Effect of nortriptyline on Ca²⁺ handling in SIRC rabbit corneal epithelial cells

To explore the effect of nortriptyline, a tricyclic antidepressant, on cytosolic free Ca²⁺ concentrations ([Ca²⁺]i) in corneal epithelial cells, [Ca²⁺]i levels in suspended SIRC rabbit corneal epithelial cells were measured by using fura-2 as a Ca²⁺-sensitive fluorescent dye. Nortriptyline at concentrations between 20-200 microM increased [Ca²⁺]i in a concentration-dependent manner. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺. Nortriptyline-induced Ca²⁺ influx was inhibited by the store-operated Ca²⁺ channel blockers econazole and SK&F96365, the phospholipase A2 inhibitor aristolochic acid, and alteration of activity of protein kinase C. In Ca²⁺-free medium, 200 microM nortriptyline pretreatment greatly inhibited the rise of [Ca²⁺]i induced by the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin. Conversely, pretreatment with thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ; another endoplasmic reticulum Ca²⁺ pump inhibitor) nearly abolished nortriptyline-induced [Ca²⁺]i rise. Inhibition of phospholipase C with U73122 decreased nortriptyline-induced [Ca²⁺]i rise by 75%. Taken together, nortriptyline induced [Ca²⁺]i rises in SIRC cells by causing phospholipase C-dependent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ influx via store-operated Ca²⁺ channels.