Expressional down-regulation of neuronal-type nitric oxide synthase I by glucocorticoids in N1E-115 neuroblastoma cells.
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 microM) or hydrocortisone (100 nM to 10 microM) 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 microM hydrocortisone. The effect of 100 nM dexamethasone was completely reversed by 1 microM of the glucocorticoid receptor antagonist mifepristone. In experiments with actinomycin D (10 microg/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.
Schwarz
P M
PM
Department of Pharmacology, Johannes Gutenberg University, 55101 Mainz, Germany. petra.schwarz@uni-mainz.de
Gierten
B
B
Boissel
J P
JP
Förstermann
U
U
D000818:Animals; D003907:Dexamethasone / Q000494:pharmacology; D015536:Down-Regulation / Q000187:drug effects; D005938:Glucocorticoids / Q000494:pharmacology*; D051379:Mice; D009447:Neuroblastoma; D009474:Neurons / Q000187:drug effects / Q000201:enzymology*; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D052248:Nitric Oxide Synthase Type I; D012333:RNA, Messenger / Q000187:drug effects / Q000378:metabolism; D014407:Tumor Cells, Cultured
D005938:Glucocorticoids; D012333:RNA, Messenger; D009569:Nitric Oxide; D003907:Dexamethasone; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496321:Nos1 protein, mouse