Abstract

Adenosine transport was characterized in human umbilical artery smooth muscle cells isolated from nondiabetic 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 (V(max)) for NBMPR-sensitive (es) saturable nucleoside transport (18 +/- 2 vs. 61 +/- 3 pmol (mu g protein)(-1) min(-1), P < 0.05) and the maximal number of binding sites (B(max)) for specific [(3)H]NBMPR binding (74 +/- 4 vs. 156 +/- 10 pmol (mu g protein)(-1), P < 0.05), with no significant changes in the Michaelis-Menten (K(m)) and dissociation (K(d)) constants, respectively. Adenosine transport was unaltered by inhibition of nitric oxide (NO) synthase (with 100 mu M N(G)-nitro-L-arginine methyl ester, L-NAME) or protein synthesis (with 1 mu M cycloheximide), but was increased by inhibition of adenylyl cyclase activity (with 100 mu M, SQ-22536) in nondiabetic cells. Diabetes-induced adenosine transport was blocked by L-NAME and associated with an increase in L-[(3)H]citrulline formation from L-[(3)H]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 mu M) did not alter the diabetes-elevated adenosine transport. However, activation of adenylyl cyclase with forskolin (1 mu M), 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.