High D ‐glucose reduces SLC29A1 promoter activity and adenosine transport involving specific protein 1 in human umbilical vein endothelium

High D ‐glucose reduces human equilibrative nucleoside transporter 1 (hENT1)‐mediated adenosine uptake involving endothelial nitric oxide synthase (eNOS), mitogen‐activated protein (MAP) kinase kinases 1 and 2/MAP kinases p42/44 (MEK/ERKs), and protein kinase C (PKC) activation in human umbilical vein endothelium (HUVEC). Since NO represses SLC29A1 gene (hENT1) promoter activity we studied whether D ‐glucose‐reduced hENT1‐adenosine transport results from lower SLC29A1 expression in HUVEC primary cultures. HUVEC incubation (24 h) with high D ‐glucose (25 mM) reduced hENT1‐adenosine transport and pGL3‐hENT1 −1114 construct SLC29A1 reporter activity compared with normal D ‐glucose (5 mM). High D ‐glucose also reduced pGL3‐hENT1 −1114 reporter activity compared with cells transfected with pGL3‐hENT1 −795 construct. N G ‐nitro‐ L ‐arginine methyl ester ( L ‐NAME, NOS inhibitor), PD‐98059 (MEK1/2 inhibitor), and/or calphostin C (PKC inhibitor) blocked D ‐glucose effects. Insulin (1 nM) and phorbol 12‐myristate 13‐acetate (PMA, 100 nM, PKC activator), but not 4α‐phorbol 12,13‐didecanoate (4αPDD, 100 nM, PMA less active analogue) reduced hENT1‐adenosine transport. L ‐NAME and PD‐98059 blocked insulin effects. L ‐NAME, PD‐98059, and calphostin C increased hENT1 expression without altering protein or mRNA stability. High D ‐glucose increased Sp1 transcription factor protein abundance and binding to SLC29A1 promoter, phenomena blocked by L ‐NAME, PD‐98059, and calphostin C. Sp1 overexpression reduced SLC29A1 promoter activity in normal D ‐glucose, an effect reversed by L ‐NAME and further reduced by S ‐nitroso‐ N ‐acetyl‐ L , D ‐penicillamine (SNAP, NO donor) in high D ‐glucose. Thus, reduced hENT1‐mediated adenosine transport in high D ‐glucose may result from increased Sp1 binding to SLC29A1 promoter down‐regulating hENT1 expression. This phenomenon depends on eNOS, MEK/ERKs, and PKC activity, suggesting potential roles for these molecules in hyperglycemia‐associated endothelial dysfunction. J. Cell. Physiol. 215: 645–656, 2008. © 2007 Wiley‐Liss, Inc.