Intracellular Ca2+ increase in Neuro-2A cells and rat astrocytes following stimulation of bradykinin B2 receptor

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 B-2 receptor antagonist, Hoe 140, but not by a B-1 receptor antagonist, des-Arg-Hoe 140, suggesting that the effect occurred via specific B-2 receptor activation. RT-PCR for bradykinin B-2 receptor mRNA showed that both Neuro-2A cells and the astrocytes expressed B-2 receptor mRNA. Binding of [H-3]bradykinin to Neuro-2A cells was assessed, and a specific binding constant of 0.75 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 B-2 receptor-mediated intracellular Ca2+ increase leading to signal transduction.