Role of Rho and Rho kinase in the activation of volume-regulated anion channels in bovine endothelial cells
Corresponding author B. Nilius: Katholieke Universiteit Leuven, Laboratorium voor Fysiologie, Campus Gasthuisberg O&N, B-3000 Leuven, Belgium. Email: firstname.lastname@example.org
- 1We 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.
- 2RT-PCR and immunoblot analysis showed that both RhoA and Rho kinase are expressed in CPAE cells.
- 3I 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.
- 4Pretreatment of CPAE cells with the Clostridium C2IN-C3 fusion toxin, which inactivates Rho by ADP ribosylation, significantly impaired the activation of ICl,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.
- 5The volume-independent activation of ICl,swell by intracellular perfusion with GTPγS 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).
- 6Activation of ICl,swell was pertussis toxin (PTX) insensitive.
- 7Y-27632, a blocker of Rho kinase, inhibited ICl,swell and delayed its activation.
- 8Inhibition of Rho and of Rho kinase by the above-described treatments did not affect the extent of cell swelling in response to HTS.
- 9These experiments provide strong evidence that the Rho-Rho kinase pathway is involved in the VRAC activation cascade.