Sidney M. Morris

Regulation of mRNA levels for five urea cycle enzymes in rat liver by diet, cyclic AMP, and glucocorticoids.

Adaptive changes in levels of urea cycle enzymes are largely coordinate in both direction and magnitude. In order to determine the extent to which these adaptive responses reflect coordinate regulatory events at the pretranslational level, measurements of hybridizable mRNA levels for all five urea cycle enzymes were carried out for rats subjected to various dietary regimens and hormone treatments. Changes in relative abundance of the mRNAs in rats with varying dietary protein intakes are comparable to reported changes in enzyme activities, indicating that the major response to diet occurs at the pretranslational level for all five enzymes and that this response is largely coordinate. In contrast to the dietary changes, variable responses of mRNA levels were observed following intraperitoneal injections of dibutyryl cAMP and dexamethasone. mRNAs for only three urea cycle enzymes increased in response to dexamethasone. Levels of all five mRNAs increased severalfold in response to dibutyryl cAMP at both 1 and 5 h after injection, except for ornithine transcarbamylase mRNA which showed a response at 1 h but no response at 5 h. Combined effects of dexamethasone and dibutyryl cAMP were additive for only two urea cycle enzyme mRNAs, suggesting independent regulatory pathways for these two hormones. Transcription run-on assays revealed that transcription of at least two of the urea cycle enzyme genes--carbamylphosphate synthetase I and argininosuccinate synthetase--is stimulated approximately four- to fivefold by dibutyryl cAMP within 30 min. The varied hormonal responses indicate that regulatory mechanisms for modulating enzyme concentration are not identical for each of the enzymes in the pathway.

Nitrite-generated NO circumvents dysregulated arginine/NOS signaling to protect against intimal hyperplasia in Sprague-Dawley rats

Vascular disease, a significant cause of morbidity and mortality in the developed world, results from vascular injury. Following vascular injury, damaged or dysfunctional endothelial cells and activated SMCs engage in vasoproliferative remodeling and the formation of flow-limiting intimal hyperplasia (IH). We hypothesized that vascular injury results in decreased bioavailability of NO secondary to dysregulated arginine-dependent NO generation. Furthermore, we postulated that nitrite-dependent NO generation is augmented as an adaptive response to limit vascular injury/proliferation and can be harnessed for its protective effects. Here we report that sodium nitrite (intraperitoneal, inhaled, or oral) limited the development of IH in a rat model of vascular injury. Additionally, nitrite led to the generation of NO in vessels and SMCs, as well as limited SMC proliferation via p21Waf1/Cip1 signaling. These data demonstrate that IH is associated with increased arginase-1 levels, which leads to decreased NO production and bioavailability. Vascular injury also was associated with increased levels of xanthine oxidoreductase (XOR), a known nitrite reductase. Chronic inhibition of XOR and a diet deficient in nitrate/nitrite each exacerbated vascular injury. Moreover, established IH was reversed by dietary supplementation of nitrite. The vasoprotective effects of nitrite were counteracted by inhibition of XOR. These data illustrate the importance of nitrite-generated NO as an endogenous adaptive response and as a pathway that can be harnessed for therapeutic benefit.

Regulation of Arginine Availability and Its Impact on NO Synthesis

Publisher Summary Arginine is the only physiological source of the nitrogen atom in nitric oxide (NO), enzymatic reactions and other processes that regulate the availability of this amino acid play crucial roles in regulating rates of NO synthesis in health and disease. The processes of arginine biosynthesis, catabolism, and transport are especially important in modulating both intracellular and plasma levels of arginine. The key players in these processes are argininosuccinate synthase, arginases, and cationic amino acid transporters (CATS), respectively. Their expression not only varies among different cell types, but is also dynamically modulated by bacterial lipopolysaccharide, cytokines and other agents. In particular, argininosuccinate synthase and the CATS are generally coinduced with inflammatory nitric oxide synthase (iNOS), apparently, to maximize cellular capacity for NO synthesis. The metabolic roles and expression of the arginases, which compete with the NOS enzymes for a common substrate, are more complex and incompletely understood. As virtually all the enzymes of arginine metabolism can be expressed within the same cell, the complex interplay between the often conflicting or competing reactions represents a considerable challenge to the ability to understand the factors that control NO synthesis in different cell types.

A cohort of supporting metabolic enzymes is coinduced with nitric oxide synthase in human tumor cell lines

Although nitric oxide (NO) has cytotoxic activity against certain tumor cell lines, some human tumor cell lines can themselves produce NO by expressing the inducible isoform of NO synthase (iNOS). As rates of cellular NO synthesis play a major role in determining whether NO has cytotoxic or cytoprotective effects at anatomic sites of NO production, identification of cellular processes which regulate rates of NO synthesis by iNOS is important for understanding the role of NO in tumor cell biology. This study demonstrates that argininosuccinate synthetase and GTP-cyclohydrolase-I, which catalyze rate-limiting steps in the synthesis of iNOS substrate (arginine) and cofactor (tetrahydrobiopterin), respectively, are coinduced with iNOS expression in two human tumor cell lines. These results indicate that coinduction of these supporting metabolic pathways helps to maximize cellular NO synthesis by iNOS in human tumor cells, suggesting that these pathways might be useful targets for pharmacologic intervention in NO-producing human tumor cells.

Induction of arginases I and II in cornea during herpes simplex virus infection

Induction of inducible nitric oxide synthase (iNOS) following corneal infection with herpes simplex virus type-1 (HSV-1) generates nitric oxide (NO), an important player in the defense against viral infection. Changes in arginine metabolism during infection are not limited to effects of iNOS but can also involve arginases, which can modulate NO synthesis and produce ornithine for the generation of polyamines and proline. The latter are important molecules involved in tissue damage and repair during inflammation. In this study we determined the responses of arginase I and II in a murine model of HSV-1-induced stromal keratitis (HSK). In the cornea iNOS and arginase II mRNA were co-induced as the initial inflammation developed at 2 days postinfection (p.i.). As stromal keratitis progressed (days 8-15 p.i.) arginase I mRNA was induced tenfold, in contrast to a moderate decrease in arginase II and a loss of iNOS expression. These results suggest that elevated expression of arginase I and II in the cornea at late stages of ocular HSV-1 infection may play a role in lesion expression in HSK.

Abundance of mRNAs encoding urea cycle enzymes in fetal and neonatal mouse liver

The relative abundances of mRNAs encoding the five urea cycle enzymes during development of mouse liver have been determined and compared with those of mRNAs encoding four other liver-specific proteins (phosphoenolpyruvate carboxykinase, tyrosine aminotransferase, alpha-fetoprotein, and albumin). Urea cycle enzyme mRNAs in fetal liver are expressed at 2-14% of the abundance in adult liver as early as 6 days before birth. Expression of the urea cycle enzyme mRNAs is not coordinate during the fetal and neonatal period. However, profiles of three urea cycle enzyme mRNAs are quite similar to that of alpha-fetoprotein mRNA, suggesting the possibility of a common response to regulatory signals during fetal development. With the exception of ornithine transcarbamylase mRNA, the urea cycle enzyme mRNAs have been shown previously to be inducible by cAMP and glucocorticoids. However, only argininosuccinate lyase mRNA exhibits any significant change in abundance at birth, resembling postnatal expression of tyrosine aminotransferase mRNA. The results indicate that the urea cycle enzyme mRNAs are potentially useful markers for elucidating various features of hepatocyte differentiation in mammals.

Nutritional and Hormonal Regulation of mRNA Abundance for Arginine Biosynthetic Enzymes in Kidney

Argininosuccinate synthetase and argininosuccinate lyase catalyze the synthesis of arginine from citrulline in kidney and also serve as components of the urea cycle in liver of ureotelic animals. Dietary and hormonal regulation of mRNAs encoding these enzymes have been well studied in liver but not in kidney. Messenger RNAs for these enzymes are localized within the renal cortex. Starvation and extreme variations in dietary protein content (0% vs 60% casein) produced 2.6- to 3.5-fold increases in mRNA abundance for these two enzymes in rat kidney. Argininosuccinate lyase mRNA was not induced by dibutyryl cAMP, dexamethasone, or a combination of the two agents. In contrast, argininosuccinate synthetase mRNA was induced 2-fold by dibutyryl cAMP but was unresponsive to dexamethasone. Thus, diet and hormones regulate levels of these mRNAs in rat kidney, but the responses are both qualitatively and quantitatively distinct from the responses previously reported for rat liver.

Thyroxine elicits divergent changes in mRNA levels for two urea cycle enzymes and one gluconeogenic enzyme in tadpole liver

Thyroxine-induced metamorphosis of the tadpole to the frog (Rana catesbeiana) is marked by increased activities of the urea cycle enzymes in liver. Cloned cDNAs for two mammalian urea cycle enzymes--carbamyl-phosphate synthetase I and argininosuccinate synthetase--were shown to cross-hybridize with the corresponding mRNAs in tadpole liver. Thyroxine treatment produced nearly 10-fold, coordinate increases in hybridizable mRNA levels for these two enzymes in tadpole liver. This increase is sufficient to account for reported increases in enzyme levels and synthesis rates, demonstrating that thyroxine largely regulates concentrations of these enzymes at a pretranslational step(s). In contrast, levels of phosphoenolpyruvate carboxykinase mRNA in tadpole liver decreased by more than 90% following thyroxine treatment. This differs from the thyroxine-induced increases in synthesis rates of enzyme and mRNA reported for phosphoenolpyruvate carboxykinase in rat liver. However, the decreased levels of this mRNA in tadpole liver may represent a secondary response due to thyroxine-stimulated release of insulin.

Differential induction of transcription for glucocorticoid-responsive genes in cultured rat hepatocytes

Dexamethasone rapidly stimulated transcription of the tyrosine aminotransferase and metallothionein-I genes--but not of the phosphoenolpyruvate carboxykinase gene--in rat hepatocytes cultured in serum-free medium. This differential response was not observed for cyclic AMP. The results suggest that the phosphoenolpyruvate carboxykinase gene--but not the tyrosine aminotransferase and metallothionein-I genes--requires a factor which is permissive for stimulation of transcription by the glucocorticoid receptor.