A prodigiosin analogue inactivates NADPH oxidase in macrophage cells by inhibiting assembly of p47phox and Rac.
Prodigiosins are natural red pigments that have multi-biological activities. Recently, we discovered a marine bacterial strain, which produces a red pigment. Extensive two-dimensional nuclear magnetic resonance and mass spectrometry analysis showed that the pigment is a prodigiosin analogue (PG-L-1). Here, we investigated the effect of PG-L-1 on NADPH oxidase activity in macrophage cells. PG-L-1 significantly inhibited superoxide anion (O(2)(-)) production by phorbol 12-myristate 13-acetate (PMA)-stimulated RAW 264.7 cells, a mouse macrophage cell line. The ED(50) value was estimated to be approximately 0.3 microM. PG-L-1 had no direct scavenging effect on O(2)(-) generated by hypoxanthine/xanthine oxidase system in electron spin resonance-spin trapping determinations, suggesting that this compound directly acts on the O(2)(-) production system, NADPH oxidase, in macrophage cells. We further investigated the effect of PG-L-1 on the behaviour of the cytosolic components of the NADPH oxidase, p67(phox), p47(phox), p40(phox), Rac and protein kinase C (PKC), in PMA-stimulated RAW 264.7 cells. Although PG-L-1 showed no effect on the activation of PKC, the immunoblotting analysis using specific antibodies showed that PG-L-1 strongly inhibits the association of p47(phox) and Rac in the plasma membrane of PMA-stimulated RAW 264.7 cells. These results suggest that PG-L-1 inactivates NADPH oxidase through the inhibition of the binding of p47(phox) and Rac to the membrane components of NADPH oxidase.
Nakashima
Takuji
T
NITE Biological Resource Center (NBRC), National Institute of Technology and Evaluation (NITE), Japan. nakashima-takuji@nite.go.jp
Iwashita
Takashi
T
Fujita
Tsuyoshi
T
Sato
Emiko
E
Niwano
Yoshimi
Y
Kohno
Masahiro
M
Kuwahara
Shunsuke
S
Harada
Nobuyuki
N
Takeshita
Satoshi
S
Oda
Tatsuya
T
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D003599:Cytoskeleton / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D008264:Macrophages / Q000187:drug effects / Q000201:enzymology*; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011353:Prodigiosin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism; D020744:rac GTP-Binding Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism
D004791:Enzyme Inhibitors; C529713:PG-L-1 compound; D013481:Superoxides; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D011493:Protein Kinase C; D020744:rac GTP-Binding Proteins; D011353:Prodigiosin