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 mm) 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 N^G-nitro-l-arginine methyl ester (l-NAME; 100 μm, an inhibitor of nitric oxide synthase (NOS)) or PD-98059 (10 μm, an inhibitor of mitogen-activated protein kinase kinase 1). 5. d-Glucose and PMA increased endothelial NOS (eNOS) activity, which was prevented by calphostin C or omission of extracellular Ca²⁺ and unaffected by PD-98059. 6. Adenosine transport was inhibited by S-nitroso-N-acetyl-l,d-penicillamine (SNAP; 100 μm, 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 μm, 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.