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

摘要

class="enumerated" style="list-style-type:decimal">The effects of elevated D-glucose on adenosine transport were investigated in human cultured umbilical vein endothelial cells isolated from normal pregnancies.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 Vmax for nitrobenzylthioinosine (NBMPR)-sensitive (es) saturable nucleoside with no significant change in Km. D-Fructose (25 mM), 2-deoxy-D-glucose (25 mM) or D-mannitol (20 mM) had no effect on adenosine transport.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.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).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.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.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.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.