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,I-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 mu M GTP gamma S. 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,I-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,I-swell by intracellular perfusion with GTP gamma S was also impaired in C2IN-C3-pretreated cells (31 +/- 7 pA pF(-1), n = 11) compared with non treated cells (132 +/- 21 pA pF(-1) n = 15). 6. Activation of I-Cl,I-swell was pertussis toxin (PTX) insensitive. 7. Y-27632, a blocker of Rho kinase, inhibited I-Cl,I-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.