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

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 B₂ receptor antagonist, Hoe 140, but not by a B₁ receptor antagonist, des−Arg−Hoe 140, suggesting that the effect occurred via specific B₂ receptor activation.RT−PCR for bradykinin 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 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 B₂ receptor−mediated intracellular Ca²⁺ increase leading to signal transduction.