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 hepatic ischemia/reperfusion (I/R) injury on asymmetric dimethylarginine (ADMA, a nitric oxide synthase inhibitor), protein methyltransferase (PRMT) and dimethylarginine dimethylaminohydrolase (DDAH) (involved, resp., in ADMA synthesis and degradation), and the cationic transporter (CAT). Male Wistar rats were subjected to 30 or 60 min hepatic ischemia followed by 60 min reperfusion. ADMA levels in serum and bile were determined. Tissue ADMA, DDAH activity, DDAH-1 and CAT-2 protein, DDAH-1 and PRMT-1 mRNA expression, GSH/GSSG, ROS production, and lipid peroxidation were detected. ADMA was found in bile. I/R increased serum and bile ADMA levels while an intracellular decrease was detected after 60 min ischemia. Decreased DDAH activity, mRNA, and protein expression were observed at the end of reperfusion. No significant difference was observed in GSH/GSSG, ROS, lipid peroxidation, and CAT-2; a decrease in PRMT-1 mRNA expression was found after I/R. Liver is responsible for the biliary excretion of ADMA, as documented here for the first time, and I/R injury is associated with an oxidative stress-independent alteration in DDAH activity. These data are a step forward in the understanding of the pathways that regulate serum, tissue, and biliary levels of ADMA in which DDAH enzyme plays a crucial role.BioMed Research International 01/2014; 2014:627434. · 2.71 Impact Factor
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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. · 2.71 Impact Factor
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ABSTRACT: Cirrhosis is associated with marked abnormalities in the circulatory function that involve a reduction in systemic vascular resistance. An important cause of this vasodilatation is the increased production or activity of nitric oxide (NO) in the splanchnic circulation. During portal hypertension and cirrhosis an increased endothelial NO synthase (eNOS) activity is demonstrated in splanchnic vessels. In contrast, the activity of eNOS in the cirrhotic liver is decreased, which suggests a different regulation of eNOS in the liver and in the splanchnic vessels. Asymmetric dimethylarginine (ADMA) is an endogenous NO inhibitor and higher plasma levels of ADMA are related to increased cardiovascular risk in both the general population and among patients with cirrhosis. It has been demonstrated that the liver is a key player in the metabolism of ADMA. This observation was further supported by investigations in human patients, showing a close correlation between ADMA plasma levels and the degree of hepatic dysfunction. ADMA is degraded to citrulline and dimethylamine by dimethylarginine dimethylaminohydrolases (DDAHs). DDAHs are expressed as type 1 and 2 isoforms and are widely distributed in various organs and tissues, including the liver. In this review, we discuss experimental and clinical data that document the effects of dimethylarginines on vascular function in cirrhosis. Our increasing understanding of the routes of synthesis and metabolism of methylarginines is beginning to provide insights into novel mechanisms of liver disease and allowing us to identify potential therapeutic opportunities.Cirrhosis: Causes, Treatment Options and Potential Complications., Edited by Ryan M. Blackwell, Arthur P. Tyson, 12/2013: chapter Asymmetric dimethylarginine (ADMA) and endotelial dysfunction in cirrhosis: pages 89-106; Nova Science Publishers., ISBN: 978-1-62948-418-1