Regulation of adenosine transport by D-glucose in human fetal endothelial cells: involvement of nitric oxide, protein kinase C and mitogen-activated protein kinase.
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 NG-nitro-L-arginine methyl ester (L-NAME; 100 microM, an inhibitor of nitric oxide synthase (NOS)) or PD-98059 (10 microM, 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 microM, 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 p44mapk (ERK1) and p42mapk (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 microM, 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-p44mapk phosphorylation. Thus, the biological actions of adenosine appear to be altered under conditions of sustained hyperglycaemia.
Montecinos
V P
VP
Cellular and Molecular Physiology Laboratory (CMPL), Department of Physiology, Faculty of Biological Sciences, University of Concepcion, PO Box 160-C, Concepcion, Chile.
Aguayo
C
C
Flores
C
C
Wyatt
A W
AW
Pearson
J D
JD
Mann
G E
GE
Sobrevia
L
L
D000241:Adenosine / Q000037:antagonists & inhibitors / Q000378:metabolism*; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000196:embryology* / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005333:Fetus / Q000502:physiology; D005419:Flavonoids / Q000494:pharmacology; D005947:Glucose / Q000494:pharmacology*; D006801:Humans; D020928:Mitogen-Activated Protein Kinases / Q000502:physiology; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009281:Naphthalenes / Q000494:pharmacology; D009569:Nitric Oxide / Q000502:physiology; D019001:Nitric Oxide Synthase / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III; D010396:Penicillamine / Q000031:analogs & derivatives / Q000494:pharmacology; D011493:Protein Kinase C / Q000502:physiology; D026423:S-Nitroso-N-Acetylpenicillamine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013868:Thioinosine / Q000031:analogs & derivatives* / Q000378:metabolism
D004791:Enzyme Inhibitors; D005419:Flavonoids; D009281:Naphthalenes; D009569:Nitric Oxide; D013868:Thioinosine; D026423:S-Nitroso-N-Acetylpenicillamine; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D011493:Protein Kinase C; D020928:Mitogen-Activated Protein Kinases; D010396:Penicillamine; C001789:4-nitrobenzylthioinosine; C058819:calphostin C; D005947:Glucose; D000241:Adenosine; D013755:Tetradecanoylphorbol Acetate; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; D019331:NG-Nitroarginine Methyl Ester