Interactions of intracellular pH and intracellular calcium in primary cultures of rabbit corneal epithelial cells

Homeostasis of intracellular calcium ([Ca++](i)) and pH (pH(i)) is important in the cell's ability to respond to growth factors, to initiate differentiation and proliferation, and to maintain normal metabolic pathways. Because of the importance of these ions to cellular functions, we investigated the effects of changes of [Ca++](i) and pH(i) on each other in primary culture of rabbit corneal epithelial cells. Digitized fluorescence imaging was used to measure [Ca++](i) with fura-2 and pH(i) with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Resting pH(i) in these cells was 7.37 +/- 0.05 (n = 20 cells) and resting [Ca++](i) was 129 +/- 10 nM (n = 35 cells) using a nominally bicarbonate-free Krebs Ringer HEPES buffer (KRHB), pH 7.4. On exposure to 20 nM NH4Cl, which rapidly alkalinized cells by 0.45 pH units, an increase in [Ca++](i) to 215 +/- 14 nM occurred. Pretreatment of the cells with 100 mu M verapamil or exposure to 1 mM ethylene bis-(oxyethylenenitrilo)-tetraacetic acid (EGTA) without extracellular calcium before addition of 20 mM NH4Cl did not abolish the calcium increase, suggesting that the source of the calcium transient was from intracellular calcium stores. On removal of NH4Cl or addition of 20 mM sodium lactate, there were minimal changes in calcium even though pH(i) decreased. Treatment of CE cells with the calcium ionophores, ionomycin and 4-bromo A23187, increased [Ca++](i), but produced a biphasic change in pH(i). Initially, there was an acidification of the cytosol, and then an alkalinization of 0.10 to 0.11 pH units above initial values. When [Ca++](i) was decreased by treating the cells with 5 mM EGTA and 20 mu M ionomycin, pH(i) decreased by 0.35 +/- 0.02 units. We conclude that an increase in pHi leads to an increase in [Ca++](i) in rabbit corneal epithelial cells; however, a decrease in pH(i) leads to minor changes in [Ca++](i). The ability of CE cells to maintain proper calcium homeostasis when pH(i) is decreased may represent an adaptive mechanism to maintain physiological calcium levels during periods of acidification, which occur during prolonged eye closure.