B. Nilius et al., Role of Rho and Rho kinase in the activation of volume-regulated anion channels in bovine endothelial cells, J PHYSL LON, 516(1), 1999, pp. 67-74
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 pu
lmonary 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 chall
enging CPAE cells with a 25% hypotonic extracellular solution (HTS) or by i
ntracellular perfusion with a pipette solution containing 100 mu M GTP gamm
a S.
4. Pretreatment of CPAE cells with the Clostridium C2IN-C3 fusion toxin, wh
ich inactivates Rho by ADP ribosylation, significantly impaired the activat
ion 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 perfu
sion 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 di
d not affect the extent of cell swelling in response to HTS.
9. These experiments provide strong evidence that the Rho-Rho kinase pathwa
y is involved in the VRAC activation cascade.