In vascular smooth muscle cells, the expression of iNOS appears to be critically dependent on the presence of both bacterial lipopolysaccharide (LPS) and interferon- (IFN-) (Baydoun et al., 1999) suggesting that activation of the JAK/STAT pathway may be critical for this process to occur. However, studies utilizing the JAK 2 selective inhibitor AG490 have proved controversial and inconclusive (Cruz et al., 1999; Marrero et al., 1998). In this study we have examined the critical requirement of JAK/STATs for the induction of iNOS by employing the new inhibitor of the JAK family of proteins referred to as JAK inhibitor I (Thompson et al., 2002). The effects of this compound together with AG490 have been examined in rat cultured aortic smooth muscle cells (RASMC) which require IFN- in combination with LPS for the expression of iNOS, and in J774.A2 macrophages which only require LPS. In parallel studies the effects of both compounds on L-arginine transport were also investigated.

Confluent monolayers of cells seeded in 96-well plates (5x10⁵ cells well^-1) were pre-treated with increasing concentration of either AG490 or JAK inhibitor I for 30 minutes prior to activation with either 1µg/ml LPS alone (J774.A2 cells) or with 100 µg/ml LPS plus 50U/ml IFN- (RASMC). Nitrite production was determined by the standard Griess assay and cell viability assessed by monitoring mitochondrial dependent reduction of MTT to formazan as described previously (Baydoun et al., 1999). In parallel studies changes in transport of L-arginine (100 µM plus 2 µCi ml^-1 L-[³H]arginine) was monitored as described (Baydoun et al., 1999).

AG490 failed to caused any statistically significant change in nitrite production in either J774.A2 macrophages or RASMC at the non cytotoxic concentrations of up to 30µM (J774.A2) or 100µM (RASMC). Similarly transport of L-arginine in both cell types was not altered by this compound. In contrast, JAK inhibitor I caused marked reductions in accumulated nitrite levels, completely abolishing the latter at 1µM and 3µM in J774.A2 and RASMCs respectively. L-arginine transport was however unaffected over the same concentration ranges that abolished nitrite production.

These results suggest that activation of JAK 2 may not be critical for the induction of iNOS in either cell type. However, the inhibitions caused by JAK inhibitor I strongly indicate that one of the other JAK family members, which remain to be identified, may be involved. Moreover, the selective inhibition of nitric oxide production but not of L-arginine transport indicates for the first time that these two pathways may be differentially regulated by the JAK/STATs.

Baydoun, A.R., Wileman, S.M., Wheeler-Jones, C.P. et al. (1999). Biochem J. 344, 265-272.
Cruz, M.T., Duarte, C.B., Gonçalo, M. et al. (1999). Am. J. Physiol., 277, C1050-C1057.
Marrero, M.B., Venema, V.J., He, H. et al. (1998). Biochem. Biophys. Res. Commun., 252, 508-512.
Thompson, J.E., Cubbon, R.M., Cummings, R.T. et al. (2002). Bioorg. Med. Chem. Lett. 12, 1219-1223.