Role of Rho and Rho kinase in the activation of volume-regulated anion channels in bovine endothelial cells

1. We have studied the modulation of volume regulated anion channels (VRACs) by the small GTPase Rho and by one of its targets, Rho kinase, in calf pulmonary artery endothelial (CPAE) cells. 2. RT-PCR and immunoblot analysis showed that both RhoA and Rho kinase are expressed in CPAE cells. 3. I(Cl,swell), the chloride current through VRACs, was activated by challenging CPAE cells with a 25% hypotonic extracellular solution (HTS) or by intracellular perfusion with a pipette solution containing 100 μM GTPγS. 4. Pretreatment of CPAE cells with the Clostridium C2IN-C3 fusion toxin, which inactivates Rho by ADP ribosylation, significantly impaired the activation of I(Cl,swell) in response to the HTS. The current density at +100 mV was 49 ± 13 pA pF^-1 (n = 17) in pretreated cells compared with 172 ± 17 pA pF^-1 (n = 21) in control cells. 5. The volume-independent activation of I(Cl,swell) by intracellular perfusion with GTPγS was also impaired in C2IN-C3-pretreated cells (31 ± 7 pA pF^-1, n = 11) compared with nontreated cells (132 ± 21 pA pF^-1 n = 15). 6. Activation of I(Cl,swell) was pertussis toxin (PTX) insensitive. 7. Y-27632, a blocker of Rho kinase, inhibited I(Cl,swell) and delayed its activation. 8. Inhibition of Rho and of Rho kinase by the above-described treatments did not affect the extent of cell swelling in response to HTS. 9. These experiments provide strong evidence that the Rho-Rho kinase pathway is involved in the VRAC activation cascade.