Purchase this article with an account.
Y. Guo, G. Wilson, S.P. Srinivas; Benzalkonium Chloride Induced ATP Release from Cultured Bovine Corneal Endothelium . Invest. Ophthalmol. Vis. Sci. 2003;44(13):4738.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose: Benzalkonium Chloride (BAK) is a disinfectant in many ophthalmic preparations for topical administration. This study has investigated BAK-induced ATP release from bovine corneal endothelial cells (BCEC) as a potential measure of plasma membrane damage to ocular surface epithelia. Methods: ATP release was assayed in realtime using Luciferase-Luciferrin (LL) bioluminescence technique. Second passage BCEC cells, grown on 35 mm Petri dishes and bathed in serum free medium (OPTIMEMTM, BRL), containing 4 mg/mL of LL were exposed to BAK at concentrations ~ 0.001%. The luminescence passing through the bottom of the Petri dish was directed to the cathode of a cooled photomultiplier tube. The output of the photomultiplier in the form of TTL pulses was counted by a high speed (80 MHz) timer-counter interface in a PC. All experiments were conducted at room temperature (24 °C). Results: Independent calibration experiments in cell-free Petri dishes showed linear increase in luminescence count with increasing ATP content over 4 orders of magnitude (10-13 moles to 10-9 moles; 66 counts/picomole; r = 0.99). The sensitivity of the assay was significantly reduced by the presence of BAK (38 counts/picomole; r = 0.99). Exposure of BCEC cells to BAK resulted in a precipitous increase in the luminescence count. This increase was transient and declined exponentially to baseline values in about 5 minutes. The magnitude of the peak was variable with photon counts ranging from 4000 to 30000, corresponding to 104 picomoles to 788 picomoles of ATP (mean ± SE= 688 ±148 picomoles; n = 8). The rate of decline in luminescence was significantly greater than the decline noticed in the calibration experiments. In the absence of BAK, ATP level in the bathing medium was 3 picomoles (n= 10). This level increased by 4-fold maximum upon stirring of the bathing medium. Conclusions: The big and immediate increase in the luminescence is indicative of ATP release through membrane damage rather than through potential mechanisms involving intracellular damage or activation of anion channels. This direct damage to the plasma membrane is consistent with the fact that BAK is a cationic surfactant. The increased rate of decline in the luminescence during BAK exposure indicates that ecto-apyrases or released phosphotases may be involved in the degradation of released ATP.
This PDF is available to Subscribers Only