+ mice, and the number of glutathione S-transferase-pi (GST-pi)-positive mature oligodendrocytes (OLG) was lower in db/db mice than in db/+ mice at 4 and 8 weeks after ischemia. There were no significant differences in the number of single-stranded DNA (ssDNA)-positive apoptotic cells in the deep white matter between the db/db and db/+ mice. We found a transient increase in the platelet-derived growth factor receptor-α (PDGFRα)-positive OPCs in white matter lesions after ischemia. However, significantly fewer PDGFRα-positive OPCs were detected in db/db than db/+ mice from 4weeks after BCAS. The number of Ki67-positive proliferating cells in the deep white matter was significantly lower in db/db mice than in db/+ mice from 4 to 8weeks after BCAS. Most of the Ki67-positive cells were PDGFRα-positive OPCs. Finally, we assessed the survival of 5-bromo-2'-deoxyuridine (BrdU)-positive proliferating cells in ischemic white matter, and found significantly poorer survival of BrdU/PDGFRα-positive OPCs or BrdU/GST-pi-positive OLGs in the db/db mice compared to the db/+ mice in the white matter after BCAS. Our findings suggest that the type 2 DM mice exhibited more severe white matter injury 8 weeks after chronic ischemia. Decreased proliferation and survival of OPCs may play an important role in the progression of white matter lesions after ischemia in diabetics.