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 pharmacological agents that activate cAMP-dependent protein kinase A (PKA) are potent inhibitors of native and ectopically expressed NHE3 in epithelial and Chinese hamster ovary AP-1 cells, respectively. Previous studies have shown that acute 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 cyclase, occurs without changes in surface expression of the exchanger but is associated with altered cytoskeletal structure. This effect resembles that obtained with cytochalasin D or latrunculin B, actin disrupting agents that also inhibit NHE3. Such similarities prompted us to further investigate the relationship between PKA-induced inhibition of the exchanger and changes in the actin cytoskeleton. Inhibition of NHE3 by cytochalasin D does not require PKA, because the inhibitory effect is preserved in a mutant NHE3 that is not phosphorylated by PKA and in cells pretreated with the PKA inhibitor H89. In contrast, involvement of actin in the effect of cAMP on the exchanger is supported by the following observations: (i) jasplakinolide, an F-actin stabilizer, prevents the inhibition caused by forskolin, and (ii) constitutively active forms of RhoA and Rho kinase interfere with actin disruption by forskolin and also decrease inhibition of the transporter. These results suggest that reorganization of the cytoskeleton by PKA is involved in mediating inhibition of NHE3.