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We studied the mechanism underlying the inhibitory effect of a benzodiazepines derivative, 4-(3-chlorophenyl)-1,3-dihydronaphtho [2,3-b][1,4]diazepin-2-one (ND700C), on superoxide anion production induced by formly-methionyl-leucyl-phenylalanine (fMLP) in human neutrophils. ND700C inhibited the fMLP-induced superoxide anion production and cathepsin G release in a concentration-dependent manner with respective IC50 values of 5.0 ± 0.5 and 8.7 ± 0.8 μM. In addition, ND700C was found to suppress fMLP-induced intracellular calcium mobilization and the phosphorylation of ERK and Akt. In another study, ND700C was observed to cause a rapid increase in intracellular cAMP level by up to threefold. Furthermore, when H89 was used to inhibit cAMP-dependent protein kinase A (PKA), we discovered that ND700C's suppressive effects on calcium mobilization, phosphorylation, and superoxide anion production were abrogated. ND700C demonstrated additive effect on the PGE1-induced increase in cAMP. However, this additive effect was not demonstrated with the IBMX-induced rise in cAMP. Our results indicated that ND700C did not directly inhibit the activity of phosphodiesterase 4. In another set of experiments, calyculin A and okadaic acid (both protein phosphatase 2A inhibitors) were found to reverse ND700C's positive effect on cAMP level. This observation suggested the involvement of protein phosphatase 2A in ND700C's cAMP-elevating mechanism. We found that the activity of protein phosphatase 2A was activated by ND700C. Furthermore, protein phosphatase 2A was co-immunoprecipitated with phosphodiesterase 4 after ND700C treatment in human neutrophils. Conclusion: ND700C inhibited fMLP-induced superoxide anion production through a PKA-dependent pathway. ND700C increased cAMP by activating protein phosphatase 2A, which subsequently inhibited phosphodiesterase 4.