Chronic ethanol treatment impairs Rac and Cdc42 activation in rat hepatocytes.
BACKGROUND
The effects of chronic ethanol feeding on rat hepatocytes have been shown to include impaired cell-extracellular matrix (ECM) adhesion events, such as decreased attachment and spreading as well as increased integrin-actin cytoskeleton association. These results, observed previously by this laboratory, are highly suggestive of impaired actin cytoskeleton reorganization, an event mediated by differential activation of the Rho family GTPases Rac, Cdc42, and RhoA. Therefore, the purpose of this study was to examine the effects of chronic ethanol administration on these GTPases.


METHODS
Male Wistar rats were pair-fed 4 to 5 weeks with a liquid diet containing either ethanol (as 36% of total calories) or isocaloric carbohydrate. Hepatocytes were isolated and plated on collagen IV up to 24 hours. At specific times, the hepatocytes were lysed and these lysates were analyzed for RhoA, Cdc42, and Rac activation.


RESULTS
In freshly isolated hepatocytes from ethanol-fed rats, the GTP-bound (active) forms of Rac and Cdc42 were significantly decreased compared with pair-fed control rats, while the GTP-bound form of RhoA was not significantly altered. These ethanol-induced impairments in Rac and Cdc42 activation persisted even after plating the hepatocytes on collagen IV. Additionally, chronic ethanol treatment did not directly affect GTP binding of Cdc42 and Rac, as incorporation of GTPgammaS was not affected.


CONCLUSIONS
Chronic ethanol administration selectively impairs Rac and Cdc42 activation in rat hepatocytes. As activation of these 2 GTPases is crucial for efficient cell attachment and spreading on ECM substrates, the results from this study suggest that the ethanol-induced impairments in Rac and Cdc42 activation are responsible for the impaired hepatocyte-ECM adhesion events observed previously by our laboratory. Furthermore, these results raise the intriguing possibility that these GTPases are involved in other ethanol-induced functional impairments, such as protein trafficking and receptor-mediated endocytosis.
Schaffert
Courtney S
CS
Department of Veterans Affairs Medical Center, Omaha, Nebraska 68105, USA. cschaffe@juno.com
Todero
Sandra L
SL
Casey
Carol A
CA
Thiele
Geoffrey M
GM
Sorrell
Michael F
MF
Tuma
Dean J
DJ
D000818:Animals; D002448:Cell Adhesion / Q000187:drug effects; D018929:Cell Culture Techniques; D024141:Collagen Type IV; D000431:Ethanol / Q000494:pharmacology*; D022781:Hepatocytes / Q000187:drug effects*; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D015398:Signal Transduction / Q000187:drug effects; D020764:cdc42 GTP-Binding Protein / Q000187:drug effects*; D020744:rac GTP-Binding Proteins / Q000187:drug effects*; D020742:rhoA GTP-Binding Protein / Q000187:drug effects
D024141:Collagen Type IV; D000431:Ethanol; D020764:cdc42 GTP-Binding Protein; D020744:rac GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein