Combination of inflammatory cytokines increases nitrite and nitrate levels in the paraventricular nucleus of conscious rats

Inflammatory cytokines stimulate glial cells in vitro to produce nitric oxide (NO) from inducible NO synthase (iNOS). Whether the stimulation with cytokines produces NO derived from iNOS has not hitherto been demonstrated in the vivo brain. Nitrite and nitrate (NOx(-)) levels in the rat paraventricular nucleus (PVN) were measured before and after intraparenchymal microinjection of cytokines with a microdialysis technique. The cytokines, tumor necrosis factor (TNF)-alpha (10 ng), interleukin (IL)-1 beta (2 ng), and interferon (IFN)-gamma (2 ng) were microinjected. None of the cytokines alone had any effect on the NOx(-) levels for 8 h. But a combination of TNF-alpha and IFN-gamma gradually increased NOx(-) levels beginning at 140 min after the microinjection, and NOx(-) levels reached 1.8 times the basal level at 380 min. A combination of TNF-alpha and IL-1 beta increased NOx(-) beginning at 340 min, reaching 1.7 times the basal level at 440 min, whereas a combination of IL-1 beta and IFN-gamma had no effect. Microinjection of a mixture of all three cytokines increased NOx(-) levels beginning at 120 min, reaching 3.3 times the basal level at 400 min. Aminoguanidine, which is a selective inhibitor of iNOS, reduced NOx(-) levels induced by the mixed cytokine treatment. Semi-quantitative RT-PCR for iNOS mRNA was done. The intensity of the iNOS mRNA band for the cytokine-treated PVN was stronger than that for the vehicle-treated PVN. These results suggest that the increased NOx(-) after the treatment with mixed cytokines were dependent on iNOS activity. This is the first report to indicate that only cytokines induce NOS in vivo in the brain.