Mechanism of Bradykinin-Induced Ca2+ Mobilization in MG63 Human Osteosarcoma Cells

Abstract

Background: The effect of bradykinin on intracellular free Ca²⁺ levels ([Ca²⁺]_i) in MG63 human osteosarcoma cells was explored using fura-2 as a Ca²⁺ dye. Methods/Results: Bradykinin (0.1 nM–1 µM) increased [Ca²⁺]_i in a concentration-dependent manner with an EC₅₀ value of 0.5 nM. The [Ca²⁺]_i signal comprised an initial peak and a fast decay which returned to baseline in 2 min. Extracellular Ca²⁺ removal inhibited the peak [Ca²⁺]_isignals by 35 ± 3%. Bradykinin (1 nM) failed to increase [Ca²⁺]_i in the absence of extracellular Ca²⁺after cells were pretreated with thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor; 1 µM). Bradykinin (1 nM)-induced intracellular Ca²⁺ release was nearly abolished by inhibiting phospholipase C with 2 µM 1-(6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122). The [Ca²⁺]_iincrease induced by 1 nM bradykinin in Ca²⁺- free medium was abolished by 1 nM HOE 140 (a B2 bradykinin receptor antagonist) but was not altered by 100 nM Des-Arg-HOE 140 (a B1 bradykinin receptor antagonist). Pretreatment with 1 pM pertussis toxin for 5 h in Ca²⁺ medium inhibited 30 ± 3% of 1 nM bradykinin-induced peak [Ca²⁺]_i increase. Conclusions: Together, this study shows that bradykinin induced [Ca²⁺]_i increases in a concentration-dependent manner, by stimulating B2 bradykinin receptors leading to mobilization of Ca²⁺ from the thapsigargin-sensitive stores in a manner dependent on inositol-1,4,5-trisphosphate, and also by inducing extracellular Ca²⁺ influx. The bradykinin response was partly coupled to a pertussis toxin-sensitive G protein pathway.