Cationic amino acid transporter gene expression in cultured vascular smooth muscle cells and in rats
Immunostimulants trigger vascular smooth muscle cells (VSMC) to express the inducible isoform of NO synthase (iNOS) and increased arginine transport activity. Although arginine transport in VSMC is considered to be mediated via the y+ system, we show here that rat VSMC in culture express the cat-1 gene transcript as well as an alternatively spliced transcript of the cat-2 gene. An RT-PCR cloning sequence strategy was used to identify a 141-base nucleotide sequence encoding the low-affinity domain of alternatively spliced CAT-2A and a 138-base nucleotide sequence encoding the high-affinity domain of CAT-2B in VSMC activated with lipopolysaccharide (LPS) in combination with interferon-gamma (IFN). With this sequence as a probe, Northern analyses showed that CAT-1 mRNA and CAT-2B mRNA are constitutively present in VSMC, and the expression of both mRNAs was rapidly stimulated by treatment with LPS-IFN, peaked within 4 h, and decayed to basal levels within 6 h after LPS-IFN. CAT-2A mRNA was not detectable in unstimulated or stimulated VSMC. Arginine transporter activity significantly increased 4-10 h after LPS-IFN. iNOS activity was reduced to almost zero in the absence of extracellular arginine uptake via system y+. Induction of arginine transport seems to be a prerequisite to the enhanced synthesis of NO in VSMC. Moreover, this work demonstrates tissue expression of CAT mRNAs with use of a model of LPS injection in rats. RT-PCR shows that the expression of CAT-1 and CAT-2B mRNA in the lung, heart, and kidney is increased by LPS administration to rats, whereas CAT-2A mRNA is abundantly expressed in the liver independent of LPS treatment. These findings suggest that together CAT-1 and CAT-2B play an important role in providing substrate for high-output NO synthesis in vitro as well as in vivo and implicate a coordinated regulation of intracellular iNOS enzyme activity with membrane arginine transport.
Available from: Plinio Richelmi
- "Nijveldt et al. provide a detailed insight into the liver's handling of dimethylarginine, showing how it plays a crucial role in the metabolism of ADMA, with DDAH : studies of gene silencing or deletion in rodents have led to the conclusion that plasma levels of ADMA are regulated by DDAH-1 isoform, whereas the predominant importance of DDAH-2 lies in preserving the endothelial function . Membrane ADMA transport occurs by cationic amino-acid transporters (CATs): liver abundantly expresses CATs, especially CAT-2A and CAT-2B, suggesting a higher uptake of ADMA in this organ as compared with heart, lung, and kidney . The aim of the present study was therefore to investigate whether obstructive cholestasis does separately affect the function of left, median, and right liver lobes via modulation of ADMA levels, DDAH activity, mRNA expression of PMRT and DDAH, and protein CAT levels. "
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ABSTRACT: We investigated the effects of obstructive cholestasis in different hepatic lobes by evaluating asymmetric dimethylarginine (ADMA) (a nitric oxide synthase inhibitor), protein methyltransferase (PRMT) and dimethylarginine dimethylaminohydrolase (DDAH) (enzymes involved, resp., in its synthesis and degradation), the cationic transporter (CAT), and metalloproteinase (MMP) activity. Sixteen male Wistar rats underwent a 3-day cholestasis by common bile duct ligation (BDL) or sham operation. Blood samples and hepatic biopsies from left lobe (LL), median lobe (ML), and right lobe (RL) were collected. Serum hepatic enzymes, tissue ADMA, DDAH activity, CAT-2 protein, mRNA expression of DDAH and PRMT, and MMP-2 and MMP-9 activity were monitored. Cholestasis was confirmed by altered serum hepatic enzymes. Higher levels of tissue ADMA were detected in RL and ML as compared with LL. PRMT mRNA expression and DDAH activity did not differ among the lobes after BDL. CAT-2 levels are higher in the RL and ML in the sham-operated group. Higher activity in MMP-2 and MMP-9 was found in RL. In conclusion, after cholestasis an increase in hepatic ADMA in RL and ML was detected as well as tissue MMP-2 and MMP-9 activation in RL, supporting the evidence of functional heterogeneity among the liver lobes also occurring in an obstructive cholestasis model.
Available from: Antonio Casella-Filho
- "Subsequent research has also suggested the involvement of the liver in the metabolism of dimethylarginines. Researchers have demonstrated that hepatocytes abundantly express y+ channels in their membranes and contain high concentrations of DDAH.34,35 A more recent study carried out in patients who underwent hepatic surgery confirmed the entrance of ADMA in the liver.36 "
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ABSTRACT: Atherosclerotic coronary heart disease is the leading cause of morbidity and mortality in industrialized countries, and endothelial dysfunction is considered a precursor phenomenon. The nitric oxide produced by the endothelium under the action of endothelial nitric oxide synthase has important antiatherogenic functions. Its reduced bioavailabilty is the beginning of the atherosclerotic process. The addition of two methyl radicals to arginine, through the action of methyltransferase nuclear proteins, produces asymmetric dimethylarginine, which competes with L-arginine and promotes a reduction in nitric oxide formation in the vascular wall. The asymmetric dimethylarginine, which is itself considered a mediator of the vascular effects of the several risk factors for atherosclerosis, can be eliminated by renal excretion or by the enzymatic action of the dimethylarginine dimethylaminohydrolases. Several basic science and clinical research studies suggest that the increase in asymmetric dimethylarginine occurs in the context of chronic renal insufficiency, dyslipidemia, high blood pressure, diabetes mellitus, and hyperhomocysteinemy, as well as with other conditions. Therapeutic measures to combat atherosclerosis may reverse these asymmetric dimethylarginine effects or at least reduce the concentration of this chemical in the blood. Such an effect can be achieved with competitor molecules or by increasing the expression or activity of its degradation enzyme. Studies are in development to establish the true role of asymmetric dimethylarginine as a marker and mediator of atherosclerosis, with possible therapeutic applications. The main aspects of the formation and degradation of asymmetric dimethylarginine and its implication in the atherogenic process will be addressed in this article.
Available from: Ruurd Van Elburg
- "Since arginine and other cationic amino acids such as ornithine and lysine and also ADMA and SDMA are transported into endothelial cells by CAT, changes in the expression of CAT mRNA by endotoxemia may influence ADMA transport (Closs et al. 1997). Hattori et al. (1999), showed that expression of CAT 1 and 2 mRNA in the lung, heart, and kidney was increased by injection of lipopolysaccharide–interferon g in rats whereas CAT 2A mRNA was abundantly expressed in the liver independent of lipopolysaccharide –interferon g treatment. The abundant expression of CAT 2 mRNA in the liver indicates a potentially high uptake of dimethylarginines in this organ. "
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ABSTRACT: Several studies have described reduced plasma concentrations of arginine, the substrate for nitric oxide synthase (NOS) in infants with necrotizing enterocolitis (NEC). No information on the plasma concentrations of the endogenous NOS inhibitor asymmetric dimethylarginine (ADMA) in patients with NEC is currently available. We investigated whether plasma concentrations of arginine, ADMA, and their ratio differ between premature infants with and without NEC, and between survivors and non-survivors within the NEC group. In a prospective case-control study, arginine and ADMA concentrations were measured in ten premature infants with NEC (median gestational age 193 d, birth weight 968 g), and ten matched control infants (median gestational age 201 d, birth weight 1102 g), who were admitted to the Neonatal Intensive Care Unit. In the premature infants with NEC, median arginine and ADMA concentrations (micromol/l), and the arginine:ADMA ratio were lower compared to the infants without NEC: 21.4 v. 55.9, P= 0.001; 0.59 v. 0.85, P=0.009 and 36.6 v. 72.3, P=0.023 respectively. In the NEC group, median arginine (micromol/l) and the arginine:ADMA ratio were lower in non-surviving infants than in surviving infants: 14.7 v. 33.8, P=0.01 and 32.0 v. 47.5, P=0.038 respectively. In premature infants with NEC not only the NOS substrate arginine, but also the endogenous NOS inhibitor ADMA and the arginine:ADMA ratio were lower than in infants without NEC. In addition, low arginine and arginine:ADMA were associated with mortality in infants with NEC. Overall, these data suggest that a diminished nitric oxide production may be involved in the pathophysiology of NEC, but this needs further investigation.
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