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.

A novel cDNA clone (20.5) which is differentially expressed between two closely related T-lymphoma cell clones was isolated by subtraction-enriched differential screening. SL12.4 cells, from which the cDNA was isolated, have characteristics of thymocytes at an intermediate stage in development. A sister cell clone derived from the same tumor, SL12.3, does not express this mRNA, has a distinct phenotype, and expresses fewer genes required for mature T-cell function. The cDNA sequence predicts a highly hydrophobic protein (approximately 49.5 kilodaltons) which contains seven putative membrane spanning domains. The gene was expressed on concanavalin A-activated T lymphocytes and was designated Tea (T-cell early activation gene). The Tea gene mapped to chromosome 8 and appeared to be conserved among mammalian and avian species. The Tea gene is distinct from, but bears extensive amino acid and DNA sequence similarity with, the murine ecotropic retroviral receptor which is encoded by the Rec-1 gene. Neither gene product displayed significant homology with other known transmembrane-spanning proteins. Thus, the Tea and Rec-1 genes establish a new family encoding multiple membrane-spanning proteins.

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.

We demonstrate that three integral membrane receptors of mammals-the ecotropic retroviral leukemia receptor (ERR), the human retroviral receptor (HRR), and the T-cell early activator (Tea)-are homologous to a family of transporters specific for amino acids, polyamines, and choline (APC), which catalyze solute uniport, so1ute:cation symport, or so1ute:solute antiport in yeast, fungi, and eubacteria. Interestingly, the ERR membrane protein was recently shown to function as a cation:amino acid cotransporter. A binary sequence similarity matrix and an evolutionary tree of the 14 members of this family, illustrating their sequence similarities and divergences, were constructed. Other proteins, including the developmentally controlled GerAII spore germination protein of Bacillus subtilis and the acetylcho~ine receptor of D r~s o~~i l a ~e I a n o~a s t e r gave sequence comparison scores of a sufficiently large magnitude to suggest (but not to establish) a common evolutionary origin with members of the APC family.We report an extended and corrected Tea cDNA sequence and show that the mammalian Tea and ERR encoding genes are differentially expressed in tissues and cell lines. Furthermore, the two mammalian cDNA sequences hybridize with other vertebrate and yeast genomic DNAs under stringent conditions. These observations support the notion that cell surface receptor proteins in mammals are transport proteins that share a common origin with transport proteins of single-ceiled organisms. Thus, permeases of essential metabolites may function pathologically as viral receptors.

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.

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.

The mKAT-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 (3). Interest in cationic AA transport regulation was stimulated by the discovery that arginine is the exclusive precursor of NO, and "de novo" arginine transport is required for NO production in some tissues (e.g., refs. 4-6).The mCAT genes were, to our knowledge, the first mammalian AA transporters cloned (reviewed in ref. 7). Their isolation permits a molecular and genetic analysis of cationic AA transporter expression and regulation (8-11). The mCAT proteins share substantial sequence (9), structural (11), and functional (12, 13) similarity when expressed and assessed in Xenopus oocytes (8,10,12,13). Based on their transport characteristics, the mCAT genes are considered to encode the y+ transport system (8,10,(12)(13)(14). Although the two genes share many similarities, they differ in their chromosomal location (9, 15) and expression patterns (7,12). Their apparent functional redundancy raises questions about mCAT gene regulation. Although little molecular information is available regarding the mechanism of AA transporter gene regulation, it is well established that AA transport systems are responsive to metabolic demands (3). We have shown that mCAT-2 mRNA accumulates during T-cell activation (9, 12) and in other tissues in response to surgical trauma (16). In contrast, mCAT-1 mRNA levels are more constant in parallel test conditions (7,12,16), although the mRNA levels are altered in response to glycogen and during liver regeneration (17).Here we report evidence that mCAT-2 gene transcription is initiated from multiple promoters. Sequence analysis of several cDNA clones revealed distinct 5' untranslated regions (UTRs) that could result from multiple promoters, alternative splicing, or trans-splicing. To analyze this further, the entire coding region of the structural gene was isolated. Moreover, the regions surrounding three of the 5' UTRs were sequenced and found to contain classical promoter and enhancer elements. We present evidence that these promoters are utilized and result in regulated gene expression in response to surgical stress. Finally, we document the differential utilization of these putative promoters in different cell types. Our data provide strong evidence that mCAT-2 transcription initiates from several distinct, widely spaced promoters.t MATERIALS AND METHODS Animals and Cell Culture. Six-to 8-week-old female AKR/J mice were purchased from The Jackson Laboratories. Tissues were harvested for RNA preparation in accordance with University of California and National Institutes of Health guidelines. The SL12.3 a...

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.