g. gene expression. p38 MAPK and c Jun N terminal kinase are members of the MAPK fam ily, and they are activated by chemical and physical stress. p38 and JNK regulate immune responses and expression of various cytokines e. g. tumor necrosis factor , inter Ivacaftor synthesis leukin 1 and interleukin 6. JNK and p38 MAPK are also involved in regulation of iNOS expression. Previous studies have shown that JNK pathway belongs to the factors that mediate the up regu lation of iNOS expression. Depending on the cell type and stimulation used, p38 MAPK has been reported to have either up regulatory role, down regulatory role or no role in iNOS expression. We have previously reported that p38 MAPK inhibitors enhance iNOS expression and NO production in LPS stimulated J774 macrophages.

The detailed mecha nism behind those stimulatory effects is not known. The aim of the present study was to investigate the mech anism by which p38 inhibition leads to increase in NO production. The results suggest that inhibition of p38 MAPK increases LPS induced JNK activity, which leads to stabilisation of iNOS mRNA and increased production of NO in activated macrophages. Results p38 MAPK inhibitor SB220025 increases LPS induced NO production and iNOS expression We have previously shown that pyridinyl imidazole inhibitor of p38 MAPK SB203580 stimulates LPS induced NO production. SB220025 is a recently developed potent and specific inhibitor of p38 MAPK with an IC50 value of 60 nM in kinase activity assay. Figure 1A shows that SB220025 had a concentration dependent stimulatory effect on LPS induced NO produc tion and maximal effect was achieved at drug con centration of 0,5 M.

The effect of SB220025 was similar to the effect of SB203580. A structurally related control compound SB202474, which does not inhibit p38 MAPK, had no effect on NO production. The stimulatory effect of SB220025 was maximal when the compound was added to cells 1 h after LPS. This result is in line with our previous report in which we production MAPK inhibitor SB220025 on LPS induced NO showed that the stimulatory effect of SB203580 was max imal when the compound was added 1 h after LPS. The levels of activated p38 peaked in 30 min after LPS, were still high at 1 h and declined gradually thereafter so that activated p38 could be detected even 4 h after LPS.

Thus, the stimulation of LPS induced iNOS pro duction by SB220025 could result from inhibition of p38, even when the compound was added to cells 1 2 h after LPS. SB220025 had a clear stimulatory effect also AV-951 on iNOS protein expression, whereas the negative control com pound SB202747 had no effect. Interestingly, SB220025 did not increase LPS induced iNOS mRNA lev els when measured 4 h after addition of LPS, whereas a hypothesized that SB220025 might stabilize iNOS mRNA. To study the effect of SB220025 on the stability of iNOS mRNA, the cells were treated with LPS or LPS SB220025 and cells were incubated for 6 h.