Insulin requires A1 adenosine receptors expression to reverse gestational diabetes-increased L-arginine transport in human umbilical vein endothelium.

Gestational diabetes mellitus (GDM) associates with increased L-arginine transport and extracellular concentration of adenosine in human umbilical vein endothelial cells (HUVECs). In this study we aim to determine whether insulin reverses GDM-increased L-arginine transport requiring adenosine receptors expression in HUVECs. Primary cultured HUVECs from full-term normal (n = 38) and diet-treated GDM (n = 38) pregnancies were used. Insulin effect was assayed on human cationic amino acid transporter 1 (hCAT1) expression (protein, mRNA, SLC7A1 promoter activity) and activity (initial rates of L-arginine transport) in the absence or presence of adenosine receptors agonists or antagonists. A1 adenosine receptors (A1AR) and A2AAR expression (Western blot, quantitative PCR) was determined. Experiments were done in cells expressing or siRNA-suppressed expression of A1AR or A2AAR. HUVECs from GDM exhibit higher maximal transport capacity (maximal velocity (V max)/apparent Michaelis Menten constant (K m), V max/K m), which is blocked by insulin by reducing the V max to values in cells from normal pregnancies. Insulin also reversed the GDM-associated increase in hCAT-1 protein abundance and mRNA expression, and SLC7A1 promoter activity for the fragment -606 bp from the transcription start point. Insulin effects required A1AR, but not A2AAR expression and activity in this cell type. In the absence of insulin, GDM-increased hCAT-1 expression and activity required A2AAR expression and activity. HUVECs from GDM pregnancies exhibit a differential requirement of A1AR or A2AAR depending on the level of insulin, a phenomenon that represent a condition where adenosine or analogues of this nucleoside could be acting as helpers of insulin biological effects in GDM.

CAT-1 as a novel CAM stabilizes endothelial integrity and mediates the protective actions of L-Arg via a NO-independent mechanism.

Interendothelial junctions play an important role in the maintenance of endothelial integrity and the regulation of vascular functions. We report here that cationic amino acid transporter-1 (CAT-1) is a novel interendothelial cell adhesion molecule (CAM). We identified that CAT-1 protein localized at cell-cell adhesive junctions, similar to the classic CAM of VE-cadherin, and knockdown of CAT-1 with siRNA led to an increase in endothelial permeability. In addition, CAT-1 formed a cis-homo-dimer and showed Ca(2+)-dependent trans-homo-interaction to cause homophilic cell-cell adhesion. Co-immunoprecipitation assays showed that CAT-1 can associate with ß-catenin. Furthermore, we found that the sub-cellular localization and function of CAT-1 are associated with cell confluency, in sub-confluent ECs CAT-1 proteins distribute on the entire surface and function as L-Arg transporters, but most of the CAT-1 in the confluent ECs are localized at interendothelial junctions and serve as CAMs. Further functional characterization has disclosed that extracellular L-Arg exposure stabilizes endothelial integrity via abating the cell junction disassembly of CAT-1 and blocking the cellular membrane CAT-1 internalization, which provides the new mechanisms for L-Arg paradox and trans-stimulation of cationic amino acid transport system (CAAT). These results suggest that CAT-1 is a novel CAM that directly regulates endothelial integrity and mediates the protective actions of L-Arg to endothelium via a NO-independent mechanism.

Nitric oxide signalling pathway in Duchenne muscular dystrophy mice: up-regulation of L-arginine transporters.

DMD (Duchenne muscular dystrophy) is an incurable rapidly worsening neuromuscular degenerative disease caused by the absence of dystrophin. In skeletal muscle a lack of dystrophin disrupts the recruitment of neuronal NOS (nitric oxide synthase) to the sarcolemma thus affecting NO (nitric oxide) production. Utrophin is a dystrophin homologue, the expression of which is greatly up-regulated in the sarcolemma of dystrophin-negative fibres from mdx mice, a mouse model of DMD. Although cardiomyopathy is an important cause of death, little is known about the NO signalling pathway in the cardiac muscle of DMD patients. Thus we used cardiomyocytes and hearts from two month-old mdx and mdx:utrophin-/- (double knockout) mice (mdx:utr) to study key steps in NO signalling: L-arginine transporters, NOS and sGC (soluble guanylyl cyclase). nNOS did not co-localize with dystrophin or utrophin to the cardiomyocyte membrane. Despite this nNOS activity was markedly decreased in both mdx and mdx:utr mice, whereas nNOS expression was only decreased in mdx:utr mouse hearts, suggesting that utrophin up-regulation in cardiomyocytes maintains nNOS levels, but not function. sGC protein levels and activity remained at control levels. Unexpectedly, L-arginine transporter expression and function were significantly increased, suggesting a novel biochemical compensatory mechanism of the NO pathway and a potential entry site for therapeutics.

L-arginine transport and nitric oxide synthesis in human endothelial progenitor cells.

Nitric oxide (NO) is an endogenous vasodilator molecule synthetized from L-arginine by a family of nitric oxide synthases. In differentiated human endothelial cells, it is well known that L-arginine uptake via cationic amino acid transporters (y(+)/CAT) or system y(+)L is required for the NO synthesis via endothelial nitric oxide synthase, but there are no reports in human endothelial progenitor cell (hEPC). Therefore, we isolated hEPCs from peripheral blood of healthy donors and cultured them for either 3 (hEPC-3d) or 14 days (hEPC-14d) to characterize the L-arginine transport and NO synthesis in those cells. L-arginine transport and NO synthesis were analyzed in the presence or absence of N-ethylmaleimide or L-nitroarginine methyl ester, as inhibitors of y(+)/CAT system and nitric oxide synthases, respectively. The results showed that L-arginine uptake is higher in hEPC-14d than in hEPC-3d. Kinetic parameters for L-arginine transport showed the existence of at least 2 transporter systems in hEPC: a high affinity transporter system (K(m)= 4.8 ± 1.1 µM for hEPC-3d and 6.1 ± 2.4 µM for hEPC-14d) and a medium affinity transporter system (K(m) = 85.1 ± 4.0 µM for hEPC-3d and 95.1 ± 8 µM for hEPC-14d). Accordingly, hEPC expressed mRNA and protein for CAT-1 (ie, system y(+)) and mRNA for 2 subunits of y(+)L system, yLAT1, and 4F2hc. Higher L-citruline production and NO bioavailability (4-fold), and endothelial nitric oxide synthase expression (both mRNA and protein) were observed in hEPC-14d compared with hEPC-3d. Finally, the high L-citruline formation observed in hEPC-14d was blocked by N-ethylmaleimide. In conclusion, this study allowed to identity a functional L-arginine/NO pathway in two hEPC differentiation stages, which improves the understanding of the physiology of these precursor cells.

Attenuated glomerular arginine transport prevents hyperfiltration and induces HIF-1α in the pregnant uremic rat.

Pregnancy worsens renal function in females with chronic renal failure (CRF) through an unknown mechanism. Reduced nitric oxide (NO) generation induces renal injury. Arginine transport by cationic amino acid transporter-1 (CAT-1), which governs endothelial NO generation, is reduced in both renal failure and pregnancy. We hypothesize that attenuated maternal glomerular arginine transport promotes renal damage in CRF pregnant rats. In uremic rats, pregnancy induced a significant decrease in glomerular arginine transport and cGMP generation (a measure of NO production) compared with CRF or pregnancy alone and these effects were prevented by l-arginine. While CAT-1 abundance was unchanged in all experimental groups, protein kinase C (PKC)-α, phosphorylated PKC-α (CAT-1 inhibitor), and phosphorylated CAT-1 were significantly augmented in CRF, pregnant, and pregnant CRF animals; phenomena that were prevented by coadministrating l-arginine. α-Tocopherol (PKC inhibitor) significantly increased arginine transport in both pregnant and CRF pregnant rats, effects that were attenuated by ex vivo incubation of glomeruli with PMA (a PKC stimulant). Renal histology revealed no differences between all experimental groups. Inulin and p-aminohippurate clearances failed to augment and renal cortical expression of hypoxia inducible factor-1α (HIF-1α) significantly increased in CRF pregnant rat, findings that were prevented by arginine. These studies suggest that in CRF rats, pregnancy induces a profound decrease in glomerular arginine transport, through posttranslational regulation of CAT-1 by PKC-α, resulting in attenuated NO generation. These events provoke renal damage manifested by upregulation of renal HIF-1α and loss of the ability to increase glomerular filtration rate during gestation.

Overexpression of cationic amino acid transporter-1 increases nitric oxide production in hypoxic human pulmonary microvascular endothelial cells.

1. The endogenous production of and/or the bioavailability of nitric oxide (NO) is decreased in pulmonary hypertensive diseases. L-arginine (L-arg) is the substrate for NO synthase (NOS). L-arg is transported into cells via the cationic amino acid transporters (CAT), of which there are two isoforms in endothelial cells, CAT-1 and CAT-2. 2. To test the hypothesis that hypoxia will decrease CAT expression and L-arg uptake resulting in decreased NO production in human pulmonary microvascular endothelial cells (hPMVEC), cells were incubated in either normoxia (21% O(2), 5% CO(2), balance N(2)) or hypoxia (1% O(2), 5% CO(2), balance N(2)). 3. The hPMVEC incubated in hypoxia had 80% less NO production than cells incubated in normoxia (P < 0.01). The hPMVEC incubated in hypoxia had significantly lower CAT-2 mRNA levels than normoxic hPMVEC (P < 0.005), and the transport of L-arg was 40% lower in hypoxic than in normoxic hPMVEC (P < 0.01). In hypoxic cells, overexpression of CAT-1 resulted in significantly greater L-arg transport and NO production (P < 0.05). 4. These results demonstrate that in hPMVEC, hypoxia decreased CAT-2 expression, L-arg uptake and NO production. Furthermore, the hypoxia-induced decrease in NO production in hPMVEC can be attenuated by overexpressing CAT in these cells. We speculate that the CAT may represent a novel therapeutic target for treating pulmonary hypertensive disorders.

Requirement of rRNA methylation for 80S ribosome assembly on a cohort of cellular internal ribosome entry sites.

Protein syntheses mediated by cellular and viral internal ribosome entry sites (IRESs) are believed to have many features in common. Distinct mechanisms for ribosome recruitment and preinitiation complex assembly between the two processes have not been identified thus far. Here we show that the methylation status of rRNA differentially influenced the mechanism of 80S complex formation on IRES elements from the cellular sodium-coupled neutral amino acid transporter 2 (SNAT2) versus the hepatitis C virus mRNA. Translation initiation involves the assembly of the 48S preinitiation complex, followed by joining of the 60S ribosomal subunit and formation of the 80S complex. Abrogation of rRNA methylation did not affect the 48S complex but resulted in impairment of 80S complex assembly on the cellular, but not the viral, IRESs tested. Impairment of 80S complex assembly on the amino acid transporter SNAT2 IRES was rescued by purified 60S subunits containing fully methylated rRNA. We found that rRNA methylation did not affect the activity of any of the viral IRESs tested but affected the activity of numerous cellular IRESs. This work reveals a novel mechanism operating on a cohort of cellular IRESs that involves rRNA methylation for proper 80S complex assembly and efficient translation initiation.

Cationic and neutral amino acid transporter transcript abundances are differentially expressed in the equine intestinal tract.

To test the hypothesis that AA transporter transcripts are present in the large intestine and similarly expressed along the intestinal tract, mRNA abundance of candidate AA transporter genes solute carrier (SLC) family 7, member 9 (SLC7A9), SLC7A1, SLC7A8, and SLC43A1 encoding for b(0,+)-type AA transporter (b(0,+)AT), cationic AA transporter-1 (CAT-1), L-type AA transporter-2 (LAT-2), and L-type AA transporter-3 (LAT-3), respectively, was determined in small and large intestinal segments of the horse. Mucosa was collected from the equine small (jejunum and ileum) and large intestine (cecum, left ventral colon, and left dorsal colon), flash frozen in liquid nitrogen, and stored at -80 degrees C. Messenger RNA was isolated from tissue samples, followed by manufacture of cDNA. Relative quantitative reverse transcription-PCR was conducted using the 2(-DeltaDeltaCT) method, with glyceraldehyde-3-phosphate dehydrogenase serving as the housekeeping gene. Compared with the jejunum, cationic and neutral AA transporter SLC7A9 mRNA abundance was similar in the ileum, cecum, and large intestinal segments. Compared with the jejunum, cationic AA transporter SLC7A1 mRNA abundance was similar in the ileum and decreased in the cecum, left ventral colon, and left dorsal colon (P < 0.001). Neutral AA transporter SLC7A8 mRNA abundance decreased from the cranial to caudal end of the intestinal tract (P < 0.001). Neutral AA transporter SLC43A1 mRNA abundance was similar in the ileum and left dorsal colon and increased in the cecum (P < 0.01) and left ventral colon (P < 0.1) compared with the jejunum. Cationic and neutral AA transporter SLC7A9 mRNA abundance was similarly expressed in the large compared with small intestine, whereas cationic AA transporter SLC7A1 was of low abundance in the large intestine; neutral AA transporters SLC7A8 and SLC43A1 were differentially expressed with decreased abundance of SLC7A8 and increased abundance of SLC43A1 in the large intestine. Results indicate that the large intestine might contribute to both cationic and neutral AA uptake and absorption predominantly via transporters LAT-3 and b(0,+)AT.

A role for insulin on L-arginine transport in fetal endothelial dysfunction in hyperglycaemia.

Endothelial cells are key in the regulation of vascular tone through the release of vasoactive molecules, including nitric oxide (NO). NO is a gas synthesized from the cationic amino acid L-arginine via the endothelial NO synthase (eNOS). The semi-essential amino acid L-arginine is a taken up by endothelial cells via systems y(+) and y(+)L in primary cultures of human umbilical vein endothelial cells (HUVEC). System y(+) is a family of membrane transporters including at least five transport systems for cationic amino acids (CAT) of which HUVEC express human CAT-1 (hCAT-1) and hCAT-2B. Exposure of HUVEC to high extracellular concentrations of D-glucose increases L-arginine transport, hCAT-1 mRNA expression and eNOS activity. These phenomena are also related with increased production of reactive oxygen species (ROS), thus supporting the possibility that changes in L-arginine/NO signalling pathway result from elevated ROS. It has been shown that insulin blocks D-glucose-increased L-arginine transport and cGMP accumulation in HUVEC, whereas in this cell type insulin also modulates high D-glucose effects by activating the transcriptional factors Sp1 and NFkappaB. These transcription factors have response elements in SLC7A1 (for hCAT-1) gene promoter region, thus representing 2 possible targets for regulation of the expression of this transporter by D-glucose and/or insulin in this cell type. Recent evidences suggest that insulin blocks the stimulatory effect of D-glucose on L-arginine transport by reducing the transcriptional activity of SLC7A1 via Sp1-, NFkappaB- and ROS-dependent mechanisms. Thus, a role for these transcription factors in response to insulin is proposed in fetal endothelial cells exposed to hyperglycaemia.

Paradoxical effect of L-arginine: acceleration of endothelial cell senescence.

We have recently shown that inhibition of nitric oxide (NO) synthesis by asymmetrical dimethylarginine (ADMA) accelerated endothelial cell (EC) senescence which was prevented by coincubation with L-arginine; however the effect of long-term treatment of l-arginine alone on senescence of ECs have not been investigated. Human ECs were cultured in medium containing different concentrations of L-arginine until senescence. L-Arginine paradoxically accelerated senescence indicated by inhibiting telomerase activity. Moreover, L-arginine decreased NO metabolites, increased peroxynitrite, and 8-iso-prostaglandin F(2alpha) formation. In old cells, the mRNA expression of human amino acid transporter (hCAT)2B, the activity and protein expression of arginase II were upregulated indicated by enhanced urea, L-ornithine, and L-arginine consumption. Inhibition of arginase activity, or transfection with arginase II siRNA prevented L-arginine-accelerated senescence. The most possible explanation for the paradoxical acceleration of senescence by L-arginine so far may be the translational and posttranslational activation of arginase II.

Lack of cholesterol mobilization in islets of hormone-sensitive lipase deficient mice impairs insulin secretion.

The observations that hormone-sensitive lipase (HSL) is located in close association to insulin granules in beta-cells and that cholesterol ester hydrolase activity is completely blunted in islets of HSL null mice made us hypothesize that the role of HSL in beta-cells is to provide cholesterol for the exocytosis of insulin. To test this hypothesis, wild type (wt) and HSL null islets were depleted of plasma membrane cholesterol using methyl-beta-cyclodextrin (mbetacd). A significant reduction in insulin secretion from HSL null islets was observed whereas wt islets were unaffected. Using synaptosomal protein of 25 kDa (SNAP-25) as indicator of cholesterol-rich microdomains, confocal microscopy was used to show that HSL null beta-cells treated with mbetacd contained fewer clusters than wt beta-cells. These results indicate that HSL plays an important role in insulin secretion by providing free cholesterol for the formation and maintenance of cholesterol-rich patches for docking of SNARE-proteins to the plasma membrane.

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

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.

Identification and expression pattern of cationic amino acid transporter-1 mRNA in small intestinal epithelia of Angus steers at four production stages.

Although dietary supplementation of cationic AA (CAA), especially l-Lys, is known to be essential for optimal growth of beef cattle, the proteins responsible for absorption of CAA by bovine intestinal epithelia have not been described. Cationic AA transporter-1 (CAT-1) is a major intestinal CAA transporter, demonstrating a high-affinity (muM) transport activity for l-Lys in other mammals, and is widely expressed by small intestinal epithelia of nonruminants, but neither sequence nor expression pattern data exist for CAT-1 in cattle. Therefore, the goal of this research was to compare the relative expression (putative) of CAT-1 mRNA by duodenal, jejunal, or ileal small intestinal epithelia across and within commercially relevant beef cattle production and development stages. Twenty-four Angus steers were assigned randomly (n = 6) to 1 of 4 treatments (suckling, weanling, growing, and finishing) after all steers were born. Duodenal, jejunal, and ileal epithelia were scraped, and total RNA was extracted after the steers were killed at 32, 184, 248, or 423 d of age. Average daily gains of the steers did not differ (1.09 +/- 0.05 kg/d) among stages, whereas the small intestinal length relative to BW decreased (P < 0.01) with steer development. Using standard reverse transcription-PCR cloning techniques, we generated a partial-length bovine CAT-1 complementary DNA (695 bp; GenBank accession no. DQ399522) from jejunal mRNA samples, which possessed 89 and 87% identities to pig and human CAT-1 orthologs, respectively. On the basis of this bovine-specific genetic data, a real-time PCR-based assay of reverse-transcribed mRNA was developed and used to measure relative changes in bovine CAT-1 mRNA abundance in intestinal epithelia as steers developed. The CAT-1 mRNA was expressed by the duodenum, jejunum, and ileum of all 4 production stages. In contrast to expression by duodenal or ileal epithelium, jejunal expression of CAT-1 mRNA by growing steers was greater (P = 0.005) than that by suckling, weanling, or finishing steers. In terms of the expression of CAT-1 mRNA within production stage, jejunal expression was greater (P = 0.002) than that by duodenum or ileum for growing steers. In contrast, no intestinal site difference was found for suckling, weanling, or finishing steers. These data indicate that previously reported Na(+)-independent uptake of Lys by jejunal and ileal epithelia likely occurred by CAT-1, and that the potential capacity for CAT-1-mediated uptake of CAA for beef steers may be greatest for the "growing" phenotype.

Polyamines upregulate the mRNA expression of cationic amino acid transporter-1 in human retinal pigment epithelial cells.

We previously showed that ornithine was mainly transported via cationic amino acid transporter (CAT)-1 in human retinal pigment epithelial (RPE) cell line, human telomerase RT (hTERT)-RPE, and that CAT-1 was involved in ornithine cytotoxicity in ornithine-delta-aminotransferase (OAT)-deficient cell produced by a OAT specific inhibitor, 5-fluoromethylornithine (5-FMO). We showed here that CAT-1 mRNA expression was increased by ornithne in OAT-deficient RPE cells, which was reversed by an inhibitor of ornithine decarboxylase (ODC), alpha-difluoromethylornithine (DFMO). Polyamines, especially spermine, one of the metabolites of ODC, also enhanced the expression of CAT-1 mRNA. ODC mRNA expression was also increased by ornithine and polyamines, and gene silencing of ODC by siRNA decreased ornithine transport activity and its cytotoxicity. In addition, the mRNA of nuclear protein c-myc was also increased in 5-FMO- and ornithine-treated hTERT-RPE cells, and gene silencing of c-myc prevented the induction of CAT-1 and ODC. Increases in expression of CAT-1, ODC, and c-myc, and the inhibition of these stimulated expression by DFMO were also observed in primary porcine RPE cells. These results suggest that spermine plays an important role in stimulation of mRNA expression of CAT-1, which is a crucial role in ornithine cytotoxicity in OAT-deficient hTERT-RPE cells.

D-glucose stimulation of L-arginine transport and nitric oxide synthesis results from activation of mitogen-activated protein kinases p42/44 and Smad2 requiring functional type II TGF-beta receptors in human umbilical vein endothelium.

Elevated extracellular D-glucose increases transforming growth factor beta1 (TGF-beta1) release from human umbilical vein endothelium (HUVEC). TGF-beta1, via TGF-beta receptors I (TbetaRI) and TbetaRII, activates Smad2 and mitogen-activated protein kinases p44 and p42 (p42/44(mapk)). We studied whether D-glucose-stimulation of L-arginine transport and nitric oxide synthesis involves TGF-beta1 in primary cultures of HUVEC. TGF-beta1 release was higher ( approximately 1.6-fold) in 25 mM (high) compared with 5 mM (normal) D-glucose. TGF-beta1 increases L-arginine transport (half maximal effect approximately 1.6 ng/ml) in normal D-glucose, but did not alter high D-glucose-increased L-arginine transport. TGF-beta1 and high D-glucose increased hCAT-1 mRNA expression ( approximately 8-fold) and maximal transport velocity (V(max)), L-[(3)H]citrulline formation from L-[(3)H]arginine (index of NO synthesis) and endothelial NO synthase (eNOS) protein abundance, but did not alter eNOS phosphorylation. TGF-beta1 and high D-glucose increased p42/44(mapk) and Smad2 phosphorylation, an effect blocked by PD-98059 (MEK1/2 inhibitor). However, TGF-beta1 and high D-glucose were ineffective in cells expressing a truncated, negative dominant TbetaRII. High D-glucose increases L-arginine transport and eNOS expression following TbetaRII activation by TGF-beta1 involving p42/44(mapk) and Smad2 in HUVEC. Thus, TGF-beta1 could play a crucial role under conditions of hyperglycemia, such as gestational diabetes mellitus, which is associated with fetal endothelial dysfunction.

Pulmonary transcription of CAT-2 and CAT-2B but not CAT-1 and CAT-2A were upregulated in hemorrhagic shock rats.

Hemorrhagic shock stimulates nitric oxide (NO) biosynthesis through upregulation of inducible NO synthase (iNOS) expression. Trans-membrane l-arginine transportation mediated by the isozymes of cationic amino acid transporters (e.g. CAT-1, CAT-2, CAT-2A, and CAT-2B) is one crucial regulatory mechanism that regulates iNOS activity. We sought to assess the effects of hemorrhage and resuscitation on the expression of these regulatory enzymes in hemorrhage-stimulated rat lungs. Twenty-four rats were randomized to a sham-instrumented group, a sustained shock group, a shock with blood resuscitation group, or a shock with normal saline resuscitation group. Hemorrhagic shock was induced by withdrawing blood to maintain MAP between 40 and 45mmHg for 60min. Resuscitation by infusing blood/saline mixtures (blood resuscitation group) or saline alone (saline resuscitation group) was then performed. At the end of the experiment (300min after hemorrhage began), rats were sacrificed and enzymes expression as well as pulmonary NO biosynthesis and lung injuries were assayed. Our data revealed that hemorrhage-induced pulmonary iNOS, CAT-2, and CAT-2B transcription which was associated with pulmonary NO overproduction and subsequent lung injury. Resuscitation significantly attenuated the hemorrhage-induced enzyme upregulation, pulmonary NO overproduction, and lung injury. Blood/saline mixtures were superior to saline as a resuscitation solution in treating hemorrhage-induced pulmonary NO overproduction and lung injury. Hemorrhage and/or resuscitation, however, did not affect the expression of pulmonary CAT-1 and CAT-2A. It is, therefore, concluded that the expression of pulmonary iNOS, CAT-2, and CAT-2B is inducible and that of CAT-1 and CAT-2A is constitutive in hemorrhagic shock rat lungs.

Intracellular accumulation of L-Arg, kinetics of transport, and potassium leak conductance in oocytes from Xenopus laevis expressing hCAT-1, hCAT-2A, and hCAT-2B.

Cationic amino acid transporters play an important role in the intracellular supply of L-Arg and the generation of nitric oxide. Since the transport of L-Arg is voltage-dependent, we aimed at determining the intracellular L-Arg concentration and describing the transport of L-Arg in terms of Michaelis-Menten kinetics, taking into account membrane voltage. The human isoforms of the cationic amino acid transporters, hCAT-1, hCAT-2A, and hCAT-2B, were expressed in oocytes from Xenopus laevis and studied with the voltage clamp technique and in tracer experiments. We found that L-Arg was concentrated intracellularly by all hCAT isoforms and that influx and efflux, in the steady state of exchange, were nearly mirror images. Conductance measurements at symmetric concentrations of L-Arg (inside/outside) allowed us to determine KM and Vmax. The empty transporter of hCAT-2B featured an unexpected potassium conductance, which was inhibited by L-Arg.

miR-122, a mammalian liver-specific microRNA, is processed from hcr mRNA and may downregulate the high affinity cationic amino acid transporter CAT-1.

These studies show that miR-122, a 22-nucleotide microRNA, is derived from a liver-specific noncoding polyadenylated RNA transcribed from the gene hcr. The exact sequence of miR-122 as well as the adjacent secondary structure within the hcr mRNA are conserved from mammalian species back to fish. Levels of miR-122 in the mouse liver increase to half maximal values around day 17 of embryogenesis, and reach near maximal levels of 50,000 copies per average cell before birth. Lewis et al. (2003) predicted the cationic amino acid transporter (CAT-1 or SLC7A1) as a miR-122 target. CAT-1 protein and its mRNA are expressed in all mammalian tissues but with lower levels in adult liver. Furthermore, during mouse liver development CAT-1 mRNA decreases in an almost inverse correlation with miR-122. Eight potential miR-122 target sites were predicted within the human CAT-1 mRNA, with six in the 3'-untranslated region. Using a reporter construct it was found that just three of the predicted sites, linked in a 400-nucleotide sequence from human CAT-1, acted with synergy and were sufficient to strongly inhibit protein synthesis and reduce mRNA levels. In summary, these studies followed the accumulation during development of miR-122 from its mRNA precursor, hcr, through to identification of what may be a specific mRNA target, CAT-1.

L-Arginine transport across the basal plasma membrane of the syncytiotrophoblast of the human placenta from normal and preeclamptic pregnancies.

Cord blood levels of nitrate/nitrite, as a measure of nitric oxide (NO), are generally increased in preeclampsia. As L-arginine is the precursor for NO synthesis, we hypothesized that L-arginine transport across the syncytiotrophoblast basal plasma membrane (BM) of placentas from preeclamptic patients is also increased. Glutamine-sensitive and -insensitive [(3)H]L-arginine uptakes into BM vesicles were measured and expressed as femtomoles per milligram of protein per minute. Total L-arginine uptake was 418 +/- 15 (mean +/- SEM; n = 9) in BM from control placentas (CBM) and 495 +/- 27 (n = 7) in BM from preeclamptic placentas (PE BM; P < 0.05, by two-tailed t test). Glutamine insensitive (system y(+)) uptake was 45 +/- 3 (n = 6) in CBM, with a significantly higher uptake of 97 +/- 23 (n = 5) into PE BM (P < 0.05, by two-tailed t test). There was no significant difference in glutamine-sensitive uptake between the two groups. The expression of mRNA for human cationic amino acid transporter (hCAT) 1, 2, and 4 (system y(+) genes) and 4F2hc (heavy chain of system y(+)L) was not different in homogenates of whole placenta from the two groups. Western blotting data showed that hCAT-1 protein expression in PE BM was higher than that in CBM. These data suggest increased activity of the BM system y(+) cationic amino acid transporter in preeclampsia. If reflected in vivo, a similar increase in transporter activity could alter the delivery of L-arginine to syncytiotrophoblast eNOS.

Five novel SLC7A7 variants and y+L gene-expression pattern in cultured lymphoblasts from Japanese patients with lysinuric protein intolerance.

Two distinct human light subunits of the heteromeric amino acid transporter, y+LAT-1 coded by SLC7A7 and y+LAT-2 coded by SLC7A6, are both known to induce transport system y+L activity. SLC7A7 has already been identified as the gene responsible for lysinuric protein intolerance (LPI). We successfully identified five novel SLC7A7 variants (S238F, S489P, 1630delC, 1673delG, and IVS3-IVS5del9.7kb) in Japanese patients with LPI by PCR amplification and direct DNA sequencing. In addition, we performed a semi-quantitative expression analysis of SLC7A7 and SLC7A6 in human tissue. In normal tissue, the gene-expression ratio of SLC7A6 to SLC7A7 was high in the brain, muscle, and cultured skin fibroblasts; low in the kidneys and small intestine; and at an intermediate level in peripheral blood leukocytes, the lungs, and cultured lymphoblasts. The gene-expression ratio of SLC7A6 to SLC7A7 in cultured lymphoblasts was significantly different between normal subjects and LPI patients with R410X and/or S238F, where the relative amount of SLC7A7 mRNA was significantly lower and the relative amount of SLC7A6 mRNA was statistically higher in affected lymphoblasts than in normal cells. Expression of SLC7A7 and SLC7A6 may thus be interrelated in cultured lymphoblasts.