A DIGITIZED FLUORESCENCE IMAGING STUDY ON THE EFFECTS OF LOCAL-ANESTHETICS ON CYTOSOLIC CALCIUM AND MITOCHONDRIAL-MEMBRANE POTENTIAL IN CULTURED RABBIT CORNEAL EPITHELIAL-CELLS

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 inter act with cytoskeletal elements. Few studies have addressed the role th at a potential disturbance of calcium homeostasis and mitochondrial fu nction may have on the toxicity caused by local anesthetics in corneal epithelial cells. This investigation was undertaken to evaluate the e ffects of tetracaine (TTC), proparacaine (PPC), and cocaine (CC) on cy tosolic calcium and mitochondrial membrane potential in primary cultur es of rabbit corneal epithelial cells. Previous studies by our laborat ory 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 concentratio ns 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 ([Ca 2+](i)) with fura-2 and mitochondrial membrane potential (Delta Psi) w ith rhodamine 123. A dose-dependent increase in [Ca2+](i) was evident after treatment with each local anesthetic. Concentrations equal or gr eater than 2.5 mM TTC dissipated Delta Psi. A rise in [Ca2+](i) preced ed 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 accurat ely because of the extensive morphological alterations that occurred a fter treatment. We conclude that TTC, PPC, and CC elevate [Ca2+](i) be fore cytotoxicity occurs and disruptions in calcium homeostasis may co ntribute to their toxicity. (C) 1994 Academic Press, Inc.