A Digitized Fluorescence Imaging Study on the Effects of Local Anesthetics on Cytosolic Calcium and Mitochondrial Membrane Potential in Cultured Rabbit Corneal Epithelial Cells

Abstract

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 (ΔΨ) 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 ΔΨ. A rise in [Ca2+]i preceded any loss of ΔΨ caused by TTC. PPC at high concentrations (4-5 mM) occasionally dissipated ΔΨ but this was not a consistent finding. The effects of CC on ΔΨ 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.