Browsing by Author "Pastor-Anglada, M."

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    Equilibrative nucleoside transporter 1 expression is downregulated by hypoxia in human umbilical vein endothelium
    (2005) Casanello, P.; Torres, A.; Sanhueza, F.; González, M.; Farías, M.; Gallardo, V.; Pastor-Anglada, M.; San Martín, R.; Sobrevía Luarte, Luis Alberto
    Reduced oxygen level (hypoxia) induces endothelial dysfunction and release of the endogenous nucleosideadenosine. Human umbilical vein endothelium (HUVEC) function in an environment with 3% to 5% O2and exhibitefficient adenosine membrane transport via human equilibrative nucleoside transporters 1 (hENT1). We studied whetheradenosine transport and hENT1 expression are altered by hypoxia in HUVEC. Hypoxia (0 to 24 hours, 2% and 1% O2)reduced maximal hENT1-adenosine transport velocity (Vmax) and maximal nitrobenzylthionosine (NBMPR, a high-affinity hENT1 protein ligand) binding, but increased extracellular adenosine concentration. Hypoxia also reducedhENT1 protein and mRNA levels, effects unaltered byN_x0001_-nitro-L-arginine methyl ester (L-NAME, nitric oxide synthase[NOS] inhibitor) or PD-98059 (inhibitor of mitogen-activated protein kinase kinase 1 and 2 [MEK1/2]). Hypoxiareduced endothelial NOS (eNOS) activity and eNOS phosphorylation at Ser1177, but increased eNOS protein level.Hypoxia increased (1 to 3 hours), but reduced (24 hours) p42/44mapkphosphorylation. Thus, hypoxia-increasedextracellular adenosine may result from reduced hENT1-adenosine transport in HUVEC. Hypoxia effect seems not toinvolve NO, but p42/44mapkmay be required for the relatively rapid effect (1 to 3 hours) of hypoxia. These results couldbe important in diseases where the fetus is exposed to intrauterine environments poor in oxygen, such as intrauterinegrowth restriction, or where adenosine transport is altered, such as gestational diabetes.
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    The transcriptional repression of equilibrative nucleoside transporter 1 by D-glucose rfsponses to transcription factor Sp1 and ZBP-89 in human foetal endothelium
    (2008) Puebla Aracena, Carlos Alberto; Farías Jofré, Marcelo Enrique; Vega Pizarro, José Luis Eduardo; Pastor-Anglada, M.; Casanello Toledo, Paola Cecilia; Sobrevía Luarte, Luis Alberto
    Objectives: Adenosine is a vasodilator in most vascular beds, an effect depending on its extracellular concentration. Uptake of this nucleoside in human umbilical vein endothelial cells (HUVEC) is mainly mediated by human equilibrative nucleoside transporters 1 (hENT1). hENT1 expression and transport activity are reduced in HUVEC exposed to high D-glucose. Since specific mechanisms for these effects of D-glucose are unknown, we examined the role of Sp1 and BP-89 transcription factors on SLC29A1 (for hENT1) promoter activity in response to D-glucose. Methods: HUVEC from normal pregnancies were isolated and exposed (24 h) to 5 mM (normal) or 25 mM (high) D-glucose. Sp1 protein levels were evaluated by western blot in nuclear fractions. Reporter activity of plasmid constructs containing a wild type promoter region of SLC29A1 (-1114 bp to ATG, pGL3-hENT1-1114), or mutations (by PCR) for Sp1 (-815/-801 bp, pGL3-hENT1-1114mutSp1) or ZBP-89 (-992/-969 bp, pGL3-hENT1-1114mutZBP) or both (-1114 bp, pGL3-hENT1-1114mutSp1/ZBP) binding sites were assayed in cells over-expressing Sp1 (using pCGN-Sp1 vector). Results: Nuclear Sp1 abundance was increased, but pGL3-hENT1-1114 transcriptional activity was reduced by high D-glucose. Sp1 over-expression reduced pGL3-hENT1-1114 transcriptional activity in normal or high D-glucose. The effect of high D-glucose or Sp1 over-expression was absent in pGL3-hENT1-1114mutSp1, pGL3-hENT1-1114mutZBP and pGL3-hENT1-1114mutSp1/ZBP cells. Conclusion: A repression of the SLC29A1 promoter activity by Sp1 and ZBP-89 could explain the reduced hENT1 expression and activity exhibited by HUVEC in high extracellular D-glucose. FONDECYT 1070865/1080534/7070249 (Chile), AECI A/5484/06 (Spain). C Puebla andJL Vega hold CONICYT fellowships. M Farías holds CONICYT and PUC-School of Medicine PhD fellowships.