A profound decrease in maternal arginine uptake provokes endothelial nitration in the pregnant rat

Abstract

While a specific role for nitric oxide (NO) in inducing the hemodynamic alterations of pregnancy is somewhat controversial, it is widely accepted that excess NO is generated during pregnancy. L-Arginine is the sole precursor for NO biosynthesis. Among several transporters that mediate L-arginine uptake, cationic amino acid transporter-1 (CAT-1) acts as the specific arginine transporter for endothelial NO synthase. The present study was designed to test the hypothesis that, during pregnancy, when arginine consumption by the fetus is significantly increased, compensatory changes in maternal arginine uptake affect the endothelium. Uptake of radiolabeled arginine (L-[3H]arginine) by freshly harvested maternal aortic rings from pregnant rats decreased by 65 and 30% in mid- and late pregnancy, respectively, compared with those obtained from virgin animals. This decrease was associated with a significant increase in endothelial protein nitration (the footprint of peroxynitrite generation), as shown by both Western blotting and immunohistochemistry utilizing anti-nitrotyrosine antibodies, reflecting endothelial damage. Northern blot analysis revealed that steady-state aortic CAT-1 mRNA levels did not change throughout pregnancy, whereas CAT-1 protein abundance was significantly increased, peaking at mid-pregnancy. Protein content of protein kinase C (PKC)-alpha, which was previously shown to decrease CAT-1 activity, increased significantly in the pregnant animals and was associated with a significant increase in CAT-1 phosphorylation. Intraperitoneal injection of alpha-tocopherol, a PKC-alpha inhibitor, prevented the decrease in arginine transport and attenuated protein nitration. In conclusion, aortic arginine uptake is reduced during pregnancy, through posttranslational modulation of CAT-1 protein, presumably via upregulation of PKC-alpha. The aforementioned findings are associated with an increase in protein nitration and, therefore, in selected individuals, may lead to the development of certain forms of endothelial dysfunction, like preeclampsia.

Protein Kinase C-dependent Ubiquitination and Clathrin-mediated Endocytosis of the Cationic Amino Acid Transporter CAT-1

“…Several studies demonstrated down-regulation of CAT-1 activity by PKC (5,6,44,45). These studies suggested that CAT-1 phosphorylation affects its functional properties.…”

Hyperuricemia Attenuates Aortic Nitric Oxide Generation, through Inhibition of Arginine Transport, in Rats

“…A possible involvement of PKCα in the regulation of L -arginine transport in different cell types has been discussed for the last several years [23,24]. We have recently reported in 4 different experimental models characterized by diminished arginine transport, namely, hypercholesterolemia, chronic renal failure, pregnancy, and aging in the male rat, a post-translational regulation of CAT-1 which was associated with upregulation of PKCα [15,16,18,25]. We have decided to focus exclusively on the membrane fraction of PKCα since this is where the interaction between PKCα and CAT-1 occurs.…”

“…Uptake of radiolabeled L -arginine in the rat aorta was measured according to previously described methods [16]. Immediately after sacrifice, the aorta was carefully excised from the left renal artery to the aortic valve ring and placed in ice-cold HEPES.…”

“…We hypothesize that delivery of transported arginine to membrane-bound eNOS, selectively by the cationic amino acid transporter-1 (CAT-1) rather than intra- or extra-cellular arginine concentration, is the predominant factor governing eNOS activity. Indeed, we have previously shown in several different animal models characterized by endothelial dysfunction and decreased eNOS activity that CAT-1 activity is attenuated [15,16,17,18]. The predominant mechanism resulting in diminished CAT-1 activity in all these experiments was post-translational modulation of CAT-1 by protein kinase C α (PKCα) [15,17,18].…”

Characterization of the L-arginine–NO–cGMP pathway in spontaneously hypertensive rat platelets: the effects of pregnancy

“…Recent evidence has come to light that maternal aortic L-arginine uptake is profoundly attenuated in pregnancy, and the researchers of that study assume that rather than supplying its own endothelium, maternal L-arginine stores are depleted due to a preferential shift to the fetus. 39 These data could explain the reduction in L-arginine transport in platelets from pregnant hypertensive rats. Both influx and availability of intracellular L-arginine are limiting factors in NO production.…”