CAT2-mediated L-arginine transport and nitric oxide production in activated macrophages.

Activated macrophages require l-arginine uptake to sustain NO synthesis. Several transport systems could mediate this l-arginine influx. Using competition analysis and gene-expression studies, amino acid transport system y+ was identified as the major carrier responsible for this activity. To identify which of the four known y+ transport-system genes is involved in macrophage-induced l-arginine uptake, we used a hybrid-depletion study in Xenopus oocytes. Cationic amino acid transporter (CAT) 2 antisense oligodeoxyribonucleotides abolished the activated-macrophage-mRNA-induced l-arginine transport. Together with expression studies documenting that CAT2 mRNA and protein levels are elevated with increased l-arginine uptake, our data demonstrate that CAT2 mediates the l-arginine transport that is required for the raised NO production in activated J774 macrophages.

Stress differentially induces cationic amino acid transporter gene expression.

The amino acid l-arginine plays a central role in several adaptive metabolic pathways and we postulate that regulated L-arginine transport contributes to important physiological responses. The majority of L-arginine flux is mediated by transport system y+ that is encoded by at least three genes, Cat1, Cat2 and Cat3. Cat2 encodes two distinct protein isoforms (CAT2/CAT2a) that differ by 10-fold in their apparent substrate affinity. Cat2 transcription is controlled by four widely spaced promoters. The expression of CAT2/2a transcripts was tested in skeletal muscle and macrophages following specific stresses or activators. Unexpectedly, CAT2a transcripts accumulated in skeletal muscle in response to surgical trauma (hepatectomy and splenectomy) as well as food deprivation, although neither high affinity CAT2 nor CAT1 were detectably altered. Activated macrophages decreased CAT1 levels, but accumulated CAT2 and iNOS mRNA and protein with parallel kinetics suggesting that CAT2 mediated L-arginine transport might regulate the L-arginine:nitric oxide pathway. In macrophages, liver and skeletal muscle, the most distal CAT2 promoter was predominant. No change in promoter usage was apparent under any stress conditions tested nor was alternate splicing of the CAT2 transcript dictated by promoter usage. The differential regulation of the Cat genes indicates their encoded transporter proteins meet different requirements for cationic amino acids in the intact animal.

IFN-gamma primes macrophage responses to bacterial DNA.

Macrophages recognize and are activated by unmethylated CpG motifs in bacterial DNA. Here we demonstrate that production of nitric oxide (NO) from murine RAW 264 macrophages and bone marrow-derived macrophages (BMM) in response to bacterial DNA is absolutely dependent on interferon-gamma (IFN-gamma) priming. Similarly, arginine uptake and expression of the inducible nitric oxide synthase (iNOS) gene in response to bacterial DNA in BMM occurred only after IFN-gamma priming. In contrast, mRNA for the cationic amino acid transporter, CAT2, was induced by plasmid DNA alone, and priming with IFN-gamma had no effect on this response. Tumor necrosis factor-alpha (TNF-alpha) release from RAW 264 and BMM in response to bacterial DNA was augmented by IFN-gamma pretreatment. In a stably transfected HIV-1 long terminal repeat (LTR) luciferase RAW 264 cell line, IFN-gamma and bacterial DNA synergized in activation of the HIV-1 LTR. Bacterial DNA has been shown to induce IFN-gamma production in vivo as an indirect consequence of interleukin-12 (IL-12) and TNF-alpha production from macrophages. The results herein suggest the existence of a self-amplifying loop that may have implications for therapeutic applications of bacterial DNA.

Induced nitric oxide synthesis is dependent on induced alternatively spliced CAT-2 encoding L-arginine transport in brain astrocytes.

The inducible isoform II of nitric-oxide synthase (iNOS) was recently cloned from brain and identified in astroglial cells. Induced nitric oxide biosynthesis occurs in brain cells only if extracellular cerebrospinal fluid contains -arginine. This study demonstrates for the first time that induced iNOS activity is strictly dependent on concomitant induction of an alternatively spliced transcript of the cat-2 gene encoding high affinity -arginine transporter System y+ in cultured rat astrocytes. Inhibition profiles of radiolabeled -arginine and -leucine uptake identified the dominance of Na+-independent transport System y+ serving cationic amino acids, with insignificant activities of Systems y+L, bo,+, or Bo,+. A reverse transcription-polymerase chain reaction/sequencing/cloning strategy was used to identify a single 123-base nucleotide sequence coding the high affinity domain of alternatively spliced CAT-2 (not CAT-2a) in astrocytes activated by lipopolysaccharide/interferon-gamma. Using this sequence as a cDNA probe, it was determined that CAT-2 mRNA, iNOS mRNA, and System y+ activity were concomitantly and strongly induced in astrocytes. Constitutive CAT-1 mRNA was weakly present in neurons and astrocytes, was not inducible in either cell type, and contributed <3% to total System y+ activity. Although astroglial iNOS Km approximately 10 microM L-arginine for intracellular substrate, hyperbolic kinetics of inducible iNOS activity measured as a function of extracellular L-arginine concentration gave Km approximately 50 microM L-arginine with intact cells. The same Km approximately 50 microM was obtained for induced membrane transport System y+ activity. iNOS activity was reduced to zero in the absence of extracellular L-arginine uptake via System y+. These findings expand the current understanding of NO biosynthesis modulation and implicate a coordinated regulation of intracellular iNOS enzyme activity with membrane L-arginine transport in brain.

Regulation of CAT: Cationic amino acid transporter gene expression.

The majority of mammalian cationic amino acid transport is mediated by the transport system y(+) which facilitates Na(+) independent cationic amino acid (arginine, lysine, & ornithine) transport and Na(+) dependent zwitterionic amino acid (glutamine & homoserine) transport. Other transport systems y(+)L, b(0,+) and B(0,+) also mediate cationic amino acid transport. Their broad substrate specificities and overlapping expression patterns confound biochemical analysis. The isolation of cDNA clones has permitted an analysis of their regulation and opens the opportunity to define the role of each protein in specific cell types. Two genes,Cat1 andCat2 encode transporters with properties similar to the y(+) transport system. Thecat2 gene from the mouse encodes two distinct proteins. mCAT2, and mCAT2A via alternate splicing; each protein has distinctly different transport properties. The regulation of mCAT1, mCAT2 and mCAT2A proteins are reviewed here. The implications of this gene specific regulation on cationic amino acid transport is discussed.

A mammalian arginine/lysine transporter uses multiple promoters.

The mCAT-2 gene encodes a Na(+)-independent cationic amino acid (AA) transporter that is inducibly expressed in a tissue-specific manner in various physiological conditions. When mCAT-2 protein is expressed in Xenopus oocytes, the elicited AA transport properties are similar to the biochemically defined transport system y+. The mCAT-2 protein sequence is closely related to another cationic AA transporter (mCAT-1); these related proteins elicit virtually identical cationic AA transport in Xenopus oocytes. The two genes differ in their tissue expression and induction patterns. Here we report the presence of diverse 5' untranslated region (UTR) sequences in mCAT-2 transcripts. Sequence analysis of 22 independent mCAT-2 cDNA clones reveals that the cDNA sequences converge precisely 16 bp 5' of the initiator AUG codon. Moreover, analysis of genomic clones shows that the mCAT-2 gene 5'UTR exons are dispersed over 18 kb. Classical promoter and enhancer elements are present in appropriate positions 5' of the exons and their utilization results in regulated mCAT-2 mRNA accumulation in skeletal muscle and liver following partial hepatectomy. The isoform adjacent to the most distal promoter is found in all tissues and cell types previously shown to express mCAT-2, while the other 5' UTR isoforms are more tissue specific in their expression. Utilization of some or all of five putative promoters was documented in lymphoma cell clones, liver, and skeletal muscle. TATA-containing and (G+C)-rich TATA-less promoters appear to control mCAT-2 gene expression. The data indicate that the several distinct 5' mCAT-2 mRNA isoforms result from transcriptional initiation at distinct promoters and permit flexible transcriptional regulation of this cationic AA transporter gene.

Multiple components of transport are associated with murine cationic amino acid transporter (mCAT) expression in Xenopus oocytes.

Expression of putative amino acid transport proteins is usually assumed to be associated with expression of a single component of transport. It is shown in this report, however, that murine cationic amino acid transporter (mCAT) expression in Xenopus oocytes is associated in important instances with expression of more than one kinetically distinguishable transport process. Accurate knowledge of the kinetics of transport continues, therefore, to be needed to understand how transport proteins function.

y(+)-type cationic amino acid transport: expression and regulation of the mCAT genes.

The transport of cationic amino acids across animal cell membranes is largely mediated by a small group of well-described transport system (y+, bo,+, Bo,+). Only recently have genes encoding transport proteins in some of these systems been isolated. Two genes, mCAT-1 and mCAT-2, encode related multiple membrane-spanning proteins that share substantial amino acid sequence identity and virtually superimposable hydrophilicity profiles. mCAT-1 and mCAT-2 proteins expressed in Xenopus oocytes are functionally indistinguishable and similar to transport system y+, but have distinct tissue distribution patterns. mCAT-1 expression is nearly ubiquitous and produces a single protein, while mCAT-2 is highly tissue-specific, has two distinct protein isoforms encoded by a single gene and is expressed in different tissues using at least two widely separated promoters. All three proteins facilitate the ion-independent transport of arginine, lysine and ornithine. Both mCAT-1 and mCAT-2 proteins have low amino acid sequence similarity but strikingly similar hydrophilicity profiles with amino acid antiporters, uniporters and symporters of yeast, fungi and eubacteria. Current work will elucidate whether any of the mCAT proteins interact with members of a newly identified family of single membrane-spanning proteins, such as rBAT, 4F2 and NAA-Tr, which are thought to modulate or activate y+L and/or bo,+ transport systems.

Special transport and neurological significance of two amino acids in a configuration conventionally designated as D.

We point out an ability of certain amino acids to be recognized at a biological receptor site as though their amino group bore, instead of an alpha relationship to a carboxylate group, a beta, gamma or delta relationship to the same or a second carboxylate group. For aspartate, the unbalanced position of its amino group between a pair of carboxylates allows its occasional biorecognition as a beta-rather than as an alpha-amino acid, whereas for proline and its homologs, their cyclic arrangement may allow the imino group, without its being replicated, to be sensed analogously as falling at either of two distances from the single carboxylate group. The greater separation might allow proline to be seen as biologically analogous to gamma-aminobutyric acid. This more remote positioning of the imino group would allow the D-form of both amino acids to present its amino group in the orientation characteristic of the natural L-form. The dual modes of recognition should accordingly be signalled by what appears to be low stereospecificity, actually due to a distinction in the enantiorecognition of the two isomers. Competing recognition for transport between their respective D- and L-forms, although it does not prove that phenomenon, has been shown for proline and, significantly, even more strongly for its lower homolog, 2-azetidine carboxylate. Such indications have so far revealed themselves rather inconspicuously for the central nervous system binding of proline, reviewed here as a possible feature of a role suspected for proline in neurotransmission.

Gene-product designations for amino acid transporters.

The molecular cloning of genes that encode amino acid transporters presents the scientific community with the opportunity to name their gene products using a scheme that could usefully recall the well-defined transport system most similar in properties to the newly identified cloned gene product. To avoid the problem of rising confusion, we propose to take advantage of established designation methods that indicate the types of amino acids transported and the co-substrate ion requirement of their transport. The economy obligated by the necessity to keep the number of symbols in a gene name to a minimum will rarely permit a listing of the full range of substrates, since amino acid transport systems have broad substrate specificities with co-substrate requirements that can differ in a substrate-specific manner. Hence, the use of established systems to codify groups of amino acid transport systems, which allow identification of the substrate range by using 1-3 letters, e.g. A, L or even ASC, could be integrated with a system used to indicate the ion-dependence of transport. The discoverers of transporters are mainly proceeding with commendable reserve and are inviting discussion, a desire which this essay urges be facilitated by more formal arrangements for further planning. These discoverers have also shown, along with an expressed desire for guidance, well-advised spontaneity in making reference to the substrate range, two trends that together suggest that a good set of designations can evolve that will be highly descriptive.(ABSTRACT TRUNCATED AT 250 WORDS)

Na(+)-independent transport (uniport) of amino acids and glucose in mammalian cells.

Recent advances have made possible the isolation of the genes and their cDNAs encoding Na(+)-independent amino acid transporters. Two classes of amino acid 'uniporters' have been isolated. One class contains the mCAT (murine cationic amino acid transporter) gene family that encodes proteins predicted to span the membrane 12-14 times and exhibits structural properties similar to the GLUT (glucose transporter) family and to other well-known transporters. The other class consists of two known genes, rBAT (related to B system amino acid transporters) and 4F2hc, that share amino acid sequence similarity with alpha-amylases and alpha-glucosidases. They are type II glycoproteins predicted to span the membrane only once, yet they mediate the Na(+)-independent transport of cationic and zwitterionic amino acids in Xenopus oocytes. Mutations in the human rBAT gene have been identified by Palacín and his co-workers in several families suffering from a heritable form of cystinuria. This important finding clearly establishes a key role for rBAT in cystine transport. The two classes of amino acid transporters are compared with the well-studied GLUT family of Na(+)-independent glucose transporters.