Gestational diabetes activates the L-arginine-nitric oxide (NO) signalling pathway in human umbilical vein endothelial cells (HUVECs, Sobrevia & Mann, 1997). We have found that HUVECs isolated from gestational diabetics have higher activity of mitogen-activated protein (MAP) kinases p42 (p42^mapk) and p44 (p44^mapk) (Vásquez et al. 2000). HUVECs isolated from non-diabetic pregnancies and exposed to elevated D-glucose exhibit higher NO synthesis and L-arginine transport paralleled by increased endothelial NO synthase (eNOS) protein, and eNOS and human cationic amino acid transporters 2B (hCAT-2B) mRNA (Flores et al. 2001). Protein kinases A (PKA), G (PKG) and C (PKC) have been shown to be involved in the modulation of eNOS activity in vitro (Butt et al. 2000) and in adenosine transport in HUVECs exposed to high D-glucose (Montecinos et al. 2000; Guijarro et al. 2001). We have investigated whether gestational diabetes-induced increase of endothelial L-arginine-NO pathway involves changes of eNOS and hCAT expression and protein kinase activity in HUVECs.

HUVECs were isolated (0.2 µg ml^-1 collagenase) from umbilical veins of normal and gestational diabetic pregnancies (ethics committee approval was obtained) and cultured in medium 199 (M199), supplemented with 10 % fetal and 10 % newborn calf serum, 3.2 mM L-glutamine and 5 mM D-glucose. Transport of L-[³H]arginine (100 µM, 2 µCi ml^-1, 37 °C, 1 min) was measured in the presence or absence of N ^G-nitro-L-arginine methylester (L-NAME, 100 µM, 30 min, eNOS inhibitor), PD-98059 (10 µM, 30 min, MAP kinase inhibitor), calphostin C (100 nM, 30 min, PKC inhibitor), KT-5720 (100 nM, 15 min, PKA inhibitor) and KT-5823 (1 µM, 15 min, PKG inhibitor). Total protein levels for eNOS, serine¹¹⁷⁷-phosphorylated eNOS and phosphorylated p42/44^mapk proteins were determined by Western blot. Activity of eNOS was estimated by conversion of L-[³H]arginine into L-[³H]citrulline (L-[³H]arginine, 100 µM, 4 µCi ml^-1, 37 °C, 30 min). Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to determine mRNA levels for hCAT-1, hCAT-2B and eNOS.

L-Arginine transport was increased (P < 0.05, Student’s unpaired t test) in cells from gestational diabetics (2.1 ± 0.2 vs. 5.6 ± 0.1 pmol (µg protein)^-1 min^-1, means ± S.E.M., n = 3-5), an effect blocked (P < 0.05) by L-NAME (2.1 ± 0.6 pmol (µg protein)^-1 min^-1), PD-98059 (1.9 ± 0.2 pmol (µg protein)^-1 min^-1), calphostin C (1.8 ± 0.4 pmol (µg protein)^-1 min^-1), KT-5720 (1.8 ± 0.4 pmol (µg protein)^-1 min^-1) and KT-5823 (2.2 ± 0.3 pmol (µg protein)^-1 min^-1). Diabetes also induced phosphorylation of p42/44^mapk, which was significantly (P < 0.05) inhibited by L-NAME (70 ± 12 %) and PD-98059 (55 ± 6 %) and calphostin C (50 ± 5 %). eNOS and hCAT-1, but not hCAT-2B mRNA levels, were increased (P < 0.05) 3.5- and 3.1-fold, respectively, in diabetic compared with non-diabetic cells. Diabetes-induced increase in eNOS mRNA was paralleled by a 3-fold increase in total eNOS protein level, with no significant changes (P > 0.05) in the serine¹¹⁷⁷-phosphorylated eNOS/total eNOS ratio (0.5 ± 0.1 vs. 0.41 ± 0.1 arbitrary units).

These results suggest that gestational diabetes-induced increase in L-arginine transport and NO synthesis could result from higher hCAT-1 and eNOS mRNA levels, respectively. The increased NO synthesis exhibited by diabetic cells is associated with higher eNOS protein levels rather than increased phosphorylation at serine¹¹⁷⁷ in eNOS, and increased L-arginine transport involves activation of p42/44^mapk, PKG, PKA and PKC.

This work was supported by FONDECYT (1000354 and 7000354) and DIUC-Iniciativa de Grupos de Investigación de Avanzada (University of Concepción)-Chile, and The Wellcome Trust (UK). C.A.F. and J.P. hold University of Concepción-MSc Fellowships, and C.A. holds a CONICYT (Chile)-PhD Fellowship.