Jpn. J. Pharmacol. 84 (2), 140-145 (2000)

Intracellular Ca²⁺ Increase in Neuro-2A Cells and Rat Astrocytes Following Stimulation of Bradykinin B₂ Receptor

Yuri Ikeda¹, Akinori Ueno¹, Hiroaki Naraba¹, Norio Matsuki² and Sachiko Oh-ishi^1,*

¹Department of Pharmacology, School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan
²Department of Pharmacology, Faculty of Pharmaceutical Sciences, Tokyo University, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
*Corresponding author.ﾊﾊPresent address for correspondence: Kitasato Institute, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8642, Japan
FAX:+81-3-5791-6120, E-mail: oh-ishi@kitasato.or.jp

Abstract: Murine neuroblastoma cell line Neuro-2A cells and rat brain astrocytes showed a dose-dependent increase in intracellular Ca²⁺ in response to bradykinin, when assessed by a single cell image analyzing system. The Ca²⁺ increase in Neuro-2A cells by bradykinin was also examined by a suspension fluorescent assay using fura-2 loading. The Ca²⁺ increase in both cases was suppressed by a bradykininﾊB₂ receptor antagonist, Hoeﾊ140, but not by a B₁ receptor antagonist, des-Arg-Hoeﾊ140, suggesting that the effect occurred via specific B₂ receptor activation. RT-PCR for bradykininﾊB₂ receptor mRNA showed that both Neuro-2A cells and the astrocytes expressed B₂ receptor mRNA. Binding of [³H]bradykinin to Neuro-2A cells was assessed, and a specific binding constant of 0.75ﾊnM was determined. Furthermore, the increase in [Ca²⁺]_i by bradykinin could be caused by a release of Ca²⁺ from storage sites in the endoplasmic reticulum, since thapsigargin and U-73122 attenuated the effect of bradykinin in Neuro-2A as well as in astrocytes. These results indicate that both astrocytes and neuroblastoma Neuro-2A cells stimulated by bradykinin could express a bradykininﾊB₂ receptor-mediated intracellular Ca²⁺ increase leading to signal transduction.

Keywords: Bradykinin, Neuro-2A, Astrocyte, Intracellular Ca²⁺, Bradykinin B₂ receptor