Stress-induced reversal of microRNA repression and mRNA P-body localization in human cells.

In metazoa, microRNAs (miRNAs) imperfectly base-pair with the 3'-untranslated region (3'UTR) of mRNAs and prevent protein accumulation by either repressing translation or inducing mRNA degradation. Examples of specific mRNAs undergoing miRNA-mediated repression are numerous, but whether the repression is a reversible process remains largely unknown. Here, we show that cationic amino acid transporter 1 (CAT-1) mRNA and reporters bearing the CAT-1 3'UTR or its fragments can be relieved from the miRNA miR-122-induced inhibition in human hepatoma cells in response to different stress conditions. The derepression of CAT-1 mRNA is accompanied by its release from cytoplasmic processing bodies (P bodies) and its recruitment to polysomes, indicating that P bodies act as storage sites for mRNAs inhibited by miRNAs. The derepression requires binding of HuR, an AU-rich-element-binding ELAV family protein, to the 3'UTR of CAT-1 mRNA. We propose that proteins interacting with the 3'UTR will generally act as modifiers altering the potential of miRNAs to repress gene expression.

Structure and function of cationic amino acid transporters (CATs).

The CAT proteins (CAT for cationic amino acid transporter) are amongst the first mammalian amino acid transporters identified on the molecular level and seem to be the major entry path for cationic amino acids in most cells. However, CAT proteins mediate also efflux of their substrates and thus may also deplete cells from cationic amino acids under certain circumstances. The CAT proteins form a subfamily of the solute carrier family 7 (SLC7) that consists of four confirmed transport proteins for cationic amino acids: CAT-1 (SLC7A1), CAT-2A (SLC7A2A), CAT-2B (SLC7A2B), and CAT-3 (SLC7A3). SLC7A4 and SLC7A14 are two related proteins with yet unknown function. One focus of this review lies on structural and functional differences between the different CAT isoforms. The expression of the CAT proteins is highly regulated on the level of transcription, mRNA stability, translation and subcellular localization. Recent advances toward a better understanding of these mechanisms provide a second focus of this review.

Dexamethasone suppresses eNOS and CAT-1 and induces oxidative stress in mouse resistance arterioles.

Long-term treatment with glucocorticoids is associated with mild to moderate hypertension. We reported previously that downregulation of endothelial NO synthase (eNOS) expression and activity is likely to contribute to this increase in blood pressure. In the present study, we tested the effects of dexamethasone on the vasodilation of microvascular arterioles using implanted dorsal skin-fold chambers in anesthetized C57BL/6J mice. Experiments were performed on control mice or on mice treated with dexamethasone (0.1-3 mg/kg of body wt). Endothelium-dependent vasodilation in response to ACh (0.1-10 microM) was reduced by dexamethasone in a dose-dependent fashion. Comparable inhibition was seen in tissues superfused with 30 microM N(G)-nitro-L-arginine methyl ester. In contrast, endothelium-independent vasodilation in response to S-nitroso-N-acetyl-D,L-penicillamine (10 microM) was not influenced by either dexamethasone or N(G)-nitro-L-arginine methyl ester. Levels of eNOS mRNA in murine hearts and NO(2)(-)/NO(3)(-) in serum were suppressed by dexamethasone (down to 63 and 50% of control values, respectively, at 3 mg/kg of body wt) along with a reduction in eNOS protein to 85.6%. Dexamethasone also concentration dependently reduced the expression of the cationic amino acid transporter-1 in murine hearts and cultured endothelial cells. The suppression by dexamethasone of the ACh-induced vasodilation could be partially reversed by dietary L-arginine (50 mg/kg of body wt) and by dietary vitamin C (10 g/kg of diet). We conclude that suppression by dexamethasone of the endothelium-mediated microvascular vasodilation involves several mechanisms including 1) downregulation of eNOS, 2) downregulation of cationic amino acid transporter-1, and 3) generation of reactive oxygen species. The demonstration that L-arginine and vitamin C can partially offset the effects of dexamethasone on microvascular arterioles suggests the potential clinical usefulness of these agents for the reduction of glucocorticoid-induced hypertension.

Regulation of intestinal phosphate cotransporter NaPi IIb by ubiquitin ligase Nedd4-2 and by serum- and glucocorticoid-dependent kinase 1.

Serum and glucocorticoid-inducible kinase 1 (SGK1) is highly expressed in enterocytes. The significance of the kinase in regulation of intestinal function has, however, remained elusive. In Xenopus laevis oocytes, SGK1 stimulates the epithelial Na(+) channel by phosphorylating the ubiquitin ligase Nedd4-2, which regulates channels by ubiquitination leading to subsequent degradation of the channel protein. Thus the present study has been performed to explore whether SGK1 regulates transport systems expressed in intestinal epithelial cells, specifically type IIb sodium-phosphate (Na(+)-P(i)) cotransporter (NaPi IIb). Immunohistochemistry in human small intestine revealed SGK1 colocalization with Nedd4-2 in villus enterocytes. For functional analysis cRNA encoding NaPi IIb, the SGK isoforms and/or the Nedd4-2 were injected into X. laevis oocytes, and transport activity was quantified as the substrate-induced current (I(P)). Exposure to 3 mM phosphate induces an I(P) in NaPi IIb-expressing oocytes. Coinjection of Nedd4-2, but not the catalytically inactive mutant (C938S)Nedd4-2, significantly downregulates I(P), whereas the coinjection of (S422D)SGK1 markedly stimulates I(P) and even fully reverses the effect of Nedd4-2 on I(P). The effect of (S422D)SGK1 on NaPi IIb is mimicked by wild-type SGK3 but not by wild-type SGK2, constitutively active (T308D,S473D)PKB, or inactive (K127N)SGK1. Moreover, (S422D)SGK1 and SGK3 phosphorylate Nedd4-2. In conclusion, SGK1 stimulates the NaPi IIb, at least in part, by phosphorylating and thereby inhibiting Nedd4-2 binding to its target. Thus the present study reveals a novel signaling pathway in the regulation of intestinal phosphate transport, which may be important for regulation of phosphate balance.

Human cationic amino acid transporter hCAT-3 is preferentially expressed in peripheral tissues.

At least five distinct carrier proteins form the family of mammalian cationic amino acid transporters (CATs). We have cloned a cDNA containing the complete coding region of human CAT-3. hCAT-3 is glycosylated and localized to the plasma membrane. Transport studies in Xenopus laevis oocytes revealed that hCAT-3 is selective for cationic L-amino acids and exhibits a maximal transport activity similar to other CAT proteins. The apparent substrate affinity and sensitivity to trans-stimulation of hCAT-3 resembles most closely hCAT-2B. This is in contrast to rat and murine CAT-3 proteins that have been reported to display a very low activity and to be inhibited by neutral and anionic L-amino acids as well as D-arginine (Hosokawa, H., et al. (1997) J. Biol. Chem. 272, 8717-8722; Ito, K., and Groudine, M. (1997) J. Biol. Chem. 272, 26780-26786). Also, in adult rat and mouse, CAT-3 has been found exclusively in central neurons. Human CAT-3 expression is not restricted to the brain, in fact, by far the highest expression was found in thymus. Also in other peripheral tissues, hCAT-3 expression was equal to or higher than in most brain regions, suggesting that hCAT-3 is not a neuron-specific transporter.

Inhibition of nitric oxide synthase abrogates lipopolysaccharides-induced up-regulation of L-arginine uptake in rat alveolar macrophages.

It was tested whether the inducible nitric oxide synthase (iNOS) pathway might be involved in lipopolysaccharides-(LPS)-induced up-regulation of L-arginine transport in rat alveolar macrophages (AM). AM were cultured in absence or presence of LPS. Nitrite accumulation was determined in culture media and cells were used to study [3H]-L-arginine uptake or to isolate RNA for RT - PCR. Culture in presence of LPS (1 microg ml(-1), 20 h) caused 11 fold increase of nitrite accumulation and 2.5 fold increase of [3H]-L-arginine uptake. The inducible NO synthase (iNOS) inhibitor 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT) present alone during culture had only marginal effects on [3H]-L-arginine uptake. However, AMT present during culture additionally to LPS, suppressed LPS-induced nitrite accumulation and LPS-stimulated [3H]-L-arginine uptake in the same concentration-dependent manner. AMT present only for the last 30 min of the culture period had similar effects on [3H]-L-arginine uptake. AMT present only during the uptake period also inhibited LPS-stimulated [3H]-L-arginine uptake, but with lower potency. The inhibitory effect of AMT could not be opposed by the NO releasing compound DETA NONOate. LPS caused an up-regulation of the mRNA for the cationic amino acid transporter CAT-2B, and this effect was not affected by AMT. AMT (100 microM) did not affect L-arginine transport studied by electrophysiological techniques in Xenopus laevis oocytes expressing either the human cationic amino acid transporter hCAT-1 or hCAT-2B. In conclusion, iNOS inhibition in rat AM abolished LPS-activated L-arginine uptake. This effect appears to be caused by reduced flow of L-arginine through the iNOS pathway.

The transport activity of the human cationic amino acid transporter hCAT-1 is downregulated by activation of protein kinase C.

1. The human cationic amino acid transporter hCAT-1 contains several consensus sequences for phosphorylation by protein kinase C (PKC). This study investigates the effect of PKC activation on hCAT-1-mediated transport. 2. When expressed in Xenopus laevis oocytes, hCAT-1-mediated L-arginine transport was reduced to 44+/-3% after a 30 min treatment of the oocytes with 100 nM phorbol-12-myristate-13-acetate (PMA). 4 alpha-phorbol-12,13-didecanoate (4 alpha-PDD, 100 nM) had no effect. 3. In EA.hy926 endothelial cells, maximal inhibition of hCAT-1-mediated L-arginine transport (to 3 -- 11% of control) was observed after treatment of the cells with 100 nM PMA for 4 h. A 20 -- 30 h exposure of the cells to 100 nM PMA led to the recovery of the L-arginine uptake rate that was now resistant to a second application of PMA. Phorbol-12,13-dibutyrate had similar effects as PMA, whereas 4 alpha-PDD had no effect. One microM bisindolylmaleimide I reduced the PMA effect significantly. 4. Interestingly, a 4 h treatment with 100 nM PMA increased the expression of hCAT-1 mRNA 3 -- 5 fold. hCAT-1 protein levels were unchanged for up to 4 h after PMA treatment and then increased slightly between 8 -- 28 h. 5. It is concluded that PMA downregulates the intrinsic activity of hCAT-1 by a pathway involving protein kinase C.

Voltage dependence of L-arginine transport by hCAT-2A and hCAT-2B expressed in oocytes from Xenopus laevis.

Membrane potential and currents were investigated with the two-electrode voltage-clamp technique in Xenopus laevis oocytes expressing hCAT-2A or hCAT-2B, the splice variants of the human cationic amino acid transporter hCAT-2. Both hCAT-2A- and hCAT-2B-expressing oocytes exhibited a negative extracellular L-arginine concentration ([L-Arg](o))-sensitive membrane potential, additive to the K(+) diffusion potential, when cells were incubated in Leibovitz medium (containing 1.45 mM L-Arg and 0.25 mM L-lysine). The two carrier proteins produced inward and outward currents, which were dependent on the L-Arg gradient and membrane potential. Ion substitution experiments showed that the hCAT-induced currents were independent of external Na(+), K(+), Ca(2+), or Mg(2+). The apparent Michaelis-Menten constant values at -60 mV, obtained from plots of L-Arg-induced currents against [L-Arg](o), were 0.97 and 0.13 mM in oocytes expressing hCAT-2A and hCAT-2B, respectively; maximal currents amounted to -194 +/- 8 and -84 +/- 2 nA, respectively. At saturating [L-Arg](o), the current-voltage relationships of hCAT-2A-expressing oocytes became steeper, yielding an additional conductance up to 2 microS/oocyte, whereas those of hCAT-2B-expressing oocytes were simply shifted to the right, resulting in voltage-independent difference currents. The distinct electrochemical properties of the two isoforms of hCAT-2 are assumed to contribute differentially to the membrane transport and the maintenance of cationic amino acids in various tissues.

Substrate supply for nitric-oxide synthase in macrophages and endothelial cells: role of cationic amino acid transporters.

The current study was designed to investigate the importance of cationic amino acid transporters (CATs) for the L-arginine supply to nitric oxide (NO) synthases in mouse J774A.1 macrophages and human EA.hy926 endothelial cells. CAT-1 was expressed in both cell types, whereas CAT-2B was only expressed in activated macrophages. Apparent K(M) values for transport of L-arginine in both cell types was consistent with the expression of the system y(+) carriers CAT-1 (and CAT-2B in macrophages). In addition, L-arginine transport was Na(+) independent and sensitive to trans-stimulation. A 2-h preincubation of activated macrophages in 2 mM L-lysine (which is exchanged for L-arginine by the CATs) reduced the intracellular L-arginine concentration from 2 mM to 160 microM. At the same time, nitric-oxide synthase (NOS) II activity was completely abolished. NOS II activity could be restored with extracellular L-arginine. No difference in NO production was seen between macrophages preincubated in L-arginine-containing buffer and incubated either with or without L-arginine during the 2-min NO assay. Incubation of endothelial cells in 2 mM L-lysine for up to 24 h decreased the intracellular L-arginine concentration from 3.5 mM to about 600 microM but did not reduce the NOS III activity. Our results suggest that both activated macrophages and endothelial cells have an L-arginine pool that is not freely exchangeable with the extracellular space. This pool seems to be accessible to NOS III in endothelial cells but not to NOS II in macrophages.

Transmembrane signalling mechanisms regulating expression of cationic amino acid transporters and inducible nitric oxide synthase in rat vascular smooth muscle cells.

The signalling mechanisms involved in the induction of nitric oxide synthase and l-arginine transport were investigated in bacterial lipopolysaccharide (LPS)- and interferon-gamma (IFN-gamma)-stimulated rat cultured aortic smooth muscle cells (RASMCs). The expression profile of transcripts for cationic amino acid transporters (CATs) and their regulation by LPS and IFN-gamma were also examined. Control RASMCs expressed mRNA for CAT-1, CAT-2A and CAT-2B. Levels of all three transcripts were significantly elevated in activated cells. Stimulated CAT mRNA expression and l-arginine transport occurred independently of protein kinase C (PKC), protein tyrosine kinase (PTK) and p44/42 mitogen-activated kinases (MAPKs), but were inhibited by the p38 MAPK inhibitor SB203580, which at 3 microM caused maximum inhibition of both responses. Induction of NO synthesis was independent of p44/42 MAPK activation and only marginally dependent on PKC, but was attenuated markedly by the PTK inhibitors genistein and herbimycin A. SB203580 differentially regulated inducible NO synthase expression and NO production, potentiating both processes at low micromolar concentrations and inhibiting at concentrations of >/=1 microM. In conclusion, our results suggest that RASMCs constitutively express transcripts for CAT-1, CAT-2A and CAT-2B, and that expression of these transcripts is significantly enhanced by LPS and IFN-gamma. Moreover, stimulation of l-arginine transport and induction of NO synthesis by LPS and IFN-gamma appear to be under critical regulation by the p38 MAPK, since both processes were significantly modified by SB203580 at concentrations so far shown to have no effect on other signalling pathways. Thus, in RASMCs, the p38 MAPK cascade represents an important signalling mechanism, regulating both enhanced l-arginine transport and induced NO synthesis.

Failure of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) to inhibit soluble guanylyl cyclase in rat ventricular cardiomyocytes.

1. The effects of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylyl cyclase (sGC), were investigated in aortic rings and ventricular cardiomyocytes from rats. The production of cyclic GMP was stimulated by NO.-donors or carbachol. Additionally, the effects of ODQ were studied in cytosolic extracts from both tissues in which the cyclic GMP production was stimulated by S-nitroso-N-acetylpenicillamine (SNAP). 2. In endothelium-intact aortic rings, SNAP (100 microM), 2,2'-(hydroxynitrosohydrazino)bis-ethana-mine (DETA NONOate; 100 microM), or carbachol (10 microM) increased cyclic GMP levels about 4 fold. These effects were abolished by ODQ (50 microM). 3. In cardiomyocytes, SNAP (100 microM), DETA NONOate (100 microM), or carbachol (10 microM) increased cyclic GMP levels about 2 fold. These effects were not affected by ODQ (50 microM). 4. In cytosolic extracts from aortic rings and cardiomyocytes, SNAP (100 microM) induced about 50 fold increases in cyclic GMP levels. ODQ (50 microM) reduced these effects by about 50%. 5. In extracts from cardiomyocytes, increases by SNAP (100 microM) of cyclic GMP levels were attenuated by myoglobin dependent on concentration: at 300 microM myoglobin, SNAP (100 microM) increased cyclic GMP levels only 3 fold. Inhibitory effects of ODQ (50 microM) were abolished by 300 microM myoglobin. 6. It is suggested that both NO. and ODQ can bind to myoglobin which, at high concentrations. can diminish their effects on sGC. Such a scavenger function of myoglobin could explain why NO. and ODQ exert only minor effects in cardiomyocytes (with high myoglobin content) but strong effects in aortic tissue (virtually devoid of myoglobin).

Coexpression of inducible NO synthase and soluble guanylyl cyclase in colonic enterocytes: a pathophysiologic signaling pathway for the initiation of diarrhea by gram-negative bacteria?

Infectious diarrhea is often caused by the exotoxins of gram-negative bacteria such as Escherichia coli. However, these organisms also contain lipopolysaccharide (LPS) endotoxin. LPS induces nitric oxide synthase II (NOS II, inducible NOS) in various types of cells. We now demonstrate by RNase protection analysis, Western blot, and immunohistochemistry that the expression of NOS II mRNA and protein is markedly induced in colonic enterocytes of mice that ingest LPS with their drinking water. Using the same techniques, significant levels of soluble guanylyl cyclase (GC-S), the effector enzyme of NO, were found constitutively expressed in the mucosa. This creates a pathophysiologic autocrine pathway producing increased levels of cyclic GMP and leading to hypersecretion and diarrhea. In fact, the LPS-induced diarrhea developed in parallel with the NOS II induction. Diarrhea could be controlled with orally administered dexamethasone, which prevented the LPS-stimulated induction of NOS II (RNase protection analysis and Western blot). Diarrhea was also blocked by oral aminoguanidine, an inhibitor of NOS II activity. These data suggest that in addition to the known heat-labile and heat-stable exotoxins, gram-negative bacteria may induce diarrhea through the release of endotoxins that induce a NOS II-GC-S autocrine pathway in mucosal epithelium.

Human cationic amino acid transporters hCAT-1, hCAT-2A, and hCAT-2B: three related carriers with distinct transport properties.

In this study, we aimed at analyzing the human homologues of the murine cationic amino acid transporters mCAT-1, mCAT-2A, and mCAT-2B. cDNAs encoding hCAT-1 had been previously reported by two independent groups [Albritton, L.M., et al. (1993) Genomics 12, 430; Yoshimoto, T., et al. (1991) Virology 185, 10]. We isolated cDNAs encoding hCAT-2A and hCAT-2B from a human liver cDNA library and from cDNA derived from the human hepatoma cell line HepG2, respectively. Analyses of the deduced amino acid sequences of both carriers demonstrated 90.9% identity with the respective murine proteins. In their functional domains (42 amino acids), both hCAT-2A and hCAT-2B differ only by one residue from the respective mouse proteins. Thus, CAT-2 proteins demonstrate a higher interspecies conservation than CAT-1 proteins that are overall 86.5% identical between mouse and human and differ by seven residues in the functional domain. The high degree of sequence conservation was reflected by the functional similarity of the human carriers with their mouse homologues. When expressed in Xenopus oocytes, hCAT-1 and hCAT-2B demonstrated transport properties consistent with y+. Unlike the mouse CAT-1 and CAT-2B, whose transport properties could hardly be distinguished, the transport properties of the human CAT-1 and CAT-2B isoforms showed clear differences: hCAT-1 had a 3-fold higher substrate affinity and was more sensitive to trans-stimulation than hCAT-2B. In contrast to the y+ carriers, hCAT-2A exhibited a 10-30-fold lower substrate affinity, a greater maximal velocity, and was much less sensitive to trans-stimulation at physiological substrate concentrations.

Interference of L-arginine analogues with L-arginine transport mediated by the y+ carrier hCAT-2B.

The inducible human cationic amino acid transporter hCAT-2B was expressed in Xenopus laevis oocytes, and this system was used to test the effect of several NO synthase (NOS) inhibitors and/or L-arginine analogues on L-arginine transport by this y+ carrier. L-NG-Methyl-L-arginine (L-NMA), asymmetrical L-NG, NG-dimethyl-L-arginine (L-ADMA), L-N5-(1-iminoethyl)-ornithine (L-NIO), L-NG-nitro-L-arginine (L-NNA), and L-NG-nitro-L-arginine methyl ester (L-NAME) all inhibited the inducible NOS II extracted from RAW 264.7 macrophages induced with bacterial lipopolysaccharide. L-NMA, L-ADMA, and L-NIO also competed with L-arginine for transport by hCAT-2B, whereas L-NNA and L-NAME did not. The two L-arginine analogues, symmetrical NG, NG-dimethyl-L-arginine (L-SDMA) and alpha-amino-delta-isothioureidovaleric acid (AITV), as well as L-lysine, did not block enzymatic activity of NOS II, but did compete for L-arginine transport mediated by hCAT-2B. L-Lysine and L-SDMA were transported efficiently by hCAT-2B and exchanged against intracellular L-arginine, resulting in an L-arginine depletion of the cells. AITV was a much poorer substrate of hCAT-2B and had only little effect on intracellular L-arginine concentrations. These data indicate that substrate recognition differs markedly between the inducible L-arginine transporter hCAT-2B and the inducible NOS II, with different L-arginine analogues having affinity to only one or both of these proteins.

Inducible NO synthase II and neuronal NO synthase I are constitutively expressed in different structures of guinea pig skeletal muscle: implications for contractile function.

The expression of NOS isoforms was studied in guinea pig skeletal muscle at the mRNA and protein level, and the effect of NO on contractile response was examined. Ribonuclease protection analyses demonstrated NOS I and NOS II mRNAs in diaphragm and gastrocnemius muscle. In Western blots, NOS I and NOS II immunoreactivities were found in the particulate but not the soluble fraction of skeletal muscle. NOS activity was found almost exclusively in the particulate fraction. About 50% of this activity was Ca2+ independent. In immunohistochemistry, the anti-NOS I antibody stained distinct membrane regions of muscle fibers. The most intense staining was seen in neuromuscular endplates identified by labeling with alpha-bungarotoxin. The anti-NOS II antibody labeled muscle fibers that contained alkali-labile myosin ATPase (type I fibers). NOS II was located to intracellular structures and was also seen in "specific pathogen-free" animals. Pretreatment of guinea pigs with bacterial lipopolysaccharide (LPS) markedly intensified NOS II staining. Significant NOS III immunoreactivity was detected only in vascular endothelium. In functional experiments, tetanic muscle contractions were induced in diaphragm and gastrocnemius muscle by electrical stimulation of the innervating nerves. Pretreatment of guinea pigs with LPS or addition of S-nitroso-N-acetyl-D,L-penicillamine to the organ bath markedly decreased tetanic contractions. N(G)-nitro-L-arginine, on the other hand, increased contractile force and reversed the effect of LPS. Our data indicate that NOS II and NOS I are expressed in different structures of skeletal muscle and are involved in the regulation of contractile response.

Cytokines and insulin induce cationic amino acid transporter (CAT) expression in cardiac myocytes. Regulation of L-arginine transport and no production by CAT-1, CAT-2A, and CAT-2B.

Cytokine-dependent production of nitric oxide (NO) by rat cardiac myocytes is a consequence of increased expression of the inducible isoform of nitric oxide synthase (iNOS or NOS2) and, in the presence of insulin, depresses the contractile function of these cells in vivo and in vitro. Experiments reported here show that L-lysine, a competitive antagonist of L-arginine uptake, suppressed NO production (detected as nitrite accumulation) by interleukin (IL)-1beta and interferon (IFN) gamma-pretreated cardiac myocytes by 70%, demonstrating that NO production is dependent on L-arginine uptake. Cardiac myocytes constitutively exhibit a high-affinity L-arginine transport system (Km = 125 microM; Vmax = 44 pmol/2 X 10(5) cells/min). Following a 24-h exposure to IL-1beta and IFNgamma, arginine uptake increases Vmax = 167 pmol/2 X 10(5) cells/min) and a second low-affinity L-arginine transporter activity appears (Km = 1.2 mM). To examine the molecular basis for these cytokine-induced changes in arginine transport, we examined expression of three related arginine transporters previously identified in other cell types. mRNA for the high-affinity cationic amino acid transporter-1 (CAT-1) is expressed in resting myocytes and steady-state levels increase by 10-fold following exposure to IL-1beta and IFNgamma. Only cytokine-pretreated myocytes expressed a second high-affinity L-arginine transporter, CAT-2B, as well as a low-affinity L-arginine transporter, CAT-2A. In addition, insulin, which potentiated cytokine-dependent NO production independent of any change in NOS activity, increased myocyte L-arginine uptake by 2-fold and steady-state levels of CAT-1, but not CAT-2A or CAT-2B mRNA. Thus, NO production by cardiac myocytes exposed to IL-1beta plus IFNgamma appears to be dependent on the coinduction of CAT-1, CAT-2A, and CAT-2B, while insulin independently augments L-arginine transport through CAT- 1.

CATs, a family of three distinct mammalian cationic amino acid transporters.

Three related mammalian carrier proteins that mediate the transport of cationic amino acids through the plasma membrane have been identified in murine and human cells (CAT for cationic amino acid transporter). Models of the CAT proteins in the membrane suggest they have 12 or 14 transmembrane domains connected by short hydrophilic loops and intracellular N- and C-termini. The transport activity of the CAT proteins is sensitive to trans-stimulation and independent of the presence of sodium ions. These features agree with the behaviour of carrier proteins mediating facilitated diffusion. The three CAT proteins, CAT-1, CAT-2A and CAT-2(B) are encoded by two different genes (CAT-1 and CAT-2). CAT-1 and CAT-2(B) exhibit transport properties consistent with system y(+), the principal mechanism for cellular uptake of cationic amino acids. In contrast, CAT-2A has tenfold lower substrate affinity, greater apparent maximal velocity and it is much less sensitive to trans-stimulation. In addition to structural and functional aspects, this review discusses the role of the CAT proteins for supplying substrate to NO synthases and the property of the rodent CAT-1 proteins to function as virus receptors.