Adenosine transport was characterized in human umbilical
artery smooth muscle cells isolated from non-diabetic and
diabetic pregnant subjects. Transport of adenosine was
mediated by a Na+-independent transport system inhibited
by nanomolar concentrations of nitrobenzylthioinosine
(NBMPR) in both cell types. Diabetes increased adenosine
transport, an effect that was associated with a higher maximal velocity
(Vmax) for NBMPR-sensitive (es) saturable nucleoside transport (18 ± 2 vs.
61 ± 3 pmol (µg protein)-1 min-1, P < 0.05) and the maximal number of
binding sites (Bmax) for specific [3H]NBMPR binding (74 ± 4 vs. 156 ± 10
pmol (µg protein)-1, P < 0.05), with no significant changes in the
Michaelis-Menten (Km) and dissociation (Kd) constants, respectively.
Adenosine transport was unaltered by inhibition of nitric oxide (NO)
synthase (with 100 µM NG-nitro-L-arginine methyl ester, L-NAME) or
protein synthesis (with 1 µM cycloheximide), but was increased by
inhibition of adenylyl cyclase activity (with 100 µM, SQ-22536) in
non-diabetic cells. Diabetes-induced adenosine transport was blocked by
L-NAME and associated with an increase in L-[3H]citrulline formation
from L-[3H]arginine and intracellular cGMP, but with a decrease in
intracellular cAMP compared with non-diabetic cells. Expression of
inducible NO synthase (iNOS) was unaltered by diabetes. Dibutyryl cGMP
(dbcGMP) increased, but dibutyryl cAMP (dbcAMP) decreased, adenosine
transport in non-diabetic cells. dbcGMP or the NO donor
S-nitrosoacetylpenicillamine (SNAP, 100 µM) did not alter the
diabetes-elevated adenosine transport. However, activation of adenylyl
cyclase with forskolin (1 µM), directly or after incubation of cells with
dbcAMP, inhibited adenosine transport in both cell types. Our findings
provide the first evidence that adenosine transport in human umbilical
artery smooth muscle cells is mediated by the NBMPR-sensitive transport
system es, and that its activity is upregulated by gestational diabetes.