alpha(1)-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 ~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 pH(i); 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.
Liu
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Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, New Hampshire 03755, USA.
Gesek
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FA
D000818:Animals; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000378:metabolism; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000502:physiology; D011493:Protein Kinase C / Q000502:physiology; D012333:RNA, Messenger / Q000378:metabolism; D011942:Receptors, Adrenergic, alpha / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology*; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics / Q000378:metabolism*
D012333:RNA, Messenger; D011942:Receptors, Adrenergic, alpha; C000613386:Slc9a3 protein, mouse; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers; C069139:growth factor-activatable Na-H exchanger NHE-1; D011493:Protein Kinase C; D020928:Mitogen-Activated Protein Kinases