Elevation of intracellular
glucose within
retinal vascular cells is believed to be an important causal factor in the development of
diabetic retinopathy. The intracellular
glucose concentration is regulated by both the rate of
glucose metabolism and
glucose transport. Because
retinal hypoxia often precedes proliferative
diabetic retinopathy, we have studied the regulation of the
glucose transport system by
hypoxia in cultured bovine
retinal endothelial cells (BRECs). Because
retinal ischemia is known to increase intracellular
adenosine levels, which subsequently regulate
hypoxia-inducible genes, such as
vascular endothelial growth factor and
erythropoietin, the role of
adenosine and its receptor-mediated pathways has also been evaluated.
Hypoxia (0.5% O2, 5% CO2, and 94.5% N2) stimulated GLUT1
mRNA expression in BRECs in a time-dependent manner with an 8.9 +/- 1.5-fold (P < 0.01) increase observed after 12 h. GLUT1
mRNA expression returned to baseline (1.4 +/- 0.3-fold of control) within 12 h after reinstitution of normoxia. N6-Cyclopentyl
adenosine (
adenosine A1 receptor agonist, Kd = 1 nmol/l) did not affect GLUT1
mRNA expression at concentrations up to 1 micromol/l, while 2-p-(2-carboxyethyl)-phenethyl-amino-5'-N-ethylcarboxamidoadenosine and 5'-(N-ethylcalboxamido)-adenosine (
adenosine A2 receptor [A2R] agonists, Kd = 15 and 16 nmol/l, respectively) increased
mRNA levels at concentrations as low as 10 nmol/l. Maximal stimulation was 2.3 +/- 0.2- and 2.1 +/- 0.2-fold, respectively (P < 0.01). The
adenosine A2a receptor antagonist
8-(3-chlorostyryl)caffeine (CSC) (Kd = 100 nmol/l for A2R) inhibited
hypoxia-stimulated GLUT1
mRNA expression by 40 +/- 8% at 100 nmo/l.
Hypoxia upregulated
GLUT1 protein expression by 3.0 +/- 0.3-fold after 12 h (P < 0.01), but this response was attenuated by CSC (P < 0.05).
Hypoxia increased
glucose transport activity by 2.1 +/- 0.3-fold (P < 0.001) after 12 h, a response inhibited 65% by CSC (P < 0.01). A
protein kinase A (
PKA) inhibitor (
H89, 20 micromol/l) suppressed
hypoxia-induced GLUT1
mRNA expression by 42 +/- 9% (P < 0.01). These data suggest that
hypoxia in BRECs upregulates
glucose transport activity through an increase of GLUT1 expression that is partially mediated by
adenosine, A2R, and the cAMP-PKA pathway.