Intracellular pH in human fibroblasts: Effect of mitogens, A23187, and phospholipase activation

Serum-deprived human fibroblasts (HSWP cells) were loaded with either the fluorescent pH indicator 6-carboxy-4',5'-dimethylfluorescein or the calcium indicator quin 2, and the fluorescence of the intracellular probes was continuously monitored with a microspectrofluorometer. Addition of a cocktail of peptide growth factors causes intracellular alkalinization, which is blocked by amiloride or by replacement of extracellular Na⁺ with choline, confirming that mitogenic stimulation activates a Na⁺-H⁺ exchanger in HSWP cells. The exchanger is also activated by A23187, acid loading the cells, and stimulation of phospholipase activity with melittin. Growth factors and melittin activation of the exchanger have been demonstrated to be dependent on the mobilization of Ca²⁺ from intracellular stores. The intracellular alkalization and increase in Ca²⁺ activity due to both melittin and growth factors is inhibited by phospholipase inhibitors, indicating that phospholipase activity is necessary for the activation of the Na⁺-H⁺ exchanger and the mobilization of intracellular Ca²⁺.