Protein kinase Cα phosphorylates a novel argininosuccinate synthase site at serine 328 during calcium-dependent stimulation of endothelial nitric-oxide synthase in vascular endothelial cells.

Endothelial nitric-oxide synthase (eNOS) utilizes l-arginine as its principal substrate, converting it to l-citrulline and nitric oxide (NO). l-Citrulline is recycled to l-arginine by two enzymes, argininosuccinate synthase (AS) and argininosuccinate lyase, providing the substrate arginine for eNOS and NO production in endothelial cells. Together, these three enzymes, eNOS, AS, and argininosuccinate lyase, make up the citrulline-NO cycle. Although AS catalyzes the rate-limiting step in NO production, little is known about the regulation of AS in endothelial cells beyond the level of transcription. In this study, we showed that AS Ser-328 phosphorylation was coordinately regulated with eNOS Ser-1179 phosphorylation when bovine aortic endothelial cells were stimulated by either a calcium ionophore or thapsigargin to produce NO. Furthermore, using in vitro kinase assay, kinase inhibition studies, as well as protein kinase Cα (PKCα) knockdown experiments, we demonstrate that the calcium-dependent phosphorylation of AS Ser-328 is mediated by PKCα. Collectively, these findings suggest that phosphorylation of AS at Ser-328 is regulated in accordance with the calcium-dependent regulation of eNOS under conditions that promote NO production and are in keeping with the rate-limiting role of AS in the citrulline-NO cycle of vascular endothelial cells.

Insulin transcriptionally regulates argininosuccinate synthase to maintain vascular endothelial function.

Diminished vascular endothelial cell nitric oxide (NO) production is a major factor in the complex pathogenesis of diabetes mellitus. In this report, we demonstrate that insulin not only maintains endothelial NO production through regulation of endothelial nitric oxide synthase (eNOS), but also via the regulation of argininosuccinate synthase (AS), which is the rate-limiting step of the citrulline-NO cycle. Using serum starved, cultured vascular endothelial cells, we show that insulin up-regulates AS and eNOS transcription to support NO production. Moreover, we show that insulin enhances NO production in response to physiological cues such as bradykinin. To translate these results to an in vivo model, we show that AS transcription is diminished in coronary endothelial cells isolated from rats with streptozotocin (STZ)-induced diabetes. Importantly, we demonstrate restoration of AS and eNOS transcription by insulin treatment in STZ-diabetic rats, and show that this restoration was accompanied by improved endothelial function as measured by endothelium-dependent vasorelaxation. Overall, this report demonstrates, both in cell culture and whole animal studies, that insulin maintains vascular function, in part, through the maintenance of AS transcription, thus ensuring an adequate supply of arginine to maintain vascular endothelial response to physiological cues.

Argininosuccinate synthase: at the center of arginine metabolism.

The levels of L-arginine, a cationic, semi-essential amino acid, are often controlled within a cell at the level of local availability through biosynthesis. The importance of this temporal and spatial control of cellular L-arginine is highlighted by the tissue specific roles of argininosuccinate synthase (argininosuccinate synthetase) (EC 6.3.4.5), as the rate-limiting step in the conversion of L-citrulline to L-arginine. Since its discovery, the function of argininosuccinate synthase has been linked almost exclusively to hepatic urea production despite the fact that alternative pathways involving argininosuccinate synthase were defined, such as its role in providing arginine for creatine and for polyamine biosynthesis. However, it was the discovery of nitric oxide that meaningfully extended our understanding of the metabolic importance of non-hepatic argininosuccinate synthase. Indeed, our knowledge of the number of tissues that manage distinct pools of arginine under the control of argininosuccinate synthase has expanded significantly.

Phosphorylation of argininosuccinate synthase by protein kinase A.

Argininosuccinate synthase (AS) is essential for endothelial nitric oxide (NO) production and its regulation in this capacity has been studied primarily at the transcriptional level. The dynamics of vascular function suggest that an acute regulation system may mediate AS function. This premise underlies our hypothesis that AS is phosphorylated in vascular endothelium. Immunoprecipitation and immobilized metal affinity chromatography demonstrated that AS is an endogenous phosphoprotein. An in vitro kinase screen revealed that protein kinase A (PKA), a kinase that enhances NO production via eNOS activation, phosphorylated AS. Vascular endothelial growth factor (VEGF) was identified as a candidate pathway for regulating AS phosphorylation since it enhanced NO production and activated PKA and eNOS. MDLA, an AS inhibitor, diminished maximal VEGF-mediated NO production. In addition, immunoprecipitation studies suggested that VEGF enhanced AS phosphorylation. Overall, these results represent the first demonstration that vascular endothelial NO production can be regulated by dynamic AS phosphorylation.

Troglitazone up-regulates vascular endothelial argininosuccinate synthase.

Vascular endothelial nitric oxide (NO) production via the citrulline-NO cycle not only involves the regulation of endothelial nitric oxide synthase (eNOS), but also regulation of caveolar-localized endothelial argininosuccinate synthase (AS), which catalyzes the rate-limiting step of the cycle. In the present study, we demonstrated that exposure of endothelial cells to troglitazone coordinately induced AS expression and NO production. Western blot analysis demonstrated an increase in AS protein expression. This increased expression was due to transcriptional upregulation of AS mRNA, as determined by quantitative real time RT-PCR and inhibition by 1-d-ribofuranosylbenzimidazole (DRB), a transcriptional inhibitor. Reporter gene assays and EMSA analyses identified a distal PPARgamma response element (PPRE) (-2471 to -2458) that mediated the troglitazone increase in AS expression. Overall, this study defines a novel molecular mechanism through which a thiazolidinedione (TZD) like troglitazone supports endothelial function via the transcriptional up-regulation of AS expression.

Endothelial nitric oxide production is tightly coupled to the citrulline-NO cycle.

Nitric oxide (NO) is an important vasorelaxant produced along with L-citrulline from L-arginine in a reaction catalyzed by endothelial nitric oxide synthase (eNOS). Previous studies suggested that the recycling of L-citrulline to L-arginine is essential for NO production in endothelial cells. However, there is no direct evidence demonstrating the degree to which the recycling of L-citrulline to L-arginine is coupled to NO production. We hypothesized that the amount of NO formed would be significantly higher than the amount of L-citrulline formed due to the efficiency of L-citrulline recycling via the citrulline-NO cycle. To test this hypothesis, endothelial cells were incubated with [14C]-L-arginine and stimulated by various agents to produce NO. The extent of NO and [14C]-L-citrulline formation were simultaneously determined. NO production exceeded apparent L-citrulline formation of the order of 8 to 1, under both basal and stimulated conditions. As further support, alpha-methyl-DL-aspartate, an inhibitor of argininosuccinate synthase (AS), a component of the citrulline-NO cycle, inhibited NO production in a dose-dependent manner. The results of this study provide evidence for the essential and efficient coupling of L-citrulline recycling, via the citrulline-NO cycle, to endothelial NO production.

Tumor necrosis factor-alpha reduces argininosuccinate synthase expression and nitric oxide production in aortic endothelial cells.

Endothelial dysfunction associated with elevated serum levels of TNF-alpha observed in diabetes, obesity, and congenital heart disease results, in part, from the impaired production of endothelial nitric oxide (NO). Cellular NO production depends absolutely on the availability of arginine, substrate of endothelial nitric oxide synthase (eNOS). In this report, evidence is provided demonstrating that treatment with TNF-alpha (10 ng/ml) suppresses not only eNOS expression but also the availability of arginine via the coordinate suppression of argininosuccinate synthase (AS) expression in aortic endothelial cells. Western blot and real-time RT-PCR demonstrated a significant and dose-dependent reduction of AS protein and mRNA when treated with TNF-alpha with a corresponding decrease in NO production. Reporter gene analysis demonstrated that TNF-alpha suppresses the AS proximal promoter, and EMSA analysis showed reduced binding to three essential Sp1 elements. Inhibitor studies suggested that the repression of AS expression by TNF-alpha may be mediated, in part, via the NF-kappaB signaling pathway. These findings demonstrate that TNF-alpha coordinately downregulates eNOS and AS expression, resulting in a severely impaired citrulline-NO cycle. The downregulation of AS by TNF-alpha is an added insult to endothelial function because of its important role in NO production and in endothelial viability.

Regulation of endothelial argininosuccinate synthase expression and NO production by an upstream open reading frame.

Argininosuccinate synthase (AS) catalyzes the rate-limiting step in the recycling of citrulline to arginine, which in endothelial cells, is tightly coupled to the production of nitric oxide (NO). In previous work, we established that endothelial AS mRNA can be initiated from multiple start sites, generating co-expressed mRNA variants with different 5'-untranslated regions (5'-UTRs). One of the 5'-UTRs, the shortest form, represents greater than 90% of the total AS mRNA. Two other extended 5'-UTR forms of AS mRNA, resulting from upstream initiations, contain an out-of-frame, upstream open reading frame (uORF). In this study, the function of the extended 5'-UTRs of AS mRNA was investigated. Single base insertions to place the uORF in-frame, and mutations to extend the uORF, demonstrated functionality, both in vitro with AS constructs and in vivo with luciferase constructs. Overexpression of the uORF suppressed endothelial AS protein expression, whereas specific silencing of the uORF AS mRNAs resulted in the coordinate up-regulation of AS protein and NO production. Expression of the full-length of the uORF was necessary to mediate a trans-suppressive effect on endothelial AS expression, demonstrating that the translation product itself affects regulation. In conclusion, the uORF found in the extended, overlapping 5'-UTR AS mRNA species suppresses endothelial AS expression, providing a novel mechanism for regulating endothelial NO production by limiting the availability of arginine.

Citrulline can preserve proliferation and prevent the loss of CD3 zeta chain under conditions of low arginine.

BACKGROUND: Arginine depletion by the enzyme Arginase I, decreases expression of the TCR zeta chain preventing T-cell activation and causing T-cell dysfunction. We hypothesized that citrulline could substitute for arginine under conditions of increased arginase expression. Thus, the goal was to establish a possible mechanism of how citrulline could overcome arginine depletion caused by arginase. METHODS: Jurkat cells were cultured, with or without arginase, in media containing different amino-acid constituents: complete RPMI containing arginine (C-RPMI) (arginine), Arginine-Free-RPMI (Arg-Free RPMI) and Citrulline-containing RPMI (Cit RPMI). Incorporation of citrulline was measured via uptake of 3H-citrulline, whereas proliferation was measured via 3H-thymidine incorporation. zeta Chain was analyzed by 2-color flow cytometry. Argininosuccinate synthase (AS) and argininosuccinate lyase expression was detected using Northern blots, RT-PCR, and Western blots. RESULTS: Jurkat cells exhibited a significant decrease in proliferation and 5 chain expression when cultured in the presence of arginase or in the absence of arginine. With citrulline, zeta chain expression and proliferation were maintained in the absence of arginine or in the presence of the enzyme arginase. Jurkat cells, cultured in the absence of arginine, were associated with a 5-fold increase in citrulline uptake. The absence of arginine was also associated with increased expression of AS. CONCLUSIONS: T cells exhibit the molecular capability of increasing citrulline membrane transport and up-regulating AS expression, thus exhibiting the necessary mechanisms for converting citrulline into arginine and escaping the ill effects of arginine depletion. Therefore, citrulline has the potential to be a substitute for supplemental arginine in diseases associated with arginase-mediated T cell dysfunction.

Argininosuccinate synthase expression is required to maintain nitric oxide production and cell viability in aortic endothelial cells.

Although cellular levels of arginine greatly exceed the apparent K(m) for endothelial nitric-oxide synthase, current evidence suggests that the bulk of this arginine may not be available for nitric oxide (NO) production. We propose that arginine regeneration, that is the recycling of citrulline back to arginine, defines the essential source of arginine for NO production. To support this proposal, RNA interference analysis was used to selectively reduce the expression of argininosuccinate synthase (AS), because the only known metabolic role for AS in endothelial cells is in the regeneration of l-arginine from l-citrulline. Western blot analysis demonstrated a significant and dose-dependent reduction of AS protein as a result of AS small interfering RNA treatment with a corresponding diminished capacity to produce basal or stimulated levels of NO, despite saturating levels of arginine in the medium. Unanticipated, however, was the finding that the viability of AS small interfering RNA-treated endothelial cells was significantly decreased when compared with control cells. Trypan blue exclusion analysis suggested that the loss of viability was not because of necrosis. Two indicators, reduced expression of Bcl-2 and an increase in caspase activity, which correlated directly with reduced expression of AS, suggested that the loss of viability was because of apoptosis. The exposure of cells to an NO donor prevented apoptosis associated with reduced AS expression. Overall, these results demonstrate the essential role of AS for endothelial NO production and cell viability.

The caveolar nitric oxide synthase/arginine regeneration system for NO production in endothelial cells.

The enzyme endothelial nitric oxide synthase (eNOS) catalyzes the conversion of arginine, oxygen and NADPH to NO and citrulline. Previous results suggest an efficient, compartmentalized system for recycling of citrulline to arginine utilized for NO production. In support of this hypothesis, the recycling enzymes, argininosuccinate synthase (AS) and argininosuccinate lyase (AL), have been shown to colocalize with eNOS in caveolae, a subcompartment of the plasma membrane. Under unstimulated conditions, the degree of recycling is minimal. Upon stimulation of NO production by bradykinin, however, recycling is co-stimulated to the extent that more than 80% of the citrulline produced is recycled to arginine. These results suggest an efficient caveolar recycling complex that supports the receptor-mediated stimulation of endothelial NO production. To investigate the molecular basis for the unique location and function of endothelial AS and AL, endothelial AS mRNA was compared with liver AS mRNA. No differences were found in the coding region of the mRNA species, but significant differences were found in the 5'-untranslated region (5'-UTR). The results of these studies suggest that sequence in the endothelial AS-encoding gene, represented by position -92 nt to -43 nt from the translation start site in the extended AS mRNA 5'-UTRs, plays an important role in differential and tissue-specific expression. Overall, a strong evidential case has been developed supporting the proposal that arginine availability, governed by a caveolar-localized arginine regeneration system, plays a key role in receptor-mediated endothelial NO production.

The protein kinase C beta II exon confers mRNA instability in the presence of high glucose concentrations.

Previous studies showed that short term exposure of cells to high glucose destabilized protein kinase C (PKC) betaII mRNA, whereas PKCbetaI mRNA levels remained unaltered. Because PKCbeta mRNAs share common sequences other than the PKCbetaII exon encoding a different carboxyl terminus, we examined PKCbetaII mRNA for a cis-acting region that could confer glucose-induced destabilization. A beta-globin/growth hormone reporter con struct containing the PKCbetaII exon was transfected into human aorta and rat vascular smooth muscle cells (A10) to follow glucose-induced destabilization. Glucose (25 mm) exposure destabilized PKCbetaII chimeric mRNA but not control mRNA. Deletion analysis and electrophoretic mobility shift assays followed by UV cross-linking experiments demonstrated that a region introduced by inclusion of the betaII exon was required to confer destabilization. Although a cis-acting element mapped to 38 nucleotides within the betaII exon was necessary to bestow destabilization, it was not sufficient by itself to confer complete mRNA destabilization. Yet, in intact cells antisense oligonucleotides complementary to this region blocked glucose-induced destabilization. These results suggest that this region must function in context with other sequence elements created by exon inclusion involved in affecting mRNA stability. In summary, inclusion of an exon that encodes PKCbetaII mRNA introduces a cis-acting region that confers destabilization to the mRNA in response to glucose.

Leptin and the adipocyte endocrine system.

Although adipose tissue has long been considered to be metabolically passive and primarily responsible for energy storage, recent scientific advances have dramatically altered our understanding of the function of this ubiquitous tissue. The fat cell is a transducer of energy supply for the changing metabolic needs of the body, modulating glucose homeostasis, hypothalamic function, sympathetic output, vascular tone, immune response, and reproduction. Through endocrine/autocrine and paracrine actions, adipocyte-derived molecules defend the body during periods of energy deficit and stress. With the development of obesity, maladaptive responses to adipose excess result in pathologic states of inflammation, coagulopathy, and altered insulin sensitivity.

Endothelial argininosuccinate synthase mRNA 5'-untranslated region diversity. Infrastructure for tissue-specific expression.

Based on the integral role that argininosuccinate synthase (AS) plays in the production of nitric oxide in vascular endothelial cells and urea in liver, an analysis was carried out to determine whether signals reside in the AS mRNA to account for tissue differences in AS function and location. Reverse transcriptase-PCR and sequence analysis showed that the AS mRNA coding region was the same for both endothelial cells and liver; however, 5'-RACE analysis (rapid amplification of cDNA ends) identified AS mRNA species in endothelial cells in addition to a major 43-nucleotide (nt) 5'-untranslated region (UTR) AS mRNA with overlapping extended 5'-UTRs of 66 and 92 nt. Comparison to the genomic sequence immediately upstream of the reported transcription start site for the human and mouse AS gene suggested that expression of all three species of bovine endothelial AS mRNA are driven by a common promoter and that 5'-UTR diversity in endothelial cells results from three transcriptional initiation sites within exon 1. RNase protection analysis and real-time reverse transcriptase-PCR verified and quantitated the differential expression of the extended 5'-UTR species relative to the major 43-nt 5'-UTR AS mRNA. In vitro translation studies showed a less pronounced but similar discordant expression. Sequential deletions starting from the 5' terminus of the 92-nt 5'-UTR construct resulted in a corresponding increase in translational efficiency, but the most pronounced effect resulted from mutation of an upstream open reading frame, which restored translational efficiency of the 92-nt 5'-UTR AS mRNA. When the different AS mRNA 5'-UTRs, cloned in front of a luciferase reporter gene, were transfected into endothelial cells, the pattern of luciferase expression was nearly identical to that observed for the different 5'-UTR AS mRNAs in endothelial cells. Given the different roles ascribed for argininosuccinate synthase, urea versus NO production, these results suggest that sequence in the AS gene represented by position -92 to -43 nt from the translation start site in the extended AS mRNA 5'-UTRs plays an important role in differential and tissue-specific expression.