Roles for protein kinases in the induction of nitric oxide synthase in astrocytes

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Abstract
Lipopolysaccharide (LPS) or a combination of interferon (IFN)‐γ and interleukin (IL)‐1β can induce a calcium‐independent nitric oxide synthase (iNOS) in astrocyte cultures (Simmons and Murphy: J Neurochem 59:897, 1992; Eur J Neurosci 5:825,1993; Galea et al: Proc Natl Acad Sci USA 89:10945,1992). This induction can be measured by assaying cyclic GMP levels in the cultures, which correlates with, but is more sensitive than, measurement of nitrite accumulation. To study potential second‐messenger systems involved in the induction of iNOS, phorbol 12‐myristate 13‐acetate (PMA), a protein kinase C (PKC) activator, and various protein kinase inhibitors were employed. PMA induced a time‐, dose‐, and L‐arginine‐dependent increase in cyclic GMP, which could be inhabited by dexamethasone or actinomycin D. This induction could be dramatically increased by concurrent treatment with IFN‐y. The presence of iNOS mRNA could be demonstrated by hybridization with a specific cDNA probe. H7 (a non‐specific serine/threonine kinase inhibitor) but not H89 (a more specific PKA inhibitor) prevented induction by all agents. However, downregulation of PKC or pretreatment with the PKC inhibitor calphostin C did not prevent the induction by LPS or cytokines, suggesting that PKC is not necessary for iNOS induction by these mediators. Additionally, genistein (a nonspecific tyrosine kinase inhibitor) could prevent induction by all agents, but the more specific inhibitor, tyrphostin, attenuated only NOS induction by LPS. These results suggest that activation ofPKC can lead to, but is not necessary for, the induction of NOS in astrocytes and that there is a potential role for tyrosine kinases in NOS induction by LPS. This complex control of induction of iNOS, seemingly requiring activation of multiple pathways for maximal effect, might represent a safeguard to prevent production of potentially toxic nitric oxide (NO) under normal physiological conditions.