Regulation of adenosine transport by D-glucose in human fetal endothelial cells: involvement of nitric oxide, protein kinase C and mitogen-activated protein kinase

1. The effects of elevated D-glucose on adenosine transport were investigated in human cultured umbilical vein endothelial cells isolated from normal pregnancies. 2. Elevated D-glucose resulted in a time- (8-12 h) and concentration-dependent (half-maximal at 10 +/- 2 mM) inhibition of adenosine transport, which was associated with a reduction in the V-max for nitrobenzylthioinosine (NBMPR)-sensitive (es) saturable nucleoside with no significant change in K-m. D-Fructose (25 mM), 2-deoxy-D-glucose (25 mat) or D-mannitol (20 mM) had no effect on adenosine transport. 3. Adenosine transport was inhibited following incubation of cells with the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA; 100 nM, 30 min to 24 h). D-Glucose induced inhibition of transport was abolished. by calphostin C(100 nM, an inhibitor of PKC), and was not further reduced by PMA. 4. Increased PKC activity in the membrane (particulate) fraction of endothelial cells exposed to D-glucose or PMA was blocked by calphostin C but was unaffected by NG-nitro-L-arginine methyl ester (L-NAME; 100 muM, an inhibitor of nitric oxide synthase (NOS)) or PD-98059 (10 muM, an inhibitor of mitogen-activated protein kinase kinase 1). 5. D-Glucose and PMA increased endothelial NOS (eNOS) activity which was presented by calphostin C or omission of extracellular Ca2+ and unaffected by PD-98059. 6. Adenosine transport mas inhibited by S-nitroso-N-acetyl-L,D-penicillamine (SNAP; 100 muM, an NO donor) but was increased in cells incubated with L-NAME. The effect of SNAP on adenosine transport was abolished by PD-98059. 7. Phosphorylation of mitogen-activated protein kinases p44(mapk) (ERK1) and p42(mapk) (ERK2) was increased in endothelial cells exposed to elevated D-glucose (25 mM for 30 min to 24 h) and the NO donor SNAP (100 muM, 30 min). The effect of D-glucose was blocked by PD-98059 or L-NAME, which also prevented the inhibition of adenosine transport mediated by elevated D-glucose. 8. Our findings provide evidence that D-glucose inhibits adenosine transport in human fetal endothelial cells by a mechanism that involves activation of PKC, leading to increased NO levels and p42-p44(mapk) phosphorylation. Thus, the biological actions of adenosine appear to be altered under conditions of sustained hyperglycaemia.