Effect of Bradykinin on Cultured Bovine Corneal Endothelial Cells

Abstract

Purpose: To clarify the effect of bradykinin on cytosolic free calcium mobilization and cell proliferation in cultured bovine corneal endothelial cells (BCEC). Methods: The cytosolic free calcium concentration (Ca²⁺]_i) was measured with the InCa^TM Imaging System after the treatment of bradykinin (10^–11 to 10^–7  M) alone or with the pretreatments of EGTA, bradykinin receptor (Bk₁ and Bk₂) antagonists and an inhibition of phospholipase C (U-73122). Also, the effect of bradykinin on cell proliferation in BCEC was evaluated using cell counts. Results: In BCEC, [Ca²⁺]_i in the resting state was 87 ± 9 n M. Bradykinin induced an increment of [Ca²⁺]_i in a concentration-dependent manner and its 50% effective concentration was approximately 5 × 10^–11  M. A [Ca²⁺]_i increment at 10^–8  M bradykinin was inhibited with the pretreatment of EGTA, an extracellular calcium chelator. U-73122 (5 × 10^–6  M) attenuated the bradykinin-induced [Ca²⁺]_i increment. The pretreatment of HOE-140 (Bk₂ antagonist) almost attenuated the bradykinin (10^–8  M)-induced [Ca²⁺]_i increase, but des-Arg⁹-[Leu⁸]-bradykinin (Bk₁ antagonist) did not suppress it. To investigate the physiological effect of bradykinin, the effect of bradykinin on cell proliferation was studied. 10^–8  M of bradykinin produced a significant increase in cell numbers. This mitogenic effect of bradykinin was inhibited by the Bk₂ antagonist. Conclusions: Bradykinin-induced stimulation of the signal transduction pathway in BCEC is coupled with the Bk₂ type receptor. Furthermore, bradykinin produces the mitogenic effect in BCEC.