title: Inhibition of neutrophil superoxide generation by shikonin is associated with suppression of cellular Ca(2+) fluxes.
abstract: Shikonin, an anti-inflammatory compound of "Shikon", inhibits the neutrophil superoxide (O2 (•-)) generation by NADPH oxidase 2 (Nox2); however, the mechanisms of how shikonin affects Nox2 activity remained unclear. We aimed to elucidate the relationship between the inhibition of Nox2 activity and influences on intracellular Ca(2+) concentration ([Ca(2+)]i) by shikonin. For this purpose, we used a simultaneous monitoring system for detecting changes in [Ca(2+)]i (by fluorescence) and O2 (•-) generation (by chemiluminescence) and evaluated the effects of shikonin on neutrophil-like HL-60 cells stimulated with N-formyl-l-methionyl-l-leucyl-l-phenylalanine (fMLP). Since fMLP activates Nox2 by elevation in [Ca(2+)]i via fluxes such as inositol 1,4,5-trisphosphate-induced Ca(2+) release (IICR) and store-operated Ca(2+) entry (SOCE), we also evaluated the effects of shikonin on IICR and SOCE. Shikonin dose-dependently inhibited the fMLP-induced elevation in [Ca(2+)]i and O2 (•-) generation (IC50 values of 1.45 and 1.12 µM, respectively) in a synchronized manner. Analyses of specific Ca(2+) fluxes showed that shikonin inhibits IICR and IICR-linked O2 (•-) generation (IC50 values: 0.28 and 0.31 µM for [Ca(2+)]i and O2 (•-), respectively), as well as SOCE and SOCE-linked O2 (•-) generation (IC50 values: 0.39 and 0.25 µM for [Ca(2+)]i and O2 (•-), respectively). These results suggested that shikonin inhibits the O2 (•-) generation by Nox2 in fMLP-stimulated neutrophils by targeting Ca(2+) fluxes such as IICR and SOCE.
authors: Central Research Laboratory, Hamamatsu Photonics K.K., 5000 Hirakuchi, Hamakita-ku, Hamamatsu 434-8601, Japan.
authors: Kimiko
authors: K
authors: Kazumura
authors: Research Institute of Pharmaceutical Sciences, Musashino University, 1-1-20 Shinmachi, Nishitokyo 202-8585, Japan.
authors: Lucia Satiko
authors: LS
authors: Yoshida
authors: Central Research Laboratory, Hamamatsu Photonics K.K., 5000 Hirakuchi, Hamakita-ku, Hamamatsu 434-8601, Japan.
authors: Akiko
authors: A
authors: Hara
authors: Central Research Laboratory, Hamamatsu Photonics K.K., 5000 Hirakuchi, Hamakita-ku, Hamamatsu 434-8601, Japan.
authors: Hiroshi
authors: H
authors: Tsuchiya
authors: Electron Tube Division, Hamamatsu Photonics K.K., 314-5 Shimokanzo, Iwata 438-0193, Japan.
authors: Naokazu
authors: N
authors: Morishita
authors: Department of Applied Biological Chemistry, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan.
authors: Hirokazu
authors: H
authors: Kawagishi
authors: Faculty of Pharmaceutical Sciences, Josai International University, 1 Gumyo, Togane 283-8555, Japan.
authors: Tomohito
authors: T
authors: Kakegawa
authors: Research Institute of Pharmaceutical Sciences, Musashino University, 1-1-20 Shinmachi, Nishitokyo 202-8585, Japan.
authors: Yasukatsu
authors: Y
authors: Yuda
authors: Research Institute of Pharmaceutical Sciences, Musashino University, 1-1-20 Shinmachi, Nishitokyo 202-8585, Japan.
authors: Hiromi
authors: H
authors: Takano-Ohmuro
keywords: NADPH oxidase; intracellular calcium; shikonin; simultaneous detection; superoxide