Fetal hypoxia has been associated with intrauterine growth restriction, a disease that reduces mRNA levels for human cationic amino acid transporters 1 (hCAT1) and 2B (hCAT2B), but increases eNOS mRNA level in human umbilical vein endothelial cells (HUVECs) (Casanello & Sobrevia, 2002), and activates protein kinase C (PKC, Langdown et al. 2001). We studied the involvement of PKC and nitric oxide (NO) in the modulation of L-arginine transport by hypoxia in HUVECs.

Cells isolated from normal 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 exposed (24 h) to normoxia (21 % O₂, 5 % CO₂) or hypoxia (2 % O₂, 5 % CO₂, 93 % N₂). L-Arginine transport (L-[2,3-³H]arginine, 36.1 Ci mmol^-1, 7.5-1000 µM, 2 µCi ml^-1, 37°C, 1 min) was determined in the absence or presence (30 min to 24 h) of S-nitroso-N-acetyl-L,D-penicillamine (SNAP, 100 µM, 30 min, NO donor), calphostin C (100 nM, PKC inhibitor), 13-acetate, 12-myristate phorbol ester (PMA, 100 nM, PKC activator) or N^G-nitro-L-arginine methyl ester (L-NAME, 100 µM, eNOS inhibitor). hCAT1, hCAT2B and eNOS mRNAs were amplified by reverse transcriptase-polymerase chain reactions. Gene expression was also assessed on cDNA microarrays (Clontech). eNOS activity was determined by L-[³H]citrulline formation from L-[³H]arginine (4 µCi ml^-1, 30 min), and eNOS protein was detected by Western blot.

Hypoxia reduced the V_max for L-arginine transport (2.8 ± 0.2 vs. 5.1 ± 0.3 pmol (µg protein)^-1 min^-1, n = 8, P < 0.05, means ± S.E.M., Student’s unpaired t test), with no significant changes in the apparent K_m (normoxia = 149 ± 15 µM, hypoxia = 125 ± 12 µM). Hypoxia reduced the hCAT-1 (50 ± 0.2 %) and hCAT-2B (52 ± 0.2 %) mRNA levels, but increased eNOS mRNA (46 ± 2 %) and protein (1.4-fold) levels. L-Citrulline synthesis was reduced (55 ± 4 %) in hypoxia. Hypoxia-induced inhibition of L-arginine transport was reversed by SNAP or calphostin C. Hypoxia increased PKCα/βII activity (2.7-fold) and PKCα gene expression (12-fold).

In summary, hypoxia-induced inhibition of the L-arginine/NO pathway could be due to reduced hCAT-1 and hCAT-2B transporter expression, an effect that may involve PKC and NO.

This work was supported by FONDECYT (1030607, 1030781, 1000354 & 7000354), DIUC-University of Concepción (201.084.003-1.0) (Chile) and The Wellcome Trust (UK).