GTP Cyclohydrolase I Is Coinduced in Hepatocytes Stimulated to Produce Nitric Oxide

Geller, David A.; Silvio, Mauricio Di; Billiar, Timothy R.; Hatakeyama, Kazuyuki

doi:10.1006/bbrc.2000.3537

Cited by 23 publications

(17 citation statements)

References 74 publications

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“…In hepatocytes, BH 4 is expressed constitutively at levels sufficient for iNOS activity (10,26). Consistent with this finding, we show here that soluble iNOS was minimally enhanced by the addition of exogenous BH 4 .…”

Section: Discussionsupporting

confidence: 89%

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Monomeric inducible nitric oxide synthase localizes to peroxisomes in hepatocytes

Loughran

Stolz

Vodovotz

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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Hepatocytes are capable of repeated inducible NO synthase (iNOS) expression, which occurs under inflammatory and stress conditions. This iNOS expression regulates a number of cellular functions as well as cell viability. To better understand the posttranslational mechanisms that regulate the fate of iNOS in these cells, we characterized the iNOS distributed within peroxisomes. The selective permeabilization of membranes (plasma vs. peroxisomal) confirmed that there are cytosolic and peroxisomal pools of iNOS in cytokine-stimulated hepatocytes and that the iNOS protein associates with peroxisome. Detergent solubilization of the membrane fraction released iNOS to the soluble fraction. iNOS localized to membrane fraction is predominantly monomeric, but dimerization is partially reconstituted rapidly upon incubation with tetrahydrobiopterin. The reconstituted iNOS exhibits a lower specific activity than iNOS isolated from the soluble pool. Depletion of intracellular tetrahydrobiopterin with an inhibitor of de novo pterin synthesis resulted in a predominance of monomeric iNOS without a greater relative distribution of iNOS to the peroxisomal pool. Thus, iNOS exists in a least two pools in hepatocytes: a soluble pool composed of both active dimer and monomer and a peroxisomal pool of monomeric iNOS. iNOS might localize to peroxisomes in long-lived cells such as hepatocytes as a protective mechanism to remove incompetent enzyme. subcellular localization ͉ inflammatory and stress conditions ͉ hepatocytes ͉ peroxisomes N itric oxide (NO) has critical signaling functions within cells but can also lead to cell dysfunction or toxicity. One mechanism for both regulating and localizing NO production is through distribution of NO synthase (NOS) to structures within the cells. For example, the association of endothelial NOS (eNOS) with caveolin localizes eNOS within structures in the plasma membrane known as caveolae and is a mechanism for regulating eNOS activity (1). The physical interaction of neuronal NOS (nNOS) with other proteins containing PDZ domains localizes this isoform to synaptic junctions in brain and motor endplates in skeletal muscle (2). Comparatively little is known about the posttranslational modifications that regulate inducible NOS (iNOS) distribution and function. In murine macrophages, iNOS has been shown to be both phosphorylated and ubiquitinated (3) and also associates with Rac1 and Rac2 as a mechanism to increase enzyme activity (4). We have previously shown that iNOS localizes to both the cytosol and peroxisomes in hepatocytes in vitro (5) and in vivo (5, 6); however, the structure and function of this cellular pool of iNOS is unknown.Proteins that localize to peroxisomes function in hydrogen peroxide breakdown, lipid metabolism, and amine synthesis (7). Peroxisomes are single-membrane organelles of 0.2-1.0 m in size that do not require protein unfolding or disruption of multimeric complexes before protein import (7). These organelles are particularly abundant in hepatocytes, constituting Ϸ1% of tota...

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Section: Discussionsupporting

confidence: 89%

“…Total biopterins in cell lysates were determined as described in ref. 10. The samples were processed for protein determination [bicinchoninic acid (BCA); Pierce], NOS activity, and immunoblots.…”

Section: Methodsmentioning

confidence: 99%

Monomeric inducible nitric oxide synthase localizes to peroxisomes in hepatocytes

Loughran

Stolz

Vodovotz

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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“…25 In contrast to the latter 2 enzymes, GTPCH activity is limiting in most tissues, 26 and it is the major regulator of BH 4 synthesis. The GCH1 gene, encoding GTPCH, is expressed in several cell types such as macrophages, 26 hepatocytes, 27 and endothelial cells. 7,8 Several in vitro studies suggested that GCH1 expression in endothelial cells or macrophages is induced by cytokines.…”

Section: Antoniades Et Al Vascular Tetrahydrobiopterin and Enos Couplmentioning

confidence: 99%

Altered Plasma Versus Vascular Biopterins in Human Atherosclerosis Reveal Relationships Between Endothelial Nitric Oxide Synthase Coupling, Endothelial Function, and Inflammation

et al. 2007

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Background-Tetrahydrobiopterin (BH 4 ) is a key regulator of endothelial nitric oxide synthase (eNOS) activity and coupling. However, the extent to which vascular and/or systemic BH 4 levels are altered in human atherosclerosis and the importance of BH 4 bioavailability in determining endothelial function and oxidative stress remain unclear. We sought to define the relationships between plasma and vascular biopterin levels in patients with coronary artery disease and to determine how BH 4 levels affect endothelial function, eNOS coupling, and vascular superoxide production. Methods and Results-Samples of saphenous veins and internal mammary arteries were collected from 219 patients with coronary artery disease undergoing coronary artery bypass grafting. We determined plasma and vascular levels of biopterins, vasomotor responses to acetylcholine, and vascular superoxide production in the presence and absence of the eNOS inhibitor N G -nitro-L-arginine methyl ester. High vascular BH 4 was associated with greater vasorelaxations to acetylcholine (PϽ0.05), whereas high plasma BH 4 was associated with lower vasorelaxations in response to acetylcholine (PϽ0.05). Furthermore, an inverse association was observed between plasma and vascular biopterins (PϽ0.05 for both saphenous veins and internal mammary arteries). High vascular (but not plasma) BH 4 was associated with reduced total and N G -nitro-L-arginine methyl ester-inhibitable superoxide, suggesting improved eNOS coupling. Finally, plasma but not vascular biopterin levels were correlated with plasma C-reactive protein levels (PϽ0.001). Conclusions-An inverse association exists between plasma and vascular biopterins in patients with coronary artery disease. Vascular but not plasma BH 4 is an important determinant of eNOS coupling, endothelium-dependent vasodilation, and superoxide production in human vessels, whereas plasma biopterins are a marker of systemic inflammation.

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“…GTP cyclohydrolase (GTPCH) is considered to be the ratelimiting step in de novo BH 4 synthesis. The simultaneous up-regulation of both GTPCH and iNOS is understood to coordinate the increased synthesis of BH 4 levels at a sufficiently high level to facilitate enzymatic NO production from iNOS protein (Gross and Levi, 1992;Geller et al, 2000).…”

mentioning

confidence: 99%

Sepiapterin Decreases Acute Rejection and Apoptosis in Cardiac Transplants Independently of Changes in Nitric Oxide and Inducible Nitric-Oxide Synthase Dimerization

Pieper¹,

Ионова²,

Cooley³

et al. 2009

J Pharmacol Exp Ther

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Tetrahydrobiopterin (BH 4 ), a cofactor of inducible nitric-oxide synthase (iNOS), is an important post-translational regulator of NO bioactivity. We examined whether treatment of cardiac allograft recipients with sepiapterin [S-(Ϫ)-2-amino-7,8-dihydro-6-(2-hydroxy-1-oxopropyl)-4-(1H)-pteridinone], a precursor of BH 4 , inhibited acute rejection and apoptosis in cardiac transplants. Heterotopic cardiac transplantation was performed in Wistar-Furth donor to Lewis recipient strain rats. Recipients were treated daily after transplantation with 10 mg/kg sepiapterin. Grafts were harvested on post-transplant day 6 for analysis of BH 4 (high-performance liquid chromatography), expression of inflammatory cytokines (reverse transcription-and real-time polymerase chain reaction), iNOS (Western blots), and NO (Griess reaction and NO analyzer). Histological rejection grade was scored, and graft function was determined by echocardiography. Apoptosis, protein nitration, and oxidative stress were determined by immunohistochemistry. Treatment of allografts with sepiapterin increased cardiac BH 4 levels by 3-fold without changing protein levels of GTP cyclohydrolase, the enzyme that regulates de novo BH 4 synthesis. Sepiapterin decreased inflammatory cell infiltrate and significantly inhibited histological rejection scores and apoptosis similar in magnitude to cyclosporine. Sepiapterin also decreased nitrative and oxidative stress. Sepiapterin caused a smaller increase in left ventricular mass versus untreated allografts but without improving fractional shortening. Sepiapterin did not alter tumor necrosis factor-␣ and interferon-␥ expression, whereas it decreased interleukin (IL)-2 expression. Sepiapterin did not change total iNOS protein or monomer levels, or plasma and tissue NO metabolites levels. It is concluded that the mechanism(s) of antirejection are due in part to decreased apoptosis, protein nitration, and oxidation of cardiomyocytes, which seems to be mediated at the immune level by limiting inflammatory cell infiltration via decreased IL-2-mediated T-lymphocyte expansion.

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GTP Cyclohydrolase I Is Coinduced in Hepatocytes Stimulated to Produce Nitric Oxide

Cited by 23 publications

References 74 publications

Monomeric inducible nitric oxide synthase localizes to peroxisomes in hepatocytes

Monomeric inducible nitric oxide synthase localizes to peroxisomes in hepatocytes

Altered Plasma Versus Vascular Biopterins in Human Atherosclerosis Reveal Relationships Between Endothelial Nitric Oxide Synthase Coupling, Endothelial Function, and Inflammation

Sepiapterin Decreases Acute Rejection and Apoptosis in Cardiac Transplants Independently of Changes in Nitric Oxide and Inducible Nitric-Oxide Synthase Dimerization

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