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

Abstract

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. 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 ìÒ GTPãS. Pretreatment of CPAE cells with the Clostridium C2IN-C3 fusion toxin, which inactivatesRho 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¢ (n = 17) in pretreated cells compared with 172 ± 17 pA pF¢ (n = 21) in control cells. 5. The volume-independent activation of ICl,swell by intracellular perfusion with GTPãS was also impaired in C2IN-C3-pretreated cells (31 ± 7 pA pF¢, n = 11) compared with nontreated cells (132 ± 21 pA pF¢, n = 15). 6. Activation of ICl,swell was pertussis toxin (PTX) insensitive. 7. Y_27632, a blocker of Rho kinase, inhibited ICl,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.

Cholesterol modulates the volume-regulated anion current in Ehrlich-Lettre ascites cells via effects on Rho and F-actin

“…If this is so, one would expect that under normal conditions, swelling-induced Rho inhibition and F-actin depolymerization tend to attenuate VRAC activity, and hence, that cholesterol depletion may increase the maximal VRAC current (which was inhibited by LB, i.e., F-actin dependent, but unaffected by Y27632, i.e., not Rho kinase dependent), and the rate of VRAC activation (which was Rho kinase but not F-actin dependent) by increasing Rho activity and causing actin polymerization. These findings are in good agreement with previous reports by us and others showing that while the Rho-ROK pathway is not the volume-sensing mechanism per se, it has a potentiating effect on swelling-induced VRAC activity (7,41,42,47). In contrast to the hypotonic VRAC current, the isotonic current, which was also stimulated by cholesterol depletion, was potentiated by F-actin disruption and unaffected by Rho kinase inhibition.…”

“…Effect of Rho kinase inhibitor Y27632 on VRAC. The RhoRho kinase pathway is a major regulator of F-actin organization, and we and others have implicated this pathway in VRAC regulation (42,47). The maximal current magnitude after hypotonic swelling is similar in control cells and in cells treated with Y27632 to inhibit Rho kinase (Fig.…”

“…49) and have been implicated in the cytoskeletal reorganization elicited by cell volume perturbations (11,13) as well as in the regulation or modulation of VRAC by cell swelling (7,14,42,47). In vascular endothelial cells (7,42) and NIH3T3 cells (47) the activity of the Rho-associated kinase (Rho kinase; ROK) is required for VRAC activation. RhoA and ROK associate with rafts and caveola and translocate to caveolae in an activationdependent manner (62,65), raising the possibility that the effect of cholesterol depletion on VRAC might involve changes in Rho kinase activity.…”

Role of volume-regulated and calcium-activated anion channels in cell volume homeostasis, cancer and drug resistance

“…21,22 Furthermore, VRAC is also activated under isotonic conditions by an increase in the intracellular concentration of GTP-g-S, presumably by a mechanism that involves the small G-protein RhoA and F-actin polymerization. 23,24 In agreement with this, VRAC is in several cell types demonstrated to be Rho and Rho-kinase regulated. [23][24][25] Activation of VRAC following a modest hypoosmotic challenge is potentiated following cholesterol depletion (Fig.…”

“…23,24 In agreement with this, VRAC is in several cell types demonstrated to be Rho and Rho-kinase regulated. [23][24][25] Activation of VRAC following a modest hypoosmotic challenge is potentiated following cholesterol depletion (Fig. 9D) 26 apparently due to an increased open probability of the channel.…”