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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.
RT-PCR and immunoblot analysis showed that both RhoA and Rho kinase are expressed in CPAE cells.
ICl,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 GTP
S.
Pretreatment 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.
The volume-independent activation of ICl,swell by intracellular perfusion with GTPS 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).
Activation of ICl,swell was pertussis toxin (PTX) insensitive.
Y-27632, a blocker of Rho kinase, inhibited ICl,swell and delayed its activation.
Inhibition of Rho and of Rho kinase by the above-described treatments did not affect the extent of cell swelling in response to HTS.
These experiments provide strong evidence that the Rho-Rho kinase pathway is involved in the VRAC activation cascade.
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