Role of the cytoskeleton in mediating cAMP-dependent protein kinase inhibition of the epithelial Na+/H+ exchanger NHE3

The Na+/H+ exchanger NHE3 isoform mediates the entry of Na+ into epithelial cells of the kidney and gastrointestinal tract. Hormones and pharmacologic al agents that activate cAMP-dependent protein kinase A (PKA) are potent in hibitors of native and ectopically expressed NHE3 in epithelial and Chinese hamster ovary AP-1 cells, respectively. Previous studies have shown that a cute inhibition is coupled to direct phosphorylation of the exchanger, but this only partly accounts for the observed effects. In this report, we show that inhibition of NHE3 activity by forskolin, an activator of adenylate c yclase, occurs without changes in surface expression of the exchanger but i s associated with altered cytoskeletal structure. This effect resembles tha t obtained with cytochalasin D or latrunculin B, actin disrupting agents th at also inhibit NHE3. Such similarities prompted us to further investigate the relationship between PKA-induced inhibition of the exchanger and change s in the actin cytoskeleton. Inhibition of NHE3 by cytochalasin D does not require PKA, because the inhibitory effect is preserved in a mutant NHE3 th at is not phosphorylated by PKA and in cells pretreated with the PKA inhibi tor H89. In contrast, involvement of actin in the effect of cAMP on the exc hanger is supported by the following observations: (i) jasplakinolide, an F -actin stabilizer, prevents the inhibition caused by forskolin, and (ii) co nstitutively active forms of RhoA and Rho kinase interfere with actin disru ption by forskolin and also decrease inhibition of the transporter. These r esults suggest that reorganization of the cytoskeleton by PKA is involved i n mediating inhibition of NHE3.