A benzodiazepines derived compound, 4-(3-chlorophenyl)-1,3-dihydronaphtho [2,3-b][1,4]diazepin-2-one (ND700C), inhibits fMLP-induced superoxide anion release by activating protein phosphatase 2A in human neutrophils.
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+/-0.8muM. 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.


CONCLUSIONS
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.
Liao
Chang-Hui
CH
Graduate Institute of Natural Products, College of Medicine, Chang-Gung University, Tao-Yuan, Taiwan. liaoch@mail.cgu.edu.tw
Lin
Shinn-Zhi
SZ
Tseng
Ching-Ping
CP
Day
Yuan-Ji
YJ
Chang
Chih-Shiang
CS
Chang
Yu-Hsun
YH
Kuo
Shen-Chu
SC
D001569:Benzodiazepines / Q000737:chemistry / Q000494:pharmacology*; D020013:Calcium Signaling / Q000187:drug effects; D056649:Cathepsin G; D002403:Cathepsins / Q000037:antagonists & inhibitors; D000242:Cyclic AMP / Q000032:analysis; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D020128:Inhibitory Concentration 50; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*; D058988:Phosphodiesterase 4 Inhibitors; D010766:Phosphorylation / Q000187:drug effects; D054648:Protein Phosphatase 2 / Q000378:metabolism*; D012697:Serine Endopeptidases; D013481:Superoxides / Q000037:antagonists & inhibitors*
D058988:Phosphodiesterase 4 Inhibitors; D013481:Superoxides; D001569:Benzodiazepines; D009240:N-Formylmethionine Leucyl-Phenylalanine; D000242:Cyclic AMP; D054648:Protein Phosphatase 2; D002403:Cathepsins; D012697:Serine Endopeptidases; C534094:CTSG protein, human; D056649:Cathepsin G