Effects of Long-Term Acidification of Extracellular pH on ATP-Induced Calcium Mobilization in Rabbit Lens Epithelial Cells

Abstract

ATP-induced calcium (Ca²⁺) mobilization was investigated in rabbit lens epithelial cells that had been cultured in a medium with pH of 7.4 (group 1), 7.2 (group 2), or 7.0 (group 3) for 10 to 21 d. Intracellular free Ca²⁺ ([Ca²⁺]_i) and pH (pH_i) were measured by using fluorescent dyes, fura-2 and BCECF, respectively. The long-term acidification decreased the pH_i to 7.15 ± 0.01, from 7.22 ± 0.01, in group 2 and to 7.09 ± 0.01 in group 3. The administration of 10 μmol/l ATP produced an initial peak followed by a sustained increase in [Ca²⁺]_i in the lens cells of group 1. Both the initial peak and the sustained increase in [Ca²⁺]_i were enhanced in groups 2 and 3. The initial peak was abolished by pretreatment with 1 μmol/l thapsigargin, an ER Ca²⁺ pump inhibitor, but was not affected by the removal of extracellular Ca²⁺. On the other hand, the sustained increase was suppressed either by the thapsigargin treatment or by the Ca²⁺ removal. Treatment with only thapsigargin caused a sustained increase in [Ca²⁺]_i that was greater in group 3 than in group 1. These results suggest that (1) the ATP-induced initial peak in [Ca²⁺]_i is due to Ca²⁺ release from the intracellular stores, (2) the sustained increase in [Ca²⁺]_i is mediated through either Ca²⁺ influx from the extracellular space or Ca²⁺ release from the store triggered by the Ca²⁺ influx, and (3) long-term, moderate acidification enhances both the initial peak and the sustained increase in [Ca²⁺]_i in rabbit lens epithelial cells. One possible mechanism of the ATP-induced Ca²⁺ influx seems to be a capacitative Ca²⁺ entry pathway.