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© Copyright 2004 The British Pharmacological Society

013P GKT, University of London
Winter Meeting December 2003

GTP cyclohydrolase 1 feedback regulatory protein over expression attenuates iNOS mediated nitric oxide generation in isolated murine endothelial cells


Manasi Nandi, Patrick Vallance and James Leiper. British Heart Foundation Laboratories, The Rayne Institute, University College London, 5 University Street, London WC1E 6JJ.

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Nandi M
Vallance P
Leiper J

GTP-cyclohydrolase 1 (GTP-CH1) catalyses the first and rate limiting step for the de novo production of tetrahydrobiopterin (BH4), an essential cofactor for nitric oxide synthase (NOS).1 GTP-CH1 is inhibited by BH4 through protein:protein interactions with GTP cyclohydrolase I feedback regulatory protein (GFRP).2 Cytokine stimulated induction of inducible NOS (iNOS) is associated with an upregulation of GTP-CH1 and BH4 and a decrease in GFRP3 but little is known about whether GFRP expression affects BH4 generation and nitric oxide (NO) pathways.

We have over-expressed N-Myc tagged recombinant human GFRP in the murine endothelial cell line sEnd 14 and have isolated GFRP over-expressing clones and empty vector transfected clones. Transfection of N-Myc-GFRP was confirmed by western blotting for the Myc epitope in cell lysates. Nitrite production, as measured by Greiss assay, was subsequently used as an indicator of nitric oxide (NO) production from these cells, basally and following cytokine stimulation (20 hours). Western blotting was used to assess iNOS protein expression.

Myc-tagged GFRP protein was present in all transfected cells and levels varied between individual clones. Basal nitrite production was detectable by Greiss assay and there was no significant difference between N-Myc GFRP over-expressing and empty vector transfected control cells.However, following cytokine stimulation, nitrite production was significantly attenuated in all GFRP overexpressing cell lines when compared to the empty vector control cells (Table1) and the extent of attenuation correlated with the expression level. As expected, there was no iNOS protein expression basally in all cell lysates but after cytokine stimulation iNOS was detectable in all cell lines. Levels of iNOS were reduced in GFRP over expressing cell lines when compared to empty vector control lines.

Cell line
Pre cytokine stimulation
Post cytokine stimulation
Nitrite†
SEM
Nitrite†
SEM
Control 1
7.36
3.03
100.00
0
Control 2
9.65
3.79
132.39
8.14
GFRP 1
3.80
1.10
17.93*
5.85
GFRP 2
10.85
5.36
53.67*
11.9
GFRP 3
7.32
2.51
63.86*
17.4
GFRP 4
9.20
3.48
39.27*
13.4

Table 1 Nitrite production pre and post cytokine treatment in GFRP overexpressing and empty vector control cells calculated as a percentage of Control 1†. *p=<0.05 unpaired T-test. Values are significantly different to cytokine stimulated nitrite production in control cells. N=5

GFRP over-expression inhibits NO generation from iNOS an effect which may be due to iNOS protein becoming rapidly degraded in the absence of BH4.

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