Schanne F A, Dowd T L, Gupta R K, Rosen J F
Department of Pediatrics, Albert Einstein College of Medicine, Montefiore Medical Center, Bronx, NY 10467.
Environ Health Perspect. 1990 Mar;84:99-106. doi: 10.1289/ehp.908499.
Lead (Pb) has been shown to perturb cellular calcium (Ca) homeostasis, altering sizes and flux rates of cellular pools of exchangeable Ca and impairing Ca-mediated cell processes. To date, however, a direct effect of Pb on intracellular-free Ca2+ has not yet been demonstrated. Heavy metals bind to the commonly used fluorescent Ca ion indicators with greater affinity than does Ca and thereby interfere with the expected Ca-dependent fluorescence. In this study, the fluorinated Ca ion indicator, 1,2-bis(2-amino-5-fluorophenoxy)ethane N,N,N',N'-tetraacetic acid (5F-BAPTA), and 19F NMR were used to measure the free intracellular Ca ion concentration ([Ca2+]i) in the rat osteoblastic bone cell line, ROS 17/2.8. Both Pb and Ca bind to 5F-BAPTA with high affinity, but the Pb-5F-BAPTA comple produces a 19F NMR signal at a chemical shift distinct from 5F-BAPTA and the Ca-5F-BAPTA complex. The apparent dissociation constants for Pb-5F-BAPTA and Ca-5F-BAPTA are 2 X 10(-10) M and 5 X 10(-7) M, respectively, at 30 degrees C, pH 7.1, and Mg2+ (0.5 mM). Thus, this methodology allows for the simultaneous identification and quantification of free Pb and free Ca ion concentrations. Determinations of [Ca2+]i were based on 19F NMR measurements of 5F-BAPTA-loaded ROS 17/2.8 osteoblastic bone cells that were attached to collagen-coated microcarrier beads. Cells were continuously superfused with freshly oxygenated medium at 30 degrees C. Under these conditions, the [Ca2+]i of ROS 17/2.8 cells was observed to be 128 +/- 14 nM.(ABSTRACT TRUNCATED AT 250 WORDS)
铅（Pb）已被证明会扰乱细胞钙（Ca）稳态，改变可交换钙的细胞池大小和通量率，并损害钙介导的细胞过程。然而，迄今为止，铅对细胞内游离Ca2+的直接影响尚未得到证实。重金属与常用的荧光钙指示剂的结合亲和力比钙更高，从而干扰预期的钙依赖性荧光。在本研究中，使用氟化钙指示剂1,2-双（2-氨基-5-氟苯氧基）乙烷N,N,N',N'-四乙酸（5F-BAPTA）和19F核磁共振来测量大鼠成骨细胞系ROS 17/2.8中的细胞内游离钙离子浓度（[Ca2+]i）。铅和钙都以高亲和力与5F-BAPTA结合，但Pb-5F-BAPTA复合物在与5F-BAPTA和Ca-5F-BAPTA复合物不同的化学位移处产生19F核磁共振信号。在30℃、pH 7.1和Mg2+（0.5 mM）条件下，Pb-5F-BAPTA和Ca-5F-BAPTA的表观解离常数分别为2×10（-10）M和5×10（-7）M。因此，这种方法可以同时鉴定和定量游离铅和游离钙离子浓度。[Ca2+]i的测定基于对附着在胶原包被微载体珠上的5F-BAPTA负载的ROS 17/2.8成骨细胞的19F核磁共振测量。细胞在30℃下用新鲜充氧培养基连续灌注。在这些条件下，观察到ROS 17/2.8细胞的[Ca2+]i为128±14 nM。（摘要截断于250字）
