Modulation of adenosine transport by insulin in human umbilical artery smooth muscle cells from normal or gestational diabetic pregnancies

1. Adenosine transport was measured in human cultured umbilical artery smooth muscle cells, isolated from non-diabetic or gestational diabetic pregnancies, under basal conditions and after pretreatment in vitro with insulin. 2. Adenosine transport in non-diabetic smooth muscle cells was significantly increased by insulin (half-maximal stimulation at 0.33 +/- 0.02 nM, 8 h) and characterized by a higher maximal rate (V-max) for nitrobenzylthioinosine (NBMPR)-sensitive (es) saturable nucleoside transport (17 +/- 5 vs. 52 +/- 12 pmol (mug protein)(-1) min(-1), control vs. insulin, respectively) and maximal binding sites (B-max) for [H-3]NBMPR (0.66 +/- 0.07 vs. 1.1 +/- 0.1 fmol (mug protein)(-1), control vs. insulin, respectively), with no significant changes in Michaelis-Menten (K-m) and dissociation (K-d) constants. 3. In contrast, in smooth muscle cells from diabetic pregnancies, where the values of V-max for adenosine transport (59 +/- 4 pmol mug protein)(-1) min(-1)) and B-max for [H-3]NBMPR binding (1.62 +/- 0.16 fmol (mug protein)(-1)) were significantly elevated by comparison with non-diabetic cells, insulin treatment (1 nM, 8 h) reduced the V-max for adenosine transport and B-max for [H-3]NBMPR binding to levels detected in non-diabetic cells. 4. In non-diabetic cells, the stimulatory effect of insulin on adenosine transport was mimicked by dibutyryl cGMP (100 nM) and reduced by inhibitors of phosphatidylinositol 3-kinase (10 nar wortmannin), nitric oxide synthase (100 muM N-G-nitro-L-arginine methyl ester, L-NAME) or protein synthesis(1 muM cycloheximide), whereas inhibition of adenylyl cyclase (100 muM SQ-22536) had no effect. 5. Wortmannin or SQ-22536, but not L-NAME or cycloheximide, attenuated the inhibitory action of insulin on the diabetes-induced stimulation of adenosine transport. 6. Protein levels of inducible NO synthase (iNOS) were similar in non-diabetic and diabetic cells, but were increased by insulin (1 nM, 8 h) only in non-diabetic smooth muscle cells. 7. Our results suggest that adenosine transport via the es nucleoside transporter is modulated differentially by insulin in either cell type. Insulin increased adenosine transport in non-diabetic cells via NO and cGMP, but inhibited the diabetes-elevated adenosine transport via activation of adenylyl cyclase, suggesting that the biological actions of adenosine may be altered under conditions of sustained hyperglycaemia in uncontrolled diabetes.