Grant R L, Acosta D
Division of Pharmacology and Toxicology, College of Pharmacy, University of Texas, Austin 78712.
Toxicol Appl Pharmacol. 1994 Nov;129(1):23-35. doi: 10.1006/taap.1994.1225.
It has been documented by several investigators that local anesthetics displace calcium from calcium binding sites and alter the functioning of different calcium regulating systems. Local anesthetics have also been shown to have adverse effects on mitochondrial function and interact with cytoskeletal elements. Few studies have addressed the role that a potential disturbance of calcium homeostasis and mitochondrial function may have on the toxicity caused by local anesthetics in corneal epithelial cells. This investigation was undertaken to evaluate the effects of tetracaine (TTC), proparacaine (PPC), and cocaine (CC) on cytosolic calcium and mitochondrial membrane potential in primary cultures of rabbit corneal epithelial cells. Previous studies by our laboratory documented that the local anesthetics produce toxicity after 30 to 60 min of treatment. In this study, the cells were treated for 15 min, a time when minimal cell damage occurred. The following concentrations of local anesthetics were used to treat the cells: TTC, 0.5-2.5 mM; PPC, 1-5 mM; and CC, 4-10 mM. We utilized the technology of digitized fluorescence imaging to measure changes in intracellular calcium ([Ca2+]i) with fura-2 and mitochondrial membrane potential (delta psi) with rhodamine 123. A dose-dependent increase in [Ca2+]i was evident after treatment with each local anesthetic. Concentrations equal or greater than 2.5 mM TTC dissipated delta psi. A rise in [Ca2+]i preceded any loss of delta psi caused by TTC. PPC at high concentrations (4-5 mM) occasionally dissipated delta psi but this was not a consistent finding. The effects of CC on delta psi could not be evaluated accurately because of the extensive morphological alterations that occurred after treatment. We conclude that TTC, PPC, and CC elevate [Ca2+]i before cytotoxicity occurs and disruptions in calcium homeostasis may contribute to their toxicity.
几位研究者已证明，局部麻醉药会从钙结合位点置换钙，并改变不同钙调节系统的功能。局部麻醉药还被证明对线粒体功能有不良影响，并与细胞骨架成分相互作用。很少有研究探讨钙稳态和线粒体功能的潜在紊乱可能在局部麻醉药对角膜上皮细胞毒性中所起的作用。本研究旨在评估丁卡因（TTC）、丙美卡因（PPC）和可卡因（CC）对兔角膜上皮细胞原代培养物中细胞溶质钙和线粒体膜电位的影响。我们实验室之前的研究记录了局部麻醉药在处理30至60分钟后会产生毒性。在本研究中，细胞处理15分钟，此时细胞损伤最小。使用以下浓度的局部麻醉药处理细胞：TTC，0.5 - 2.5 mM；PPC，1 - 5 mM；CC，4 - 10 mM。我们利用数字化荧光成像技术，用fura - 2测量细胞内钙（[Ca2 + ]i）的变化，用罗丹明123测量线粒体膜电位（δψ）。用每种局部麻醉药处理后，[Ca2 + ]i呈剂量依赖性增加。浓度等于或大于2.5 mM的TTC使δψ消失。TTC导致的δψ丧失之前，[Ca2 + ]i先升高。高浓度（4 - 5 mM）的PPC偶尔会使δψ消失，但这不是一个一致的发现。由于处理后发生广泛的形态学改变，无法准确评估CC对δψ的影响。我们得出结论，TTC、PPC和CC在细胞毒性发生前会升高[Ca2 + ]i，钙稳态的破坏可能导致它们的毒性。
