Inhibition of formyl-methionyl-leucyl-phenylalanine-stimulated respiratory burst by cirsimaritin involves inhibition of phospholipase D signaling in rat neutrophils

Abstract.

In this study, the cellular localization of the inhibitory effect of a natural flavonoid cirsimaritin against formyl-methionyl-leucyl-phenylalanine (fMLP)-induced respiratory burst in rat neutrophils was investigated. Cirsimaritin concentration-dependently inhibited the superoxide anion $\left( {{\rm O}_2^{ \bullet - } } \right)$ generation and O₂ consumption (IC₅₀ 11.5±2.2 µM and 17.0±3.9 µM, respectively) of neutrophils. Cirsimaritin did not reduce, but slightly enhanced the ${\rm O}_2^{ \bullet - }$ generation in phorbol 12-myristate 13-acetate (PMA)-activated or arachidonic acid-stimulated NADPH oxidase preparation as well as during the autoxidation of dihydroxyfumaric acid. Cirsimaritin did not elevate cellular cAMP levels, and only partially inhibited the fMLP-induced [Ca²⁺]_i changes in the presence or absence of extracellular Ca²⁺. The phosphorylation of protein tyrosine, extracellular signal-regulated protein kinase and Akt caused by fMLP were attenuated by cirsimaritin in a concentration-dependent manner. In contrast, cirsimaritin had no effect on the phosphorylation of p38 mitogen-activated protein kinase. Cirsimaritin produced a concentration-dependent reduction in the formation of phosphatidic acid and phosphatidylethanol, in the presence of ethanol, from fMLP-stimulated neutrophils (IC₅₀ 15.1±6.5 µM and 15.6±3.4 µM, respectively), but did not affect the phosphatidylethanol formation in response to PMA. Under the similar concentration range, cirsimaritin attenuated the membrane translocation of ARF and Rho A. However, the GTPγS-stimulated membrane-associated ARF and Rho in cell lysate were unaffected by cirsimaritin. Collectively, these results indicate that the inhibition of fMLP-induced respiratory burst by cirsimaritin in rat neutrophils is likely mainly through the blockade of phospholipase D signaling pathway.