Abstract
Our previous studies have demonstrated that nitric oxide (NO) leads to nitric oxide synthase (NOS) uncoupling and an increase in NOSderived superoxide. However, the cause of this uncoupling has not been adequately resolved. The pteridine cofactor tetrahydrobiopterin (BH4) is a critical determinant of endothelial NOS (eNOS) activity and coupling, and GTP cyclohydrolase I (GCH1) is the rate-limiting enzyme in its generation. Thus the initial purpose of this study was to determine whether decreases in BH 4 could underlie, at least in part, the NO-mediated uncoupling of eNOS we have observed both in vitro and in vivo. Initially we evaluated the effect of inhaled NO levels on GCH1 expression and BH4 levels in the intact lamb. Contrary to our hypothesis, we found that there was a significant increase in both plasma BH4 levels and peripheral lung GCH1 protein levels. Furthermore, in vitro, we found that exposure to the NO donor spermine NONOate (SPNONO) led to an increase in GCH1 protein and BH4 levels in both COS-7 and pulmonary arterial endothelial cells. However, SPNONO treatment also caused a significant increase in phospho-cAMP response element binding protein (CREB) levels, as detected by Western blot analysis, and significantly increased cAMP levels, as detected by enzyme immunoassay. Furthermore, utilizing GCH1 promoter fragments fused to a luciferase reporter gene, we found that GCH1 promoter activity was enhanced by SPNONO in a CREB-dependent manner, and electromobility shift assays revealed an NO-dependent increase in the nuclear binding of CREB. These data suggest that NO increases BH4 levels through a cAMP/CREBmediated increase in GCH1 transcription and that the eNOS uncoupling associated with exogenous NO does not involved reduced BH4 levels.
| Original language | English (US) |
|---|---|
| Pages (from-to) | L309-L317 |
| Journal | American Journal of Physiology - Lung Cellular and Molecular Physiology |
| Volume | 297 |
| Issue number | 2 |
| DOIs | |
| State | Published - Aug 1 2009 |
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Keywords
- Adenosine 3′,5′-cyclic monophosphate response element binding protein
- Cell signaling
- Rebound pulmonary hypertension
- Tetrahydrobiopterin
ASJC Scopus subject areas
- Physiology
- Pulmonary and Respiratory Medicine
- Physiology (medical)
- Cell Biology
Cite this
GTP cyclohydrolase I expression is regulated by nitric oxide : Role of cyclic AMP. / Kumar, Sanjiv; Sun, Xutong; Sharma, Shruti; Aggarwal, Saurabh; Ravi, Kandasamy; Fineman, Jeffery R.; Black, Stephen M.
In: American Journal of Physiology - Lung Cellular and Molecular Physiology, Vol. 297, No. 2, 01.08.2009, p. L309-L317.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - GTP cyclohydrolase I expression is regulated by nitric oxide
T2 - Role of cyclic AMP
AU - Kumar, Sanjiv
AU - Sun, Xutong
AU - Sharma, Shruti
AU - Aggarwal, Saurabh
AU - Ravi, Kandasamy
AU - Fineman, Jeffery R.
AU - Black, Stephen M.
PY - 2009/8/1
Y1 - 2009/8/1
N2 - Our previous studies have demonstrated that nitric oxide (NO) leads to nitric oxide synthase (NOS) uncoupling and an increase in NOSderived superoxide. However, the cause of this uncoupling has not been adequately resolved. The pteridine cofactor tetrahydrobiopterin (BH4) is a critical determinant of endothelial NOS (eNOS) activity and coupling, and GTP cyclohydrolase I (GCH1) is the rate-limiting enzyme in its generation. Thus the initial purpose of this study was to determine whether decreases in BH 4 could underlie, at least in part, the NO-mediated uncoupling of eNOS we have observed both in vitro and in vivo. Initially we evaluated the effect of inhaled NO levels on GCH1 expression and BH4 levels in the intact lamb. Contrary to our hypothesis, we found that there was a significant increase in both plasma BH4 levels and peripheral lung GCH1 protein levels. Furthermore, in vitro, we found that exposure to the NO donor spermine NONOate (SPNONO) led to an increase in GCH1 protein and BH4 levels in both COS-7 and pulmonary arterial endothelial cells. However, SPNONO treatment also caused a significant increase in phospho-cAMP response element binding protein (CREB) levels, as detected by Western blot analysis, and significantly increased cAMP levels, as detected by enzyme immunoassay. Furthermore, utilizing GCH1 promoter fragments fused to a luciferase reporter gene, we found that GCH1 promoter activity was enhanced by SPNONO in a CREB-dependent manner, and electromobility shift assays revealed an NO-dependent increase in the nuclear binding of CREB. These data suggest that NO increases BH4 levels through a cAMP/CREBmediated increase in GCH1 transcription and that the eNOS uncoupling associated with exogenous NO does not involved reduced BH4 levels.
AB - Our previous studies have demonstrated that nitric oxide (NO) leads to nitric oxide synthase (NOS) uncoupling and an increase in NOSderived superoxide. However, the cause of this uncoupling has not been adequately resolved. The pteridine cofactor tetrahydrobiopterin (BH4) is a critical determinant of endothelial NOS (eNOS) activity and coupling, and GTP cyclohydrolase I (GCH1) is the rate-limiting enzyme in its generation. Thus the initial purpose of this study was to determine whether decreases in BH 4 could underlie, at least in part, the NO-mediated uncoupling of eNOS we have observed both in vitro and in vivo. Initially we evaluated the effect of inhaled NO levels on GCH1 expression and BH4 levels in the intact lamb. Contrary to our hypothesis, we found that there was a significant increase in both plasma BH4 levels and peripheral lung GCH1 protein levels. Furthermore, in vitro, we found that exposure to the NO donor spermine NONOate (SPNONO) led to an increase in GCH1 protein and BH4 levels in both COS-7 and pulmonary arterial endothelial cells. However, SPNONO treatment also caused a significant increase in phospho-cAMP response element binding protein (CREB) levels, as detected by Western blot analysis, and significantly increased cAMP levels, as detected by enzyme immunoassay. Furthermore, utilizing GCH1 promoter fragments fused to a luciferase reporter gene, we found that GCH1 promoter activity was enhanced by SPNONO in a CREB-dependent manner, and electromobility shift assays revealed an NO-dependent increase in the nuclear binding of CREB. These data suggest that NO increases BH4 levels through a cAMP/CREBmediated increase in GCH1 transcription and that the eNOS uncoupling associated with exogenous NO does not involved reduced BH4 levels.
KW - Adenosine 3′,5′-cyclic monophosphate response element binding protein
KW - Cell signaling
KW - Rebound pulmonary hypertension
KW - Tetrahydrobiopterin
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UR - http://www.scopus.com/inward/citedby.url?scp=67651221996&partnerID=8YFLogxK
U2 - 10.1152/ajplung.90538.2008
DO - 10.1152/ajplung.90538.2008
M3 - Article
C2 - 19447893
AN - SCOPUS:67651221996
VL - 297
SP - L309-L317
JO - American Journal of Physiology - Heart and Circulatory Physiology
JF - American Journal of Physiology - Heart and Circulatory Physiology
SN - 0363-6135
IS - 2
ER -