Angiotensin II stimulates NHE3 activity by exocytic insertion of the transporter: role of PI 3-kinase.
BACKGROUND
Low-concentration angiotensin II (Ang II) stimulates Na+/H+ exchanger 3 (NHE3) activity in renal proximal tubule mainly via angiotensin II type 1 (AT1) receptors. The mechanisms that mediate the increase in NHE3 activity elicited by Ang II remain incompletely settled.


METHODS
To assess a potential role of NHE3 trafficking in the Ang II effect, NHE3 activity was measured by H+-driven initial rate of 22Na uptake resistant to 50 micromol/L of the Na+/H+ exchange inhibitor cariporide (HOE642), and sensitive to 300 micromol/L ethyl isopropyl amiloride (EIPA), in a model of cultured proximal tubular cells (MKCC), in which functional apical NHE3 and AT receptors are normally present. Apical expression of NHE3 protein was determined by cell surface biotinylation and immunoblotting.


RESULTS
Ang II (10-10 mol/L, 43 minutes) increased NHE3 activity and biotinylated NHE3 protein without any change in total amount of NHE3 protein. Both effects were suppressed by specific AT1 receptor antagonists. When 2-mercaptoethanesulphonic acid (MESNA) was used to cleave biotin from all apical proteins, intracellular biotinylated NHE3 protein remained unchanged after Ang II incubation compared to control. When sulfo-N-hydrosuccinimide (NHS)-acetate was used first to block all apical reactive sites, an increase in biotinylated NHE3 protein was observed following Ang II incubation. To evaluate the role of phosphatidylinositol 3-kinase (PI 3-kinase), the specific inhibitor wortmannin was used. It suppressed Ang II-induced increase in NHE3 activity and trafficking. Furthermore, latrunculin B, inhibitor of actin filament polymerization, prevented both Ang II stimulatory effects.


CONCLUSIONS
Ang II stimulates NHE3 activity, at least in part, by exocytic insertion of the protein into the apical membrane. This effect is mediated by PI 3-kinase and required integrity of actin cytoskeleton.
du Cheyron
Damien
D
Institut National de la Santé et de la Recherche Médicale, Paris, France. ducheyron-d@chu.caen.fr
Chalumeau
Cecile
C
Defontaine
Nadia
N
Klein
Christophe
C
Kellermann
Odile
O
Paillard
Michel
M
Poggioli
Josiane
J
D000199:Actins / Q000378:metabolism; D000804:Angiotensin II / Q000008:administration & dosage / Q000502:physiology*; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D004705:Endocytosis / Q000502:physiology; D005089:Exocytosis / Q000502:physiology*; D007672:Kidney Cortex / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology*; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism*
D000199:Actins; C000613386:Slc9a3 protein, mouse; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers; D000804:Angiotensin II; D019869:Phosphatidylinositol 3-Kinases