ACTIVATION OF THE OSMO-SENSITIVE CHLORIDE CONDUCTANCE INVOLVES P21(RHO) AND IS ACCOMPANIED BY A TRANSIENT REORGANIZATION OF THE F-ACTIN CYTOSKELETON

Hypo-osmotic stimulation of human Intestine 407 cells rapidly activate d compensatory Cl- and K+ conductances that limited excessive cell swe lling and, finally, restored the original cell volume. Osmotic eel swe lling was accompanied by a rapid and transient reorganization of the F -actin cytoskeleton, affecting both stress fibers as well as apical ru ffles. In addition, an increase in total cellular F-actin was observed . Pretreatment of the cells with recombinant Clostridium botulinum C3 exoenzyme, but not with mutant enzyme (C3-E173Q) devoid of ADP-ribosyl transferase activity, greatly reduced the activation of the osmo-sensi tive anion efflux, suggesting a role for the ras-related GTPase p21(rh o). In contrast, introducing dominant negative N17-p21(rac) into the c ells did not affect the volume-sensitive efflux. Cell swelling-induced reorganization of F-actin coincided with a transient, C3 exoenzyme-se nsitive tyrosine phosphorylation of p125 focal adhesion kinase (p125(F AK)) as well as with an increase in phosphatidylinositol-3-kinase (Ptd Ins-3-kinase) activity. Pretreatment of the cells with wortmannin, a s pecific inhibitor of PtdIns-3-kinase, largely inhibited the volume-sen sitive ion efflux. Taken together, our results indicate the involvemen t of a p21(rho) signaling cascade and actin filaments in the activatio n of volume-sensitive chloride channels.