6533b873fe1ef96bd12d4dec

RESEARCH PRODUCT

Expressional down-regulation of neuronal-type nitric oxide synthase I by glucocorticoids in N1E-115 neuroblastoma cells.

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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.