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First published online on April 26, 2002.
 Copyright © 2002 by The Physiological Society
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Received February 14, 2002
Accepted after revision March 27, 2002

Rho family GTP binding proteins are involved in the regulatory volume decrease process in NIH3T3 mouse fibroblasts

Stine F. Pedersen1, Kristine H. Beisner1, Charlotte Hougaard1, Berthe M. Willumsen2, Ian H. Lambert1, and E. K. Hoffmann3*

1 Department of Biochemistry, August Krogh Institute, University of Copenhagen, Denmark
2 Department of Molecular Cell Biology, University of Copenhagen, Denmark
3 August Krogh Institute,13, Universitetsparken, Dk-2100 Copenhagen, Denmark

* To whom correspondence should be addressed. E-mail: ekhoffmann{at}aki.ku.dk.

The role of Rho GTPases in the regulatory volume decrease (RVD) process following osmotic cell swelling is controversial and has so far only been investigated for the swelling-activated Cl- efflux. We investigated the involvement of RhoA in the RVD process in NIH3T3 mouse fibroblasts, using wild-type cells and three clones expressing constitutively active RhoA (RhoAV14). RhoAV14 expression resulted in an up to fourfold increase in the rate of RVD, measured by large-angle light scattering. The increase in RVD rate correlated with RhoAV14 expression. RVD in wild-type cells was unaffected by the Rho kinase inhibitor Y-27632 and the phosphatidyl-inositol 3 kinase (PI3K) inhibitor wortmannin. The maximal rates of swelling-activated K+ (86Rb+ as tracer) and taurine ([3H]taurine as tracer) efflux after a 30 % reduction in extracellular osmolarity were increased about twofold in cells with maximal RhoAV14 expression compared to wild-type cells, but were unaffected by Y-27632. The volume set points for activation of release of both osmolytes appeared to be reduced by RhoAV14 expression. The maximal taurine efflux rate constant was potentiated by the tyrosine phosphatase inhibitor Na3VO4, and inhibited by the tyrosine kinase inhibitor genistein. The magnitude of the swelling-activated Cl- current (ICl,swell ) was higher in RhoAV14 than in wild-type cells after a 7.5 % reduction in extracellular osmolarity, but, in contrast to 86Rb+ and [3H]taurine efflux, similar in both strains after a 30 % reduction in extracellular osmolarity. ICl,swell was inhibited by Y-27632 and strongly potentiated by the myosin light chain kinase inhibitors ML-7 and AV25. It is suggested that RhoA, although not the volume sensor per se, is an important upstream modulator shared by multiple swelling-activated channels on which RhoA exerts its effects via divergent signalling pathways.




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