Casanello & sobrevia

Intrauterine Growth Retardation Is Associated With Reduced Activity and Expression of the Cationic Amino Acid Transport Systems y /hCAT-1 and y /hCAT-2B and Lower Activity of Nitric Oxide Synthase in Human Umbilical Vein Endothelial Cells
Paola Casanello, Luis Sobrevia
Abstract—Intrauterine growth retardation (IUGR) is associated with vascular complications leading to hypoxia and abnormal fetaldevelopment. The effect of IUGR on L-arginine transport and nitric oxide (NO) synthesis was investigated in cultures of human umbilical vein endothelial cells (HUVECs). IUGR was associated with membrane depolarization and reduced L-arginine transport (Vmax 5.8 0.2 versus 3.3 0.1 pmol/ g protein per minute), with no significant changes in transport affinity (Km 159 15 versus 137 14 mol/L).L-Arginine transport was transstimulated (8- to 9-fold) in cells from normal and IUGR pregnancies. IUGR was associated with reduced production of 3 3 L-[ H]citrulline from L-[ H]arginine, lower nitrite and intracellular L-arginine, L-citrulline, and cGMP. IUGR decreased hCAT-1 and hCAT-2B mRNA, and increased eNOS mRNA and protein levels. IUGR-associated inhibition of L-arginine transport and NOsynthesis, and membrane depolarization were reversed by the NO donor S-nitroso-Nacetyl-L,D-penicillamine. In summary, endothelium from fetuses with IUGR exhibit altered L-arginine transport and NO synthesis (L-arginine/NO pathway), reduced expression and activity of hCAT-1 and hCAT-2B and reduced eNOS activity. Alterations in L-arginine/NO pathway could be critical for the physiological processes involvedin the etiology of IUGR in human pregnancies. (Circ Res. 2002;91:127-134.) Key Words: L-arginine intrauterine growth retardation nitric oxide human endothelium

ntrauterine growth retardation (IUGR) is associated with prenatal disturbances, including prematurity and fetal asphyxia.1 Fetal growth and development depend on fetal tissue oxygenation and substrate delivery.2 The human fetoplacentalcirculation exhibits a low vascular resistance and lacks autonomic innervation.3 Thus, circulating and locally released vasoactive molecules are therefore likely to be involved in the control of fetoplacental hemodynamics. The release of local vasoactive molecules, such as nitric oxide (NO), from endothelium maintains appropriate placental blood flow, fetal nutrition, and oxygenation leading tonormal fetal development and growth.4 Endothelial NO synthesis results from conversion of 2 L-arginine into L-citrulline by the Ca /calmodulin-dependent NO synthase (eNOS), a process associated with L-arginine transport via system y /CATs (Cationic Amino acid Transporters) in human umbilical vein endothelial cells (HUVECs).5–8 L-Arginine transport is preferentially mediated by system y /CAT-1, a Na-independent, high-affinity transport system (Km 100 mol/L), sensitive to changes in membrane potential in HUVECs.5,6 Long-term inhibition of NO synthase mimics IUGR in gravid rats,9 and eNOS-targeted mutagenesis is associated

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with fetal growth retardation in mouse.10 Other studies show that eNOS protein levels are reduced in rat placental microvasculature of IUGR pregnancies.11Furthermore, L-arginine supplementation prevents fetal growth retardation in animal models of IUGR,12,13 and L-arginine infusion improved vasorelaxation in IUGR pregnant women with increased uterine resistance.14 Therefore, L-arginine/NO signaling pathway could play a crucial role in IUGR pregnancies. We characterized L-arginine transport and NO synthesis in HUVECs from normal pregnancies or pregnanciescomplicated with IUGR. IUGR is associated with reduced activity of L-arginine/NO pathway, due to reduced expression of hCAT-1 and hCAT-2B transporters, and membrane depolarization. Inhibition of NO synthesis in cells from IUGR pregnancies was associated with elevated eNOS protein and mRNA levels.

Materials and Methods
IUGR Samples
Umbilical cords were collected after delivery from full-term...