Effect of Latanoprost Acid and Pilocarpine on Cultured Rabbit Ciliary Muscle Cells

Abstract

Objective: To study the interaction between latanoprost and pilocarpine on cultured rabbit ciliary muscle (RCM) cells, and investigate the time courses of the two drugs, when given alone or in combination. Methods: Cultured RCM cells were treated for 24 h with different concentrations of latanoprost acid, pilocarpine and mixtures of latanoprost acid and pilocarpine. RNA was extracted, expressions of matrix metalloproteinase 1 (MMP-1), tissue inhibitor of metalloproteinase 1 (TIMP-1) and TIMP-2 were detected by reverse transcription-polymerase chain reaction (RT-PCR), and the optimum concentrations of those drugs were found. Then the cells were treated with the optimum concentrations of those drugs for various periods. RNA was extracted after the treatment and expressions of MMP-1, TIMP-1 and TIMP-2 were detected by RT-PCR again. Changes in [Ca²⁺]_i were estimated by fluorescence measurement using the Ca²⁺ indicator Fluo-3 AM with a laser scanning confocal microscope. [Ca²⁺]_i of each cell was monitored continually after administration of the drugs. Gray values at 5 s and 2, 4, 6, 8 and 10 min were chosen for statistical analysis, and the influence and time-effect relationship of those drugs on [Ca²⁺]_i of the cultured cells were evaluated. Results: Exposure of the cells to increasing concentrations of latanoprost acid induced increased MMP-1 mRNA and decreased TIMP-1 and TIMP-2 mRNA in a dose-dependent manner. After 24 h of treatment, the optimum concentration of latanoprost acid for maximal changes in MMP-1 and TIMP-2 expression was 2 × 10^–7M, and for maximal changes in TIMP-1 expression, the optimum concentration was 5 × 10^–7M. When the optimum concentrations of latanoprost acid were chosen to treat the cells for various periods, the optimum time of the peak MMP-1 expression and trough TIMP-1 expression was 24 h, and of the trough TIMP-2 expression, it was 36 h after initiation of treatment. No significant expression changes of MMP-1, TIMP-1 and TIMP-2 mRNA were found when the cells were treated with pilocarpine at any concentration or at any time. Exposure of the cells to the mixtures of latanoprost acid and pilocarpine induced the same changes and time course of MMP-1, TIMP-1, and TIMP-2 mRNA expression as exposure of the cells to latanoprost acid alone. Exposure of ciliary muscle cells to pilocarpine induced an increase in [Ca²⁺]_i, with the peak of increase observed at 5 s after initiation of treatment; then [Ca²⁺]_i gradually decreased near to baseline level within 10 min. Exposure of the cells to latanoprost acid did not significantly change [Ca²⁺]_i. Exposure of the cells to the mixtures of latanoprost acid and pilocarpine induced the same [Ca²⁺]_i change as exposure to pilocarpine alone. Conclusion: Latanoprost and pilocarpine have no interaction in their various effects on the cultured RCM cells.