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 Ki values for inhibition of Na+ flux by these agents (10 microM and 18 microM, respectively) are comparable to their Ki 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.