Cationic amino acid transporter gene expression in cultured vascular smooth muscle cells and in rats
ABSTRACT 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.
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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.BioMed Research International 01/2014; 2014:327537. DOI:10.1155/2014/327537 · 2.71 Impact Factor
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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.British Journal Of Nutrition 06/2007; 97(5):906-11. DOI:10.1017/S0007114507669268 · 3.34 Impact Factor