Adenosine transport in cultured human umbilical vein endothelial cells is reduced in diabetes
Sobrevía, L.; Jarvis, S.M.; Yudilevich, D.L.
American Journal of Physiology 267(1 Pt 1): C39-C47
1994
ISSN/ISBN: 0002-9513 PMID: 8048491 Document Number: 435276
Adenosine transport in cultured human umbilical vein endothelial cells (HUVEC) was characterized and shown to be mediated by a single facilitated diffusion mechanism. Initial rates of adenosine influx at 22 degree C were saturable (apparent Michaelis constant, 69 +- 10 mu-M; maximum velocity (V-max), 600 +- 70 pmol cntdot 10-6 cells-1 cntdot s-1) and inhibited by nitrobenzylthioinosine (NBMPR). Formycin B had an unusually high affinity (inhibitory constant (K-i), 18 +- 4.3 mu-M), whereas inosine had a low affinity (K-i, 440 +- 68 mu-M) and nucleobases were without effect on adenosine influx. The number of transporters (1.2 times 10-6 sites/cell) was estimated by NBMPR equilibrium binding (apparent dissociation constant, 0.11 +- 0.01 nM; maximum binding, 2.0 +- 0.15 pmol/ 10-6 cells). In addition, we compared these endothelial cells with those obtained from cords from pregnancies complicated by diabetes (HUVEC-D), since embryopathy may occur in these conditions. HUVEC-D exhibited a 2.3-fold reduction in both the V-max for adenosine influx and the maximum number of NBMPR binding sites (260 +- 40 pmol cntdot 10-6 cells-1 cntdot s-1 and 0.86 +- 0.08 pmol/10-6 cells, respectively). However, the turnover number for each nucleoside transporter in normal and diabetic HUVEC was similar ( apprx 300 adenosine molecules/s). Adenosine metabolism at 10 mu-M in HUVEC-D was modified compared with normal cells. Intracellular phosphorylation ( gt 90%) was the predominant pathway in normal HUVEC, whereas in HUVEC-D, substantial levels of adenine and adenosine were detected. The present results demonstrate therefore the downregulation of the NBMPR-sensitive nucleoside transporter and changes in adenosine metabolism in HUVEC from diabetic pregnancies.