TETRAHYDROBIOPTERIN AND GTP CYCLOHYDROLASE I IN A RAT MODEL OF ENDOTOXIC SHOCK: RELATION TO NITRIC OXIDE SYNTHESIS

Transcription

1 Experimental Physiology (1996), 81, Printed in Great Britain TETRAHYDROBIOPTERIN AND GTP CYCLOHYDROLASE I IN A RAT MODEL OF ENDOTOXIC SHOCK: RELATION TO NITRIC OXIDE SYNTHESIS Y. HATTORI, N. NAKANISHI*, K. KASAI, Y. MURAKAMI AND S. SHIMODA Department of Endocrinology, Dokkyo University School of Medicine, Mibu, Tochigi and *Department ofbiochemistry, Meikai University School ofdentistry, Sakado, Saitama, Japan (MANUSCRIPT RECEIVED 5 FEBRUARY 1996, ACCEPTED 20 MARCH 1996) SUMMARY Induction of the inducible isoform of nitric oxide synthase (inos) in various types of cells is implicated as the cause of septic shock. We evaluated the concentration of tetrahydrobiopterin (BH4), a cofactor of NOS, in plasma and various other tissues of rats treated with lipopolysaccharide (LPS; 10 mg/kg i.v.). The activity of GTP cyclohydrolase I (GTPCH), the first and rate-limiting enzyme in the de novo synthesis of BH4, in rat tissues was also determined. Three hours after administration of LPS, rats showed plasma levels of BH4 and NOx (NO3 and NO2-) that were elevated by 137 and 206 %, respectively. GTPCH was expressed in liver and, to a lesser extent, in the lung, heart and kidney of control rats. In control rats, although a high concentration of BH4 was detected in the liver, its level was lower in lung, heart, kidney and aorta. Three hours after LPS administration, a significant increase in BH4 concentration and/or GTPCH activity was observed in all tissues examined except the liver. Our results demonstrate that the de novo synthesis of BH4 is upregulated by LPS in the rat in vivo, which may, at least in part, account for the increases in plasma level and tissue concentration of BH4 after the administration of LPS. INTRODUCTION The overproduction of nitric oxide (NO) by an inducible isoform of NO synthase (inos) within the vascular wall is involved in the profound hypotension and resistance to vasoconstrictor agents that characterize endotoxic shock. The release of NO is enhanced in rats treated with lipopolysaccharide (LPS), the main component of bacterial endotoxin; the widespread induction of inos has been well documented under these conditions (Thiemermann & Vane, 1990; Knowles, Salter, Brooks & Moncada, 1990; Szabo, Mitchel, Thiemermann & Vane, 1993). Since tetrahydrobiopterin (BH4) appears to be an essential cofactor for NO formation (Nathan, 1992), inhibition of its biosynthesis represents a feasible therapeutic target (Gross & Levi, 1992; Schoedon, Schneemann, Hofer, Guerrero, Blau & Schaffner, 1993; Muhl & Pfeilschifter, 1994; Nakayama, Geller, Di Silvio, Bloomgarden, Davies, Pitt, Hatakeyama, Kagamiyama, Simmons & Billiar, 1994). LPS causes a rise in BH4 levels, as well as the induction of inos in vascular smooth muscle (VSM) cells of the rat (Gross & Levi, 1992). The synthesis of BH4 occurs via two distinct pathways: a de novo synthetic pathway that uses guanosine triphosphate (GTP) as a precursor and a salvage pathway for pre-existing dihydropterins (Nichol, Smith & Duch, 1985). GTP cyclohydrolase I (GTPCH), the first and rate-limiting enzyme in the de novo synthesis of BH4, causes the formation of dihydroneopterin 1473

2 666 Y. HATTORI AND OTHERS triphosphate from GTP. Dihydroneopterin triphosphate is subsequently metabolized to BH4 by 6-pyruvoyltetrahydropterin synthase and sepiapterin reductase, with the formation of tetrahydropterin intermediates. The de novo synthesis of BH4 is required for the formation of NO by LPS-stimulated VSM, in which the induction of GTPCH mrna precedes BH4 synthesis (Gross & Levi, 1992; Hattori & Gross, 1993). We recently demonstrated the increased levels of GTPCH mrna, as well as of inos mrna, in the tissues of a model based on LPS injection into rats (Hattori, Oka, Kasai, Nakanishi & Shimoda, 1995a). This may be a mechanism for the increase in BH4 synthesis in endotoxic shock. However, little is known about the alterations in the biosynthesis of BH4 under these conditions. The present study investigated whether LPS administration to rats affects plasma levels or the tissue concentration of BH4 and, if so, whether the effect is due to a change in GTPCH activity. METHODS Animal treatment Male Wistar rats, weighing g, were anaesthetized (with thiopentone sodium; 120 mg/kg i.p.; supplementary doses given when necessary) and injected intravenously with either LPS (E. coli type, Serotype 0111: B4; 10 mg/kg; Sigma) or an equal volume (1 ml/kg) of saline for the endotoxin-treated and control groups, respectively. Three hours later, the animals were killed by exsanguination and various organs were removed, frozen in liquid nitrogen, and stored at -70 C until assayed. NO, (nitrate and nitrite) assay Concentrations of NOX in rat plasma were determined by an autoanalyser (TCI-NOx 1000; Tokyo Kasei Kogyo Co., Tokyo, Japan). Diluted samples were passed through a copperized cadmium reduction column in which nitrate was reduced to nitrite, which reacts with Griess reagent (I Oo sulphanilamide and 011% naphthylethylenediamide in 5 % HCI) to form a purple azo dye. Absorbance at 540 nm was detected by a flow-through visible spectrophotometer. Biopterin assay Biopterin (tetrahydrobiopterin and more oxidized species) was measuied essentially as described by Fukushima & Nixon (1980). Samples of rat tissue in 50 mm Tris-HCI (ph 7 8) were homogenized at 0 C in a glass-teflon homogenizer and then centrifuged for 20 min at 12(000g. The supernatant of the tissue homogenate or a rat plasma sample was treated with 0.2 M perchloric acid and oxidized by exposure to 02 %0 2, and 0 4 % KI in 0-2 M perchloric acid, for l h at room temperature in the dark. Ascorbate (2 %) was added to remove residual free 12 and the mixture was centrifuged for 10 min at 10000g. Biopterin ill the supernatant was quantitated by C18 reversed-phase high-performance liquid chromlatography (HPLC) with fluorescence detection; authentic BH4 ((6R)-5,6,7,8-tetrahydrobiopterin; Dr Schircks Co., Jona, Switzerland) was used as a standard. GTPCH activity The activity of GTPCH was assayed as previously described (Nakanishi, Ozawa, Iwanaga, Akatsuka, Sawada, Asaumi, Hasegawa & Yamada, 1990). Briefly, dihydroneopterin triphosphate, the product of the GTPCH reaction, wais oxidized to neopterin triphosphate, which was then dephosphorylated by alkaline phosphatase to form neopterin (NP). NP content was measured by HPLC as the activity of GTPCH in the supernat,ant of each tissue homogenate. Materials All chemicals were purchased from Sigma Chemical Co. (St Louis, MO, USA) unless other-wise stated.

3 NITRIC OXIDE IN ENDOTOXIC SHOCK 667 Statistical evaluation Values aire expressed as means + S.D. Student's unpaired t test was used to assess the statistical significance of differences. A P value less than 0.05 was considered statistically significant. RESULTS In anaesthetized rats intravenous administration of E. coli endotoxin induces a rapid (within 5 min) hypotension. Blood pressure then remains below the baseline value for several hours. In this model, prolonged periods of endotoxaemia (3 h) result in a significant induction of calcium-independent (inducible) NOS activity, which contributes to the vascular hyporeactivity to noradrenaline and the circulatory failure seen after prolonged periods of endotoxic shock (Szabo et al. 1993; Hattori, Szabo, Gross, Thiemermann & Vane, 1995b). Thus, we first evaluated rat plasma concentrations of NOX (nitrate and nitrite) and biopterin (BH4 and more oxidized forms) 3 h after intravenous injection of LPS (10 mg/kg; Fig. 1). Plasma NOx concentrations were significantly higher in the LPS-treated vs. the control rats (Fig. 1 A). Plasma biopterin levels in control rats averaged UM and ranged from 49 3 to 66.3 AM. In contrast, the systemic administration of LPS to anaesthetized rats for 3 h produced a substantial increase in plasma concentrations of biopterin, which averaged am (Fig. I B). The concentration of biopterin was higher in the lung and kidney than in the heart and aorta of control animals, but it was even higher in the tissues of animals treated with LPS than in the corresponding tissues of control animals (Fig. 2). The concentration of biopterin in the liver of control animals was much higher than in other tissues and was unaffected by LPS administration. Extracts of rat liver contained considerable GTPCH activity that was unaffected by administering LPS. Extracts of lung, heart and kidney also contained low levels of GTPCH activity; a significant increase in enzyme activity was observed in the lung and kidney after LPS administration (Fig. 3). However, GTPCH activity in tissues other than the liver was far 200( Control LPS Control LPS Fig. 1. ElIects ofl LPS treaitmiient in vivo on the plasma- concentrations of NO. and hiopterin in rats. The concentrations of NO, (nitraite plus nitrite; A) and biopterin (BH4 and more oxidized species; B) in plasma of' riats 3 h alter intravcoiolis administraition of saline (control) or LPS (10 tng/k-g) were measured. Data are mcans + S.D. (o1 = 9). P < 0-01 I's. control rats.

4 668 Y. HATTORI AND OTHERS 12 10m 8 E Lun HeatA Lung Heart Liver Kidney Aorta Fig. 2. Eflects of LPS treattment in v'ivo on the concentration of BH4 in lung, heart, liver, kidney and aorta of rats. Samples were prepared from tissues of rats 3 h after intravenous addministration of saline (controls; D2) or LPS (10 mg/kg; 1), and were measured by HPLC with authentic BH4 used as a standard. Data are means + S.D. (n = 6). P < 0(05 and ** P < 01 's. control rats. lower, even after LPS dosage, than that in liver. was below the limit of detection of the assay. GTPCH activity in the aorta as a whole tissue DISCUSSION The results of this study confirmed earlier findings that prolonged periods of endotoxaemia (3 h) lead to the induction of a calcium-independent NOS (inos) activity that contributes to the profound hypotension and resistance to vasoconstrictor agents in a model based on injection of LPS into rats (Szabo et al. 1993; Hattori et al b). Although inos activity in all tissues studied in control rats was very low, a substantial induction of inos activity occurred in the lung, liver, spleen, mesentery, heart and aorta, most pronouncedly in the lung (Szabo et al. 1993; Hattori et al b). In contrast, GTPCH was expressed in liver and, to a much lesser extent, in the lung, heart and kidney of control rats. A substantial amount of BH4 was detected in the liver of control rats; other tissues contained lower levels of BH4. An important finding was that a significant increase in BH4 concentration and/or GTPCH activity was observed in all but the liver after LPS injection. Plasma levels of biopterin were significantly increased in the rats injected with LPS. The de novo synthesis of BH4 appeared to be upregulated by LPS in the rats in vivo consistent with our previous findings that LPS administration in vivo induces the tissue expression of GTPCH mrna (Hattori et al a). This fact may, at least in part, account for the increase in the plasma and tissue concentrations of BH4 in the LPS-

5 NITRIC OXIDE IN ENDOTOXIC SHOCK U~~~~~ Lung Heart Liver Kidney Aorta Fig. 3. Effects of LPS treatment in vivo on activity of GTPCH in lung, heart, liver, kidney and aorta of rats. Samples were prepared from tissues of rats 3 h after intravenous administration of saline (controls; O) or LPS (10 mg/kg; 0), and were assayed by measuring the neopterin (NP) formed as a result of the GTPCH reaction. Note that the aorta was not dissected to determine activities in smooth muscle and/or endothelial cells, but a whole section of aorta was used after fat tissues surrounding it had been trimmed off. Data are means + S.D. (n = 4). * P < 0-05 and ** P < 0-01 vs. control rats. injected rats. Werner-Felmayer and co-workers showed that a single dose of LPS given to rats increases the activity of GTPCH and levels of biopterin in several tissues, including the cerebellum and adrenal glands (Werner-Felmayer, Prast, Werner, Philippu & Wachter, 1993). They suggested that an increase in BH4 synthesis and, therefore, an increase in BH4 levels after LPS dosage might act on NO formation, as well as on the biosynthesis of catecholamines and serotonin; the key enzyme for that biosynthesis requires BH4 as a cofactor. This view is supported, with respect to septic circulatory failure, by the increase in GTPCH activity and/or BH4 levels in lung, the tissue in which inos induction is most marked (Szabo et al. 1993; Hattori et al b) and in cardiovascular tissues, as well as in plasma BH4 levels, found in the present study. Interestingly, Gross recently used inhibitors of BH4 synthesis to show that BH4 availability limits the LPS-induced killing and NO synthesis in rats (Gross, 1995). He found that the protective effect arose specifically from a blocking of dihydropterin salvage, and thus of BH4 synthesis, rather than from an inhibition of GTPCH in de novo synthesis of BH4. This finding suggests that the NO that mediates vascular dysfunction arises mainly in cells that produce BH4 by the salvage of dihydrobiopterin rather than by de novo biosynthesis (Gross, 1995). Conversely, this view may be supported by recent reports showing that BH4 is a secretory product of vascular endothelial cells (Schaffner, Blau, Schneemann, Steurer, Edgell & Schoedon, 1994; Walter, Schaffner, Blau, Kierat & Schoedon, 1994). While LPS induces de

6 670 Y. HATTORI AND OTHERS novo synthesis of BH4 in VSM, an event that is essential for the induction of NO release- (Gross & Levi, 1992), BH4 produced by LPS-activated VSM was actively secreted instead of being effectively kept within the cells (Hattori, Nakanishi, Kasai, Shimoda & Gross, 1996). These findings suggest that LPS increases the in vivo biosynthesis and secretion of BH4, which may, in turn, serve as a transcellular metabolite that can be produced by one cell type for another. In the present study, we demonstrated that the de novo synthesis of BH4 is upregulated by LPS in the rat in vivo. This may, at least in part, account for the increases in plasma level and tissue concentration of BH4 after the administrtion of LPS; however, BH4 production by the salvage of dihydrobiopterin may also occur under these conditions. Interrupting the availability of BH4 may be a useful therapeutic strategy for selectively limiting the overproduction of NO in septic shock. This work was supported in part by a grant from the Japan Private School Promotion Foundation. REFERENCES FUKUSHIMA, T. & NIXON, J. C. (1980). Analysis of reduced forms of biopterin in biological tissues and fluids. Analytical Biochemistry 102, GROSS, S. S. (1995). Tetrahydrobiopterin availability limits LPS-induced killing and NO synthesis in rats. In Fourth International Meeting: Biology of Nitric Oxide. Endothelium 3 (suppl.), s1 18. GROSS, S. S. & LEVI, R. (1992). Tetrahydrobiopterin synthesis: An absolute requirement for cytokineinduced nitric oxide generation by vascular smooth muscle. Journal of Biological Chemistry 267, HATrORI, Y. & GROSS, S. S. (1993). GTP cyclohydrolase I mrna is induced by LPS in vascular smooth muscle: characterization, sequence and relationship to nitric oxide synthase. Biochemical and Biophysical Research Communications 195, HATTORI, Y., NAKANISHI, N., KASAI, K., SHIMODA, S. & GROSS, S. S. (1996). Pyrrolidine dithiocarbamate inhibits immunostimulant-induced tetrahydrobiopterin synthesis in rat vascular smooth muscle. European Journal of Pharmacology 296, HA7rORI, Y., OKA, M., KASAI, K., NAKANISHI, N. & SHIMODA, S. (1995 a). Lipopolysaccharide treatment in vivo induces tissue expression of GTP cyclohydrolase I mrna. FEBS Letters 368, HATTORI, Y., SZABO, C., GROSS, S. S., THIEMERMANN, C. & VANE, J. R. (1995 b). Lipid A and the lipid A analogue anti-tumour compound ONO-4007 induce nitric oxide synthase in vitro and in vivo. European Journal of Pharmacology 291, KNOWLES, R. G., SALTER, M., BROOKS, S. L. & MONCADA, S. (1990). Anti-inflammnatory glucocorticoids inhibit the induction by endotoxin of nitric oxide synthase in the lung, liver, and aorta of' the rat. Biochemical and Biophysical Research Communications 172, MUHL, H. & PFEILSCHIFTER, J. (1994). Tetrahydrobiopterin is a limiting factor of nitric oxide generation in interleukin 1/3-stimulated rat glomerular mesangial cells. Kidney International 46, NAKANISHI, N., OZAWA, S., IWANAGA, M., AKATSUKA, I., SAWADA, E., ASAUMI, R., HASEGAWA, H. & YAMADA, S. (1990). Simultaneous determination of GTP cyclohydrolase activity and biopterin content in pheochromocytoma PC I2h cells. Journal of Meikai University School of Dentistry 19, NAKAYAMA, D. K., GELLER, D. A., Di SILvIo, M., BLOOMGARDEN, G., DAvIES, P., PITr, B. R., HATAKEYAMA, K., KAGIMIYAMA, H., SIMMONS, R. L. & BILLIAR, T. R. (1994). Tetrahydrobiopterin synthesis and inducible nitric oxide production in pulmonary artery smooth muscle. Amer-ican Journal of Physiology 266, L NATHAN, C. (1992). Nitric oxide as a secretory product of mammalian cells. FASEB.Journal 6, NICHOL, C. A., SMITH, G. K. & DUCH, D. S. (1985). Biosynthesis and metabolismii of tetr.ahydrobiopterin and molybdopterin. Annual Review of Biochemistry 54,

7 NITRIC OXIDE IN ENDOTOXIC SHOCK 671 SCHAFFNER, A., BLAU, N., SCHNEEMANN, M., STEURER, J., EDGELL, C. J. S. & SCHOEDON, G. (1994). Tetrahydrobiopterin as another EDRF in man. Biochemical and Biophysical Research Communications 205, SCHOEDON, G., SCHNEEMANN, M., HOFER, S., GUERRERO, L., BLAU, N. & SCHAFFNER, A. (1993). Regulation of the L-arginine-dependent and tetrahydrobiopterin-dependent biosynthesis of nitric oxide in murine macrophages. European Journal of Biochemistry 213, SZABO, C., MITCHEL, J. A., THIEMERMANN, C. & VANE, J. R. (1993). Nitric oxide-mediated hyporeactivity to noradrenaline precedes the induction of nitric oxide synthase in endotoxin shock. British Journal of Pharmacology 108, THIEMERMANN, C. & VANE, J. R. (1990). Inhibition of nitric oxide synthesis reduces the hypotension induced by bacterial lipopolysaccharides in the rat in vivo. European Journal of Pharmacology 182, WALTER, R., SCHAFFNER, A., BLAU, N., KIERAT, L. & SCHOEDON, G. (1994). Tetrahydrobiopterin is a secretory product of murine vascular endothelial cells. Biochemical and Biophysical Research Communications 203, WERNER-FELMAYER, G., PRAST, H., WERNER, E. R., PHILIPPU, A. & WACHTER, H. (1 993). Induction of GTP cyclohydrolase I by lipopolysaccharide in the rat. FEBS Letters 322,