Constitutively active phosphatidylinositol 3-kinase and AKT are sufficientto stimulate the epithelial Na+/H+ exchanger 3

Phosphatidylinositol 3-kinase (PI 3-kinase) is a cytoplasmic signaling mole cule that is recruited to activated growth factor receptors and has been sh own to be involved in regulation of stimulated exocytosis and endocytosis. One of the downstream signaling molecules activated by PI 3-kinase is the p rotein kinase Akt. Previous studies have indicated that PI 3-kinase is nece ssary for basal Na+/H+ exchanger 3 (NHE3) transport and for fibroblast grow th factor-stimulated NHE3 activity in PS120 fibroblasts. However, it is not known whether activation of PI 3-kinase is sufficient to stimulate NHE3 ac tivity or whether Akt is involved in this PI 3-kinase effect. We used an ad enoviral infection system to test the possibility that activation of PI 3-k inase or Akt alone is sufficient to stimulate NHE3 activity. This hypothesi s was investigated in PS120 fibroblasts stably expressing NHE3 after somati c gene transfer using a replication-deficient recombinant adenovirus contai ning constitutively active catalytic subunit of PI 3-kinase or constitutive ly active Akt. The adenovirus construct used was engineered with an upstrea m ecdysone promoter to allow time-regulated expression. Adenoviral infectio n was nearly 100% at 48 h after infection. Forty-eight hours after infectio n (24 It after activation of the ecdysone promoter), PI 3-kinase and Akt am ount and activity were increased. Increases in both PI 3-kinase activity an d Akt activity stimulated NHE3 transport. In addition, a membrane-permeant synthetic 10-mer peptide that binds polyphosphoinositides and increases PI 3-kinase activity similarly enhanced NHE3 transport activity and also incre ased the percentage of NHE3 on the plasma membrane. The magnitudes of stimu lation of NHE3 by constitutively active PI 3-kinase, PI 3-kinase peptide, a nd constitutively active Akt were similar to each other. These results demo nstrate that activation of PI 3-kinase or Akt is sufficient to stimulate NH E3 transport activity in PS120/NHE3 cells.