Evidence for a role of phospholipase activity in the serum stimulation of Na⁺ influx in human fibroblasts

Serum stimulation of cultured human fibroblasts activates a Na⁺ influx via an amiloride-sensitive Na⁺/H⁺ exchange system. Evidence is presented which indicates that phospholipase activity is an important component of the mechanism by which mitogen receptor occupation leads to activation of Na⁺/H⁺ exchange. Serum stimulation of Na⁺ flux is effectively blocked by inhibitors of phospholipase activity such as mepacrine and U-1002. The K(i) values for inhibition of Na⁺ flux by these agents (10 μM and 18 μM, respectively) are comparable to their K(i) values for inhibition of serum-stimulated arachidonic acid release. In contrast, the activation of Na⁺ influx produced by the divalent cation A23187 is not affected by phospholipase inhibitors indicating that these agents specifically block mitogen activation of Na⁺ flux rather than nonspecifically disrupting the membrane's ability to perform Na⁺/H⁺ exchange. The phospholipase activator melittin stimulates Na⁺ influx in the absence of mitogens at concentrations that cause a comparable stimulation of arachidonic acid release. The melittin-stimulated Na⁺ influx is inhibited by amiloride and mepacrine, suggesting that melittin activation of phospholipase activity leads to the activation of the Na⁺/H⁺ exchange system. In addition, chronic treatment of cells with dexamethasone, which is known to induce an endogenous phospholipase inhibitor in human fibroblasts, leads to a substantial inhibition of the serum stimulation of both Na⁺ influx and arachidonic acid release. When taken together, these data support the involvement of phospholipase activity in the serum stimulation of the Na⁺/H⁺ exchange system.