Regulatory role of arginase I and II in nitric oxide, polyamine, and proline syntheses in endothelial cells.

Endothelial cells (EC) metabolize L-arginine mainly by arginase, which exists as two distinct isoforms, arginase I and II. To understand the roles of arginase isoforms in EC arginine metabolism, bovine coronary venular EC were stably transfected with the Escherichia coli lacZ gene (lacZ-EC, control), rat arginase I cDNA (AI-EC), or mouse arginase II cDNA (AII-EC). Western blots and enzymatic assays confirmed high-level expression of arginase I in the cytosol of AI-EC and of arginase II in mitochondria of AII-EC. For determining arginine catabolism, EC were cultured for 24 h in DMEM containing 0.4 mM L-arginine plus [1-(14)C]arginine. Urea formation, which accounted for nearly all arginine consumption by these cells, was enhanced by 616 and 157% in AI-EC and AII-EC, respectively, compared with lacZ-EC. Arginine uptake was 31-33% greater in AI-EC and AII-EC than in lacZ-EC. Intracellular arginine content was 25 and 11% lower in AI-EC and AII-EC, respectively, compared with lacZ-EC. Basal nitric oxide (NO) production was reduced by 60% in AI-EC and by 47% in AII-EC. Glutamate and proline production from arginine increased by 164 and 928% in AI-EC and by 79 and 295% in AII-EC, respectively, compared with lacZ-EC. Intracellular content of putrescine and spermidine was increased by 275 and 53% in AI-EC and by 158 and 43% in AII-EC, respectively, compared with lacZ-EC. Our results indicate that arginase expression can modulate NO synthesis in bovine venular EC and that basal levels of arginase I and II are limiting for endothelial syntheses of polyamines, proline, and glutamate and may have important implications for wound healing, angiogenesis, and cardiovascular function.

Arginase I: a limiting factor for nitric oxide and polyamine synthesis by activated macrophages?

Because arginase hydrolyzes arginine to produce ornithine and urea, it has the potential to regulate nitric oxide (NO) and polyamine synthesis. We tested whether expression of the cytosolic isoform of arginase (arginase I) was limiting for NO or polyamine production by activated RAW 264.7 macrophage cells. RAW 264.7 cells, stably transfected to overexpress arginase I or beta-galactosidase, were treated with interferon-gamma to induce type 2 NO synthase or with lipopolysaccharide or 8-bromo-cAMP (8-BrcAMP) to induce ornithine decarboxylase. Overexpression of arginase I had no effect on NO synthesis. In contrast, cells overexpressing arginase I produced twice as much putrescine after activation than did cells expressing beta-galactosidase. Cells overexpressing arginase I also produced more spermidine after treatment with 8-BrcAMP than did cells expressing beta-galactosidase. Thus endogenous levels of arginase I are limiting for polyamine synthesis, but not for NO synthesis, by activated macrophage cells. This study also demonstrates that it is possible to alter arginase I levels sufficiently to affect polyamine synthesis without affecting induced NO synthesis.

Salicylate-enhanced activation of transcription factors induced by interferon-gamma.

Salicylate enhanced the interferon-gamma-dependent activation of two transcription factors in a murine macrophage cell line: signal transducer and activator of transcription (STAT)1 and interferon-gamma-responsive factor 1. Salicylate alone did not activate these transcription factors. This enhancement was reflected by increased DNA-binding activities and was the consequence of prolonged tyrosine phosphorylation of these transcription factors following interferon-gamma treatment. However, salicylate did not directly inhibit protein-tyrosine phosphatase activity in nuclear extracts of interferon-gamma-treated cells. The enhanced activation of STAT1 resulted in increased induction of mRNA encoding interferon regulatory factor-1. These results not only demonstrate that aspirin and its metabolite salicylate may have pro-inflammatory as well as anti-inflammatory effects but also raise the possibility that new cellular targets may be identified for modulating the Janus kinase-STAT signalling pathway.

Differential regulation of arginases and inducible nitric oxide synthase in murine macrophage cells.

Activated macrophages avidly consume arginine via the action of inducible nitric oxide synthase (iNOS) and/or arginase. In contrast to our knowledge regarding macrophage iNOS expression, the stimuli and mechanisms that regulate expression of the cytosolic type I (arginase I) or mitochondrial type II (arginase II) isoforms of arginase in macrophages are poorly defined. We show that one or both arginase isoforms may be induced in the RAW 264.7 murine macrophage cell line and that arginase expression is regulated independently of iNOS expression. For example, 8-bromo-cAMP strongly induced both arginase I and II mRNAs but not iNOS. Whereas interferon-gamma induced iNOS but not arginase, 8-bromo-cAMP and interferon-gamma mutually antagonized induction of iNOS and arginase I mRNAs. Dexamethasone, which did not induce either arginase or iNOS, almost completely abolished induction of arginase I mRNA by 8-bromo-cAMP but enhanced induction of arginase II mRNA. Lipopolysaccharide (LPS) induced arginase II mRNA, but 8-bromo-cAMP plus LPS resulted in synergistic induction of both arginase I and II mRNAs. In all cases, increases in arginase mRNAs were sufficient to account for the increases in arginase activity. These complex patterns of expression suggest that the arginase isoforms may play distinct, although partially overlapping, functional roles in macrophage arginine metabolism.

Structure of the murine arginase II gene.

Mammals contain two genes encoding distinct isoforms of arginase (arginases I and II), both of which catalyze the conversion of arginine to ornithine and urea. However, their subcellular localization and tissue-specific patterns of expression are very different, indicating that they perform distinct physiologic roles. As an initial step in elucidating the regulation and physiologic roles of arginase II, this report describes the characterization of a mammalian arginase II gene. The murine arginase II gene contains eight exons like the arginase I gene. The six internal exons have intron/exon boundaries that are identical to the arginase I gene; however, exon three of the arginase II gene has obtained a three-base-pair insertion. The identity of the exon/intron boundaries is consistent with a gene duplication as the origin of the arginase isozymes with the small insertion occurring after the duplicative event. The promoter region of the arginase II gene, which bears no resemblance to that of the arginase I genes, contains numerous potential binding sites for enhancer and promoter elements but does not contain a TATA box.

Human type II arginase: sequence analysis and tissue-specific expression.

A full-length cDNA encoding type II arginase was isolated from a human kidney cDNA library and its sequence compared to those of vertebrate type I arginases as well as to arginases of bacteria, fungi and plants. The predicted sequence of human type II arginase is 58% identical to the sequence of human type I arginase but is 71% identical to the sequence of Xenopus type II arginase, suggesting that duplication of the arginase gene occurred before mammals and amphibians diverged. Seven residues known to be essential for activity were found to be conserved in all arginases. Type II arginase mRNA was detected in virtually all human and mouse RNA samples tested whereas type I arginase mRNA was found only in liver. At least five mRNA species hybridizing to type II arginase cDNA were found in the human RNA samples whereas only a single type II arginase mRNA species was found in the mouse. This raises the possibility that the multiple type II arginase mRNAs in humans arise from differential RNA processing or usage of alternative promoters.

Novel actions of aspirin and sodium salicylate: discordant effects on nitric oxide synthesis and induction of nitric oxide synthase mRNA in a murine macrophage cell line.

Aspirin and sodium salicylate each inhibit to a similar extent the production of nitric oxide (NO) in the RAW 264.7 murine macrophage cell line following stimulation by either lipopolysaccharide (LPS) or interferon-gamma (IFN-gamma). The similar potencies of aspirin and sodium salicylate indicate that acetylation of cellular macromolecules is not essential for the observed effects. The failure of added prostaglandin E2 to overcome the effects of aspirin or sodium salicylate indicates that these effects are not simply the result of inhibition of prostaglandin synthesis. The inhibition of NO production occurs irrespective of the effect of these agents on induction of nitric oxide synthase (iNOS) mRNA by LPS or IFN-gamma. Aspirin and sodium salicylate inhibit iNOS mRNA induction in LPS-stimulated cells but enhance iNOS mRNA induction in IFN-gamma-stimulated cells. In contrast, these agents consistently inhibit induction of argininosuccinate synthetase mRNA in both LPS- and IFN-gamma-stimulated cells. Concentrations of aspirin in the 3-10 mM range inhibit induced NO production and expression of iNOS protein without inhibiting induction of iNOS mRNA. Discordances between effects on NO synthesis and induction of iNOS mRNA indicate that aspirin and sodium salicylate have multiple sites of action in their effects on pathways that are involved in the production of NO by stimulated RAW 264.7 cells.

Specific disruption of renal function and gene transcription by cyclosporin A.

The effects of cyclosporin A (CsA) are cell-specific, ranging from its immunosuppressive action on cells of the immune system to a variety of nonimmunologic toxic side effects. The predominant undesirable side effects of CsA occur in the kidney. Although many toxic renal effects of CsA have been described, the molecular basis of the nephrotoxicity is unknown. Elucidation of the molecular basis for the renal action of CsA may shed light on the function of cyclophilin in nonimmune cell types. The present study demonstrates that CsA selectively reduces the gluconeogenic capacity of rat proximal tubules via a decrease in activity of phosphoenolpyruvate carboxykinase (GTP:oxaloacetate carboxy-lyase (transphosphorylating), E.C. 4.1.1.32; PEPCK). The decrease in renal PEPCK activity occurs within 3 days and reflects a corresponding reduction in renal PEPCK mRNA abundance. This, in turn, is due to a selective inhibition of renal PEPCK gene transcription. Expression of several other renal genes is unaffected by CsA, as is expression of the PEPCK gene in liver. Thus, the effects of CsA are organ-specific and do not represent a general cytotoxic effect on proximal tubule cells. These results suggest that selective inhibition of the activity of a transcription factor(s) required for expression of specific genes in renal tubules may play a role in CsA-induced nephrotoxicity.