Expressional Down-Regulation of Neuronal-Type Nitric Oxide Synthase I by Glucocorticoids in N1E-115 Neuroblastoma Cells
Abstract
Neuronal-type nitric oxide synthase (NOS I) is involved in ischemia-induced brain damage, and glucocorticoids have been reported to protect from brain damage. This prompted us to investigate if the activity or expression of NOS I was influenced by glucocorticoids. We used the murine neuroblastoma cell line N1E-115 as our experimental model. Short-term incubation (30 min) of the N1E-115 cells with dexamethasone (10 nm to 1 μm) or hydrocortisone (100 nm to 10 μm) did not change the enzymatic activity of NOS I. However, the glucocorticoids inhibited NOS I mRNA expression in a concentration-dependent fashion (down to 53.3 ± 2.5% of control). In time-course experiments with 100 nm dexamethasone, maximum down-regulation of NOS I mRNA was seen after 24 hr (55.6 ± 6.3% of control). Similar effects were seen with 10 μm hydrocortisone. The effect of 100 nm dexamethasone was completely reversed by 1 μm of the glucocorticoid receptor antagonist mifepristone. In experiments with actinomycin D (10 μg/ml), the half-life of the NOS I mRNA was determined to be approximately 12 hr and remained unchanged after glucocorticoid incubation. Nuclear run-on analyses indicated that the decrease in NOS I mRNA was the result of a glucocorticoid-induced inhibition of NOS I gene transcription. In Western blots, the 160-kDa NOS I protein band was down-regulated to 68.5 ± 8.4% of control after an incubation of the N1E-115 cells with 100 nm dexamethasone for 26 hr. Similarly, NO production was down-regulated to 57.8 ± 8.7% of control. These data demonstrate that glucocorticoids reduce the expression of NOS I without changing its activity.
Footnotes
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Send reprint requests to: Dr. Petra M. Schwarz, Department of Pharmacology, Johannes Gutenberg University, Obere Zahlbacher Strasse 67, 55101 Mainz, Germany. E-mail:petra.schwarz{at}uni-mainz.de
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This work was supported by Grants Fo 144/3–2 and SFB 553 (Project A1) from the Deutsche Forschungsgemeinschaft (Bonn, Germany) and by a grant from the Ministry of the Environment of the State of Rhineland-Palatinate (Mainz, Germany). This article is part of the thesis work of B.G.
- Abbreviations:
- NO
- nitric oxide
- NOS
- nitric oxide synthase
- NOS I
- neuronal-type nitric oxide synthase
- NOS II
- inducible-type nitric oxide synthase
- NOS III
- endothelial-type nitric oxide synthase
- DMEM
- Dulbecco’s modified Eagle’s medium
- DMSO
- dimethylsulfoxide
- PBS
- phosphate-buffered saline
- SSC
- saline-sodium citrate buffer
- TBS
- Tris-buffered saline
- nt
- nucleotide
- PIPES
- piperazine-N,N′-bis(2-ethanesulfonic acid)
- SDS
- sodium dodecyl sulfate
- CHAPS
- 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate
- HEPES
- 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
- IBMX
- 3-isobutyl-1-methylxanthine
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- Received January 22, 1998.
- Accepted May 5, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
