Activity of the human equilibrative nucleoside transporter 1 (hENT1) (sensitive to inhibition by nanomolar nitrobenzylthioinosine, NBMPR) is down-regulated in human umbilical vein endothelial cells (HUVECs) isolated from pregnancies with gestational diabetes (Sobrevia et al. 1994). hENT1 expression and activity is also down-regulated in HUVECs from normal pregnancies exposed to 25 mM D-glucose, an effect associated with increased activity of protein kinase C (PKC), endothelial nitric oxide synthase (eNOS) and mitogen-activated protein kinases (MAPK) p44 and p42 (p42/44^mapk) (Montecinos et al. 2000; Parodi et al. 2002). We now report the involvement of PKC, eNOS and p42/44^mapk on expression of human ENT1 in HUVECs from gestational diabetes.

Cells isolated (0.2 µg ml^-1 collagenase) from normal or gestational diabetic pregnancies (ethics committee approval and informed patient consent were obtained) were cultured in medium 199, containing 20 % bovine sera, 3.2 mM L-glutamine, and 5 mM D-glucose. hENT1 mRNA was amplified by reverse transcriptase-polymerase chain reactions on total RNA extracted (Trizol), and quantified by real time PCR. Cells were also exposed (30 min) to PD-98059 (10 µM, MAPK kinase inhibitor), N^G-nitro-L-arginine methyl ester (L-NAME, 100 µM, eNOS inhibitor), calphostin C (100 nM, PKC inhibitor) or RO-320432 (50 or 100 nM, PKC inhibitor), and phorbol 12-myristate, 13-acetate (PMA, 100 nM, PKC activator). hENT1, eNOS and p42/44^mapk protein levels were determined by Western blots. eNOS activity was monitored by measuring L-[³H]citrulline formation from L-[³H]arginine (4 µCi ml^-1, 100 µM L-arginine, 30 min, 37 °C).

hENT1 mRNA level was significantly lower (70 ± 12 %, P < 0.05, Student’s unpaired t test, means ± S.E.M., n = 8-17) in diabetic compared with normal pregnancies. In addition, gestational diabetes reduced (47 ± 5 %) hENT1 protein level. Maximal NBMPR-sensitive adenosine transport velocity (V_max) in diabetic cells was lower (211 ± 45 pmol (10⁶ cells)^-1 s^-1) compared with normal cells (712 ± 83 pmol (10⁶ cells)^-1 s^-1), with no significant (P > 0.05) changes in the apparent K_m (85 ± 12 and 101 ± 23 µM, for normal and diabetic, respectively). Parallel experiments show that maximal binding (B_max) was also reduced (P < 0.05) in diabetic (0.7 ± 0.1 pmol (10⁶ cells)^-1) compared with normal pregnancies (2.1 ± 0.2 pmol (10⁶ cells)^-1), with no significant changes in apparent K_d (0.21 ± 0.01 and 0.19 ± 0.02 nM, for normal and diabetic, respectively). Gestational diabetes is also associated with increased PKC activity (4.5-fold), eNOS activity (2.7-fold) and protein (3.1-fold) and mRNA (2.4-fold) levels, and p42/44^mapk phosphorylation. The effect of gestational diabetes on hENT1 mRNA, NBMPR binding, and adenosine transport was blocked by calphostin C, PD-98059, L-NAME and RO-320432. Calphostin C, L-NAME and RO-320432, but not PD-98059, blocked the effect of gestational diabetes on eNOS.

These results suggest that reduced adenosine transport induced by gestational diabetes could be due to reduced hENT1 expression, via activation of signalling pathways involving PKC, p42/44^mapk and eNOS in HUVECs.

This work was supported by FONDECYT (1030781, 1030607, 7030004, 7030109) and DIUC, University of Concepción (201.084.003-1.0), Chile, and The Welcome Trust (UK).