Suppression of manganese-dependent production of nitric oxide in astrocytes: implications for therapeutic modulation of glial-derived inflammatory mediators

Primary cultured astrocytes were treated with Mn in the absence and presence of proinflammatory cytokines to determine their effect upon stimulation of nitric oxide (NO) production. Treatments of manganese and cytokines raised NO production to intermediate levels, whereas combined treatment raised NO creation to much greater levels. Furthermore, this combined treatment differed from control only in its ability to elevate cellular NO levels at 24 hours, but not at earlier time points. Combined exposure in astrocytes derived from mice lacking the nos2 gene prevented any increase in production of NO. Thus, manganese and cytokines enhance NO production through activation of the nos2 gene. Additionally, pharmacologic ligands of the peroxisome proliferator-activated receptor gamma (PPAR?) were used to test the role of this orphan nuclear receptor in modulating Mn-dependent production of NO. The agonist, 1,1-Bis(3?-indolyl)-1-(p-trifluormethylphenyl) methane (cDIM1) diminished NO in a dose-dependent manner, whereas addition of the PPAR? antagonist, GW 9662, amplified cellular NO production, also in a dose-dependent fashion. Moreover, it was observed that NO production was both attenuated and augmented at similar rates, suggesting the agonist and antagonist work through similar mechanisms. To clarify the means by which NO levels are manipulated by PPAR?, we measured activation levels of the transcription factor NF-?B, a primary factor resulting in expression of NOS2. We found that NF-?B was slightly activated in cells treated solely with manganese or cytokines, whereas cells treated with both manganese and cytokines showed the highest levels of activation. Also, we found that these ligands function through an NF-?B dependent mechanism. Treatment of cDIM1 to astrocytes already treated with manganese and cytokines caused decreased activation of NF-?B, while addition of GW9662 to similarly treated cells resulted in increased activation of NF-?B. While these compounds were effective at manipulating induction of the nos2 gene, they had no effect on induction of guanosine tri-phosphate cyclohydrolase (GTPCH) the rate limiting enzyme for the production of tetrahydrobiopterin (BH4), a cofactor essential to the conversion of arginine to NO, Thus, these novel PPAR? ligands can influence manganese- and cytokine-induced production of NO by an NF-?B dependent mechanism.