Blau, Nenad

Kunz, Dieter

Pfeilschifter, Josef

Plüss, Christoph

Redweik, Udo

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Pluss et al. : GTP '::- c;e,hydrolase I in mesangial cells Pteridines Vol. 6,1995, pp. 129-131 Short Communication Co-ordinate Expression of GTP Cyclohydrolase I and Inducible Nitric Oxide Synthase in Rat Mesangial Cells Christoph Pliiss#, Dieter Kunz#, Udo Redweikt, Nenad Blau t and Josef Pfeilschifter#§ # Department of Pharmacology, Biozentrum, University of Basel, Basel, Switzerland t Division of Cinical Chemistry, Department of Pediatrics, University of Zurich, Zurich, Switzerland (Received June 15, 1995) Introduction The synthesis of NO from L-arginine by NO syn-thase (NOS) is an important intracellular and in-tercellular signalling pathway originally discovered in macrophages and endothelial cells (1). Molecular cloning and sequence analyses revealed the exist-ence of at least 3 main types of NOS isoforms. The brain and endothelial enzmes are constitutively ex-pressed and are regulated by Ca2+ -mobilizing ago-nists that trigger an increase in intracellular free Ca2+, which binds to calmodulin, and thus activates NOS. A third type of NOS has been cloned from murine macrophages and is not constitutively exp-ressed, but is induced by y-interferon and lipopo-lysaccharide, and is Ca2+ -independent. The macro-phage NOS is regulated on a transcriptional level and once induced is active for hours and days. Not surprisingly, therefore, the inducible macrophage enzyme produces amounts of NO that are several hundred times higher than the ones produced by the constitutive endothelial cell and brain enzymes which are only active during the short periods of elevated intracellular Ca2+ observed after exposure of cells to Ca2+ -mobilizing agonists (1). We have shown that cytokines, such as interleu-kin 1 (IL-I) and tumour necrosis factor a (TNFa), ~ Author to whom correspondence should be addressed. Prof. Josef M. Pfeilschifter: Department of Pharmacology Biozentrum, University of Basel, Kingelbergstrasse 70, CH-4056 Basel, Switzeriand, Tel: +41/612672223, Fax: +41/61 267 22 08 Pteridines I Vol. 6 I No.3 induce a macrophage type of NOS in mesangial cells with subsequent elevation of cellular cGMP concentrations (3,4). The excessive formation of NO and cGMP in mesangial cells not only alters cor-ractile responses of the cells, but may also cause tissue injury and thus contribute to the pathogenesis of certain forms of glomerulonephritis. Inducible NOS is expressed in renal mesangial cells in res-ponse to two principal classes of activating signals that interact in a synergistic fashion. These two groups of activators comprise inflammatory cyto-kines such as IL-113 or TNFa and agents that elevate cellular levels of cAMP (6,7). Tetrahydrobiopterin (BH4) is an essential cofactor for inducible NOS and its biosynthesis occurs via two distinct pathways: a de novo pathway which uses GTP as a substrate and a salvage pathway using the intracellular pool of preexisting dihydro-pterins (8). Cytokines like y-interferon or TNFa have been reported to potently stimulate the de novo synthesis of BH4 in human and murine macropha-ges, fibroblasts and endothelial cells (8). This in-creased production of BH4 may be requried in order to ensure a sufficient supply of cofactor for long-term synthesis of NO via the inducible NOS (8). We have shown previously that mesangial cells predo-minantly synthesize BH4 by de novo synthesis from GTP. BH4 synthesis is an absolute requirement for, and limits IL-I 13 induction of NO synthesis in mesangial cells (9). Since GTP cyclohydrolase I (GTP-CH) is the rate-limiting enzyme for BH4 syn-thesis in mesangial cells (9), we investigated whe-ther the identified stimulators of inducible NOS 130 expression in mesangial cells co-ordinately induce GTP-CH expression. Materials and Methods Rat glomerular mesangial cells were cultured as described previously (3,4). The GTP-CH cDNA probe was constructed by subcloning a 189-nuceo-tide fragment (NcollNotl) from the cDNA clone GTP-CH (a gift from Dr. Hatakeyama, Osaka, Ja-pan) into the pSPTBM2l transcription vector (Boe-rhinger Mannheim). pGTP-CH corresponds to nu-cleotide 1-189 of the rat GTP-CH eDNA sequence (10). GTP-CH mRNA was determined by nuclease protection assay. 20 Ilg of total RNA was hvbridized at 45't for 16h with 1 fmol of GTP-CH antisense probe transcribed from the plasmid pGTP-CH con-taining 19 base pairs of GTP-CH coding sequence and 0.5 fmol of 32 P-Iabelled GAPDH antisense probe transcribed from the pasmid pGAPDH con-taining 159 base pairs of GAPDH coding sequence. Nonhybridized single-stranded radiolabeled probe was then digested with 25 units of T1 and 0.5 ng of A ribonuclease for Ih at 30't and then protein digested with 50 fJg of proteinase K for 30 min at 37°C. Samples were then electrophoresed in dena-turing 8% polyacryamide gels containing 8M urea. Gels were dried and quantified by phosphor ima-ging. GTP-CH mRNA levels were expressed as % of control. GTP-CH activity was assessed as previously de-scribed (11). Mesangial cells were disrupted by rapid freeze-thawing, extracts were centrifuged at 10,000 X g for 10 min and aliquots of the supernatant were freed from low molecular weight compounds by chromatography on sephadex G-25. The sephadex G-25 eluate (5-6 mg of cell protein) was incubated with 2 mM GTP for 90 min at 37°C in a total volume of 100 fJl. The dihydroneopterin triphosphate thus formed was then oxidized to neopterin triphosphate with iodine s01ution, containing 2.5% (w/v) 12 , 5% (w/v) KI, in 20 mM HCI, Excessive iodine was de-stroyed with 0.5% (w/v) ascorbic acid. For HPLC analysis, the sample was applied to a Partisil-1O SAX column (120 mm X 6 mm). Neopterin tri-phosphate was eluted with a solvent of 250 mM NH.H 2PO., 250 mM KC1, and 50 mM Na.P 20 7 , pH 5.9, at a flow rate of 1 ml/ min, and the products were detected fluorometrically (excitation 350 nm; emission 450 nm). Results Incubation of mesangial cells for 24 h with IL-ll3 Pteridines / Vol. 6 / No. 3 Pluss et al. : GTP cyclohydrolase I in mesangial cells e c o 2000 (J 1500 "0 ~ 1000 a: E :c u 500 a.. t-o (5 1400 .g 1200 0 u "0 1000 ~ 800 « z a: 600 E :c 400 u "- LUU I-" e 3000 c: o u ~ 2000 « z a: E :c 1000 u "-I-" Time (h) (a) Time (h) (b) Time (h) (c) 12 24 12 24 12 24 Figure 1. Time course of induction of GTP-CH mRNA in mesangial cells Mesangial cells were treated with IL-I ~(2 nM) (A) Bt2 cAMP (O.SmM), (B) or a combination of IL-I~ (2 nM) plus forskolin(lO 11M) (C) for the indicated time per-iods. RNA was extracted and GTP-CH mRNA was quan-tified by nuclease protection assay as described in Materials and Methods. (2 nM) or N 6 , 0-2'-dibutyryladenosine 3',5'-phos-phate (Bt2 cAMP, 0.5 mM) resulted in a 1.6-fold and 1.2-fold increase in GTP-CH activity, respectivly. Combination ofIL-ll3 and forskolin (10 fJM), a com-pound which increases cellular levels of cAMP by directly activating the catalytic moiety of adenylate cyclase, revealed a strong synergy in terms of GTP-CH activation-(5.l-fold increase). Because GTP-CH  C. Pliiss et al. : GTP cyc1ohydrolase I in mesangial cells is the rate-limiting enzyme in BH. biosynthesis in mesangial cells (9), the changes in GTP-CH activity reported here likely reflect an increase in GTP-CH enzyme levels. As we do not have access to an an-tibody for quantitative Westemblot analysis we measured next GTP-CH mRNA steady-state levels in mesangial cells. With the cloning of a cDNA encoding rat GTP-CH (10) we were able to set up a highly sensitive and quantitative nuclease protec-tion assay for GTP-CH mRNA. As shown in Figure 1, exposure of mesangial cells to IL-I ~(A) or Bt2 cAMP (B) caused a marked increase in GTP-CH mRNA levels as early as lh after addition of the stimuli. Maximal effects were seen after 4h for IL-I~ and after lh for Bh cAMP. Stimulation of mesangial cells with a combination of IL-I ~ plus forskolin (C) reveals an additive induction profile. Discussion Previously, we reported that the inflammatory cytokines IL-I ~ and TNFa as well as agents that elevate cellular concentrations of cAMP are capable of inducing NOS expression in mesangial cells (6,7). In the present study, we obseved that IL-I~ and cAMP-induced NOS expression is associated with an increase in GTP-CH mRNA and GTP-CH ac-tivity. These data are compatible with the view that an increased de novo synthesis of GTP-CH is es-sential for optimal activity of inducible NOS in mesangial cells (9). Nuclear run-on transcription experiments suggested that IL-l ~ and cAMP syne-rgistically interact to incerase inducible NOS gene expression at the transcriptional level. Furthermore, cAMP exposure markedly prolonged the half-life of inducible NOS mRNA (7). Comparable experi-ments have to be performed to assess the exact mo-lecular mechanism of IL-l ~ and cAMP induction of GTP-CH in mesangial cell. However, an impor-tant difference between GTP-CH and inducible NOS regulation can already be derived from our present data (Figure lA-C). Whereas IL-l ~ and cAMP cause a strong synergistic induction of in-ducible NOS mRNA and activity (6,7) there is only an additive effect on GTP-CH mRNA which is however combined with a synergistic stimulation of GTP-CH activity. This may suggest that in addition to transcriptional regulation there is a prominent posttranscriptional or even posttranslational mo-131 dulation of GTP-CH expression and activity. This hypothesis is under evaluation in our laboratory. Acknowledgments This work was supported by Swiss National Science Foundation grant 31-33653.92. c.P. is sup-ported by a grant of the Krebsliga beider Basel. We thank Dr. K Hatakeyama for kindly providing the rat GTP-CH cDNA clone and Dr. Christiane Ror-dorf for the generous gift of recombinant human IL-II3. References 1. Knowles RG and Moncada S: Nitric oxide synthase in mammals. Biochem J. 1994; 298: 249-258. 2. Pfeilschifter J : Cross-talk between transmembrane sig-nalling systems: a prerequisIte for the delicate regulation of glomerular haemodynamics by mesangial cells. Eur. J. Clin. Invest 1989; 19: 347-361 3. Pfeilschifter J , Schwarzenbach H : Interleukin 1 and tu-mor necrosis factor stimulate cGMP in rat renal mesan-gial cells. FEBS Lett. 1990;273:185-187. 4. Pfeilschifter J, Rob P. Miilsch A, Fandrey J , Vosbeck K, Busse R : Interleukin 1 t3 and tumour necrosis factor a induce a macrophage-type of nitric oxide synthase in rat renal mesangial cells. Eur. J. Biochem 1992; 203: 251-255. 5. Pfeilschifter J, Kunz D ,-Muhl H : Nitric oxide: an inf-lammatory mediator of glomerular mesangial cells. Ne-phron 1993; 64: 518-525. . 6. Miihl H. Kunz D, Pfeilschifter J : Expression of nitric oxide svnthase in glomenular mesangial cells mediated by cAMP. Bf. J. Pharmacol 1994; 112: 1-8 7. Kunz D. Miihl H, Walker G, Pfeilschifter J : Two distinct pathways trigger the expression of inducible nitric oxide synthase in rat renal mesangial cells. Proc. Nati. Acad. Sci. USA 1994; 91:5387-5391. 8. Werner ER, Wemer-Felmayer G , Wachter H : Tetrahy-drobiopterin and cytokines. Proc. Soc. Exp. BioI. Med. 1993; 203 : 1-12. 9. Miihl H , Pfeilschifter J : Tetrahydrobiopterin is a limiting factor of nitric oxide generation in interleukin 1t3-sti-mulated rat glomerular mesangial cells. Kidney Int 1994; 46: 1302-1306. 10. Hatakeyama K, Inoue Y, Harada T, Kagamiyama H: Cloning and sequencing of cDNA encoding rat GTP-cyc1ohydrolase I . J. BioI. Chern. 1991; 266: 765-769. 11. Blau N, Niederwieser A: Guanosine triphosphate cy-c1ohydrolase I assay in Human and rat liver using high-performance liquid chromatography of neopterin phosphates and guanine nuc1eotides. Anal. Biochem. 1983; 128: 446-452. Pteridines / Vol. 6 / No.3 

Co-ordinate Expression of GTP Cyclohydrolase I and Inducible Nitric Oxide Synthase in Rat Mesangial Cells

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