Aims: In this study we investigated the effects of P2 receptors in the regulation of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) in human umbilical vein endothelial cells (HUVEC). Methods: Cytosolic Ca²⁺ concentration ([Ca²⁺]_i) was measured using fura-2/AM, and MAPK/ ERK phosphorylation using Western blot analysis. Results: ATP, 2-meSATP, UTP and UDP cause a rapid and transitory increase in the phosphorylation of MAPK/ERK. In contrast, negligible response was seen for a,ß-meATP, a general P2X receptors agonist. ATPdependent activation of MAPK/ERK was prevented by pretreatment of HUVEC with pertussis toxin or MEK inhibitor PD98059. In addition, activation of the MAPK/ ERK cascade by ATP was blocked in cells pretreated with wortmannin and LY294002, but not by U73122, BAPTA or a Ca²⁺-free medium. Furthermore, an inhibition of ATP-dependent MAPK/ERK phosphorylation was observed in HUVEC pretreated with high doses of GF109203X or myristoylated PKC- ζ pseudosubstrate. Similar results were observed when cells were pretreated with the Src tyrosine kinase inhibitor PP2. However, ATP-stimulated MAPK/ERK activation was unaffected in cells pretreated with AG1478 or perillic acid. We also found that ATP stimulates both the phosphorylation of 3- phosphoinositide-dependent protein kinase-1 (PDK1) and its translocation to plasma membrane in a timedependent manner. Conclusion: These observations suggest that the effects mediated by ATP in HUVEC occur via PTX-sensitive G-protein-coupled P2Y receptors through PI3K-dependent mechanisms, in which PDK1 and PKC-ζ are two key molecules within signal cascade leading to MAPK/ERK activation.

No abstract

No abstract

Aims: In this study we investigated the effect of cisplatin on endothelial cell migration, an essential process for vascular remodeling and regeneration in several physiological and pathological situations. Material and Methods: Human umbilical vein endothelial cells (HUVEC) were treated with cisplatin and endothelial cell migration analyzed by fluorescence and scratch-wound migration assay. MMP2 and MMP9 activity were determined by zymographic assay, and MAPK activation by Western blotting analysis. Results: We demonstrated that cisplatin provoked a time- and dose-dependent decrease of HUVEC migration; this effect was clearly independent from its well known cytotoxic activity. In addition, cisplatin markedly reduced MMP2 activity in both conditioned media and cell lysates, increased p38 MAPK and JNK phosphorylation, but did not affect ERK phosphorylation. Endothelial cell migration was attenuated by treatment of cells with GM6001, a non-specific inhibitor of MMPs, or by a selective anti-MMP2 antibody. However, treatment of cells with SB202190 or SP600125, inhibitors of p38 MAPK and JNK respectively, did not affect HUVEC migration. Conclusion: These results suggested that cisplatin induced a reduction of endothelial cell migration through an inhibition of MMP2 activity by downstream signal transduction pathways independent of JNK and p38 MAPK activation.

33 Un ejemplar similar a este sello, aunque fragmentado, faltándole gran parte de las ansae, fue catalogado como inédito en nuestro estudio sobre los materiales de un vertedero extramuros situado en la C/Maestro Copín c/v San Salvador del Nido, datado a mediados del siglo II d. C. (FERNÁNDEZ FREI-LE, 2003: 37-38, lám. 6, nº 5). Sin embargo, con la excavación del acueducto, previa a esta publicación, pudimos comprobar la similitud de esta estampilla latericia hasta el momento única, con el numeroso grupo que aparecía en los ladrillos de la conducción hidráulica, aspecto que hacemos constar en Ibid. p. 38, nota 51. 34 La presencia de veteranos asentados en Legio es escasa según constata Palao (1998: 186), a tenor de los datos proporcionados fundamentalmente por las fuentes epigráficas. 40 Sobre la diferente composición demográfica de este núcleo y características de la vida social y religiosa, a partir de diversas fuentes epigráficas, vid. GARCÍA MARTÍNEZ, 1999. 41 El papel del anfiteatro en la vida del soldado romano ha sido tratado ampliamente por LE ROUX (1990). FIG. 7: Vertederos localizados en Legio(Elaboración propia sobre plano general de V. García Marcos) 66 Para establecer las diferencias, en la estructura entre las letrinas privadas de las domus y las situadas en los espacios públicos, vid.