The purposes of this study are to characterize the pathophysiological effects of benzalkonium chloride and gentamicin on corneal epithelial cells and to determine the concentration dependent effect of these agents on membrane currents of these cells. Rabbit corneal epithelial cells were isolated and subdivided into small, medium and large cells according to their cell capacitance. Using whole cell clamp technique, potassium current of corneal epithelial cells was recorded. Transmembrane current was measured again after bathing in benzalkonium chloride 1, 3, 10, 30 and 100 micrometer ml(-1)for 3 min. The effect of gentamicin was tested at concentrations of 0.4, 1.0, 2.0, 4.0 and 10 mg ml(-1). Synergistic effect of gentamicin of the above mentioned concentrations in the presence of benzalkonium chloride 1 microgram ml(-1)was also measured. We found that small corneal epithelial cells had the highest depolarization-gated, outward potassium current density and large cells had the lowest current density, while medium cells had a current density in between. Benzalkonium chloride induced a concentration dependent increase in the leak current with increasing concentration from 1 to 100 microgram ml(-1). The increase of leak current in medium and large cells was less than that in the small cells. Gentamicin also caused a concentration dependent increase in leak current density from 0.4 to 10.0 mg ml(-1). The increase in leak current density was statistically significant when the concentration was 2.0 mg ml(-1)or higher in small corneal epithelial cells and 1.0 mg ml(-1)or higher in the medium and large cells. Benzalkonium chloride 1.0 microgram ml(-1)augmented the effect of gentamicin on epithelial cell membrane. The extent of enhancement was more prominent in larger than smaller cells. Using whole-cell clamp technique, we were able to determine the threshold concentration of gentamicin and benzalkonium chloride on the integrity of corneal epithelial cell membrane. The toxic action of both agents is mediated by an increase in leak current. We propose that the whole-cell clamp technique is a sensitive and useful tool in determining cytotoxic effects of various agents.
Copyright 2000 Academic Press.