ABSTRACT
The sodium hydrogen exchanger isoform 1 (NHE1) is present in nearly all cells. Regulation of proton flux via the exchanger is a permissive step in cell growth and tumorgenesis and is vital in control of cell volume. The regulation of NHE1 by growth factors involves the Ras-extracellular signal regulated kinase (ERK) pathway, however, the mechanism for G protein-coupled receptor (GPCR) activation of NHE1 is not well established. In this report, the relationship between GPCRs, ERK, and NHE1 in CCL39 cells is investigated. We give evidence that two agonists, the specific alpha(1)-adrenergic agonist, phenylephrine and the water-soluble lipid mitogen, lysophosphatidic acid (LPA) activate NHE1 in CCL39 cells. Activation of ERK by phenylephrine and LPA occurs in a dose- and time-dependent manner. Optimal ERK activation was observed at 10 min and displayed a maximum stimulation at 100 microM phenylephrine and 10 microM LPA. alpha(1)-Adrenergic stimulation also led to a rise in steady-state pH(i) of 0.16+/-0.02 pH units, and incubation with LPA induced a 0.43+/-0.06 pH unit increase in pH(i). Phenylephrine-induced activation of NHE1 transport and ERK activity was inhibited by pretreating the cells with the MEK inhibitor PD98059. While only half of the LPA activatable exchange activity was abolished by PD98059 and U0126. To further demonstrate the specificity of the phenylephrine and LPA regulation of NHE1 and ERK, CCL39 cells were transfected with a kinase inactive MEK. The data indicate that ERK activation is essential for phenylephrine stimulation of NHE1, and that ERK and RhoA are involved in LPA stimulation of NHE1 by more than one mechanism. In addition, evidence of the convergence of these two pathways is shown by the loss of NHE1 activity when both pathways are inhibited and by the partial additivity of the two agonists on ERK and NHE1 activity. These studies indicate a direct involvement of ERK in the alpha(1)-adrenergic activation of NHE1 and a significant role for both ERK and RhoA in LPA stimulation of NHE1 in CCL39 fibroblasts.
