α1-Adrenergic receptors activate NHE1 and NHE3 through distinct signaling pathways in epithelial cells

The Na+/H+ exchanger (NHE) regulates intracellular pH, cell volume, Na+ absorption and H+ secretion in epithelial cells of the renal proximal tubule (PT). alpha (1)-Adrenergic receptors (ARs) increase NHE activity in PT cells. The purpose of this study was to determine the mechanism of alpha (1)-AR activation of NHE isoforms expressed in PT cells. Northern and Western blotting demonstrate transcripts and protein expression of NHE1 and NHE3 in PT cells. An anti-NHE1 antibody predominately labels protein expressed at basal and lateral membranes. In contrast, NHE3 protein is expressed exclusively at the apical membrane. To determine NHE isoforms regulated by alpha (1)-ARs, antisense oligodeoxynucleotides (AS-ODNs) specific for NHE1 and NHE3 isoforms were introduced into cells with streptolysin O permeabilization. Cells incubated with AS-ODNs a total of three times exhibited a reduction in protein expression of similar to 85%. Na uptake and changes in intracellular pH (pH(i)) were used as measures of NHE activity in PT cells. alpha (1)-AR stimulation increased Na uptake from 8.5 to 13.8 nmol . min(-1) . mg protein(-1). AS-ODNs to NHE3 significantly reduced alpha (1)-AR stimulated Na uptake and increases in pHi; no effect was observed in sense-ODN-treated cells. Inhibition of NHE1 but not NHE3 expression abolishes amiloride-suppressible NHE activity. alpha (1)-AR stimulation of NHE1 is inhibited by the protein kinase C (PKC) inhibitor calphostin C whereas NHE3 activity is abolished by the mitogen-activated protein kinase (MAPK) inhibitor PD-98059. In PT cells transfected with MAPK kinase MEKK1(COOH), a truncated version of MEKK1 that activates MAPK, NHE3 but not NHE1 activity is stimulated. We conclude that alpha (1)-ARs activate distinct signaling pathways to regulate specific NHE isoforms localized on opposite membranes in polarized renal epithelial cells. alpha (1)-AR activation of NHE1 is regulated by PKC whereas NHE3 is controlled by MAPK and serves to separately regulate pH(i), Na absorption, and proton excretion in PT cells.