Handlogten M E, Shiraishi N, Awata H, Huang C, Miller R T
Division of Nephrology, Department of Medicine, University of Florida, Gainesville, Florida 32610, USA.
Am J Physiol Renal Physiol. 2000 Dec;279(6):F1083-91. doi: 10.1152/ajprenal.2000.279.6.F1083.
The extracellular Ca(2+)-sensing receptor (CaR) responds to polycations, including Ca(2+) and neomycin. This receptor is a physiological regulator of systemic Ca(2+) metabolism and may also mediate the toxic effects of hypercalcemia. A number of divalent cations, including Pb(2+), Co(2+), Cd(2+), and Fe(2+), are toxic to the kidney, brain, and other tissues where the CaR is expressed. To determine which divalent cations can activate the CaR, we expressed the human CaR in HEK-293 cells and measured activation of phospholipase A(2) (PLA(2)) and the mitogen-activated protein kinase p42ERK in response to potential agonists for the receptor. HEK-293 cells expressing the nonfunctional mutant CaR R796W served as controls. Extracellular Ca(2+), Ba(2+), Cd(2+), Co(2+), Fe(2+), Gd(3+), Ni(2+), Pb(2+), and neomycin activated the CaR, but Hg(2+) and Fe(3+) did not. We analyzed the kinetics of activation of p42ERK and PLA(2) by the CaR in response to Ca(2+), Co(2+), and Pb(2+). The EC(50) values ranged from approximately 0.1 mM for Pb(2+) to approximately 4.0 mM for Ca(2+). The Hill coefficients were >3, indicating multiple cooperative ligand binding sites or subunits. Submaximal concentrations of Ca(2+) and Pb(2+) were additive for activation of the CaR. The EC(50) for Ca(2+) or Pb(2+) was reduced four- to fivefold by the presence of the other ion. These divalent cations also activated PLA(2) via the CaR in Madin-Darby canine kidney cells that stably express the CaR. We conclude that many divalent cations activate the CaR and that their effects are additive. The facts that the CaR is a promiscuous polycation sensor and that the effects of these ions are additive to activate it suggest that the CaR may contribute to the toxicity of some heavy metals such as Pb(2+), Cd(2+), Co(2+), and Fe(2+) for the kidney and other tissues where it is expressed.
细胞外钙敏感受体（CaR）对包括Ca²⁺和新霉素在内的多价阳离子有反应。该受体是全身钙代谢的生理调节因子，也可能介导高钙血症的毒性作用。包括Pb²⁺、Co²⁺、Cd²⁺和Fe²⁺在内的多种二价阳离子对表达CaR的肾脏、大脑和其他组织有毒性。为了确定哪些二价阳离子能激活CaR，我们在HEK - 293细胞中表达了人CaR，并测量了磷脂酶A₂（PLA₂）的激活以及丝裂原活化蛋白激酶p42ERK对该受体潜在激动剂的反应。表达无功能突变体CaR R796W的HEK - 293细胞用作对照。细胞外Ca²⁺、Ba²⁺、Cd²⁺、Co²⁺、Fe²⁺、Gd³⁺、Ni²⁺、Pb²⁺和新霉素激活了CaR，但Hg²⁺和Fe³⁺没有。我们分析了CaR对Ca²⁺、Co²⁺和Pb²⁺激活p42ERK和PLA₂的动力学。半数有效浓度（EC₅₀）值范围从Pb²⁺的约0.1 mM到Ca²⁺的约4.0 mM。希尔系数大于3，表明存在多个协同配体结合位点或亚基。亚最大浓度的Ca²⁺和Pb²⁺对CaR的激活具有加和性。另一种离子的存在使Ca²⁺或Pb²⁺的EC₅₀降低了四到五倍。这些二价阳离子还通过CaR在稳定表达CaR的马-达二氏犬肾细胞中激活了PLA₂。我们得出结论，许多二价阳离子激活CaR，且它们的作用具有加和性。CaR是一种混杂的多价阳离子传感器以及这些离子激活它的作用具有加和性这一事实表明，CaR可能导致某些重金属如Pb²⁺、Cd²⁺、Co²⁺和Fe²⁺对其表达所在的肾脏和其他组织产生毒性。
