In brain, activation of astrocytes can induce calcium independent nitric oxide synthase (NOS-2) expression, which can contribute to the damage during ischemia, demyelinating disease, and viral infection. The applicants have previously characterized factors which activate astroglial NOS-2 expression, and two methods for potently blocking that expression, namely stimulation of b-ARs, and pretreatment with inhibitors of protein tyrosine kinases. They have also found that heat shock of glial cells reduces NOS-2 expression. Whether these methods of suppression represent distinct mechanisms, or share common features, is not known. Since activation of the transcription factor NFkB is required for NOS-2 expression, it is possible that all three suppressive means interfere with NFkB activation. This grant therefore proposes to test and characterize NFkB activation in astrocytes as it relates to NOS-2 induction. Differential NFkB activation by different inducers and/or in different cell types (e.g. astrocytes versus C6 cells) may explain differences in levels of NOS-2 induction. Since protein phosphorylation plays a key role in NFkB activation, the applicants will test if NE effects are mediated via a protein kinase, and if so if that kinase modifies NFkB activation. They will also test if NE induces IkB expression. They will fully characterize the heat shock response in astrocyte and C6 cells with the purpose of defining which heat shock proteins can block NOS-2 induction, and if that is due to blocking NFkB activation. They will test if heat shock proteins physically bind to the NFkB and/or IkB proteins, if they block IkB phosphorylation and/or degradation, or if they simply block NFkB movement into the nucleus. They will test if NE effects are due in part to induction of heat shock proteins. Some experiments (e.g. heat shock) will be carried out in animals to test if this suppressive method also works in vivo. These experiments will define multiple pathways to block NOS-2 expression that may be useful in preventing pathological NOS-2 expression in brain.
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