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Role of Rho and Rho kinase in the activation of volume‐regulated anion channels in bovine endothelial cells
Author(s) -
Nilius Bernd,
Voets Thomas,
Prenen Jean,
Barth Holger,
Aktories Klaus,
Kaibuchi Kozo,
Droogmans Guy,
Eggermont Jan
Publication year - 1999
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1111/j.1469-7793.1999.067aa.x
Subject(s) - rhoa , rho kinase inhibitor , rho associated protein kinase , chemistry , extracellular , fasudil , intracellular , pertussis toxin , kinase , microbiology and biotechnology , biophysics , g protein , biochemistry , biology , signal transduction
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 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 non‐treated 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.