Use of a murine cell line for identification of human nitric oxide synthase inhibitors.
BACKGROUND
Nitric oxide (NO) has been implicated in a wide range of physiological and pathological processes. Low concentrations of this mediator play homeostatic roles, whereas many acute and chronic responses are associated with excessive production of NO. This upregulation is due in part to the induction of inducible nitric oxide synthase (iNOS) by proinflammatory cytokines in several different cell types, including macrophages and their CNS derivative, microglia.


METHODS
The crystal structures of the oxygenase domains of mouse and human iNOS were superimposed using the "align by homology" feature in Sybyl (SYBYL 7.0, Tripos Inc.). NOS isoform expression was assessed by TaqMan, Western blotting, and activity assays.


RESULTS
We demonstrate that there is a high degree of three-dimensional overlap between the mouse and human iNOS active centers and propose that the murine isoform can serve as a suitable substitute for the human in assays. We also demonstrate that LPS stimulation of the mouse macrophage cell line RAW 264.7 induces the expression of iNOS, but not nNOS or eNOS, at the levels of mRNA transcription and protein expression. Furthermore, the pharmacology and calcium dependency of the NO formation support the finding that it is due to iNOS alone. Also reported is the demonstration of LPS-induced RAW 264.7 macrophages in simple cell-based and cell-free screening assays for iNOS inhibitors. Both assays were reproducible, as demonstrated by Z' factors of 0.69 and 0.71, and had high signal to noise ratios of 11- and 6-fold for the cell-based and cell-free assay, respectively.


CONCLUSIONS
Our computational analyses indicate that there is a high degree of three-dimensional overlap between the oxygenase domains of human and murine iNOS. This observation together with the selective induction of murine iNOS in RAW 264.7 macrophages demonstrates the potential utility of the mouse iNOS assay to identify inhibitors of the human enzyme.
Naureckiene
Saule
S
Neuroscience Discovery Research, Wyeth Research, CN8000, Princeton, NJ 08543, USA. naurecs@wyeth.com
Edris
Wade
W
Ajit
Seena K
SK
Katz
Alan H
AH
Sreekumar
Kodangattil
K
Rogers
Kathryn E
KE
Kennedy
Jeffrey D
JD
Jones
Philip G
PG
D000818:Animals; D015153:Blotting, Western; D002118:Calcium; D002460:Cell Line; D003198:Computer Simulation; D016207:Cytokines; D005786:Gene Expression Regulation; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008264:Macrophages / Q000378:metabolism; D051379:Mice; D017628:Microglia / Q000378:metabolism; D009569:Nitric Oxide / Q000096:biosynthesis / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020033:Protein Isoforms; D012333:RNA, Messenger; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D013045:Species Specificity; D040681:Structural Homology, Protein; D014158:Transcription, Genetic
D016207:Cytokines; D008070:Lipopolysaccharides; D020033:Protein Isoforms; D012333:RNA, Messenger; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; D002118:Calcium