Submitted on May 18, 2004
Accepted on September 8, 2004
ROLE OF MICROTUBULES IN ESTRADIOL-17
-D-GLUCURONIDE-INDUCED ALTERATION OF CANALICULAR MRP2 LOCALIZATION AND ACTIVITY
Aldo D. Mottino1, Fernando A. Crocenzi2, Enrique J. Sanchez Pozzi2, Luis M. Veggi2, Marcelo G. Roma2, and Mary Vore3*
1 Graduate Center for Toxicology, University of Kentucky, Lexington, Kentucky, USA; Institute of Experimental Physiology, National University of Rosario, Argentina
2 Institute of Experimental Physiology, National University of Rosario, Argentina
3 Graduate Center for Toxicology, University of Kentucky, Lexington, Kentucky, USA
* To whom correspondence should be addressed. E-mail: maryv{at}pop.uky.edu.
Estradiol-17
-D-glucuronide (E2-17G) induces a marked but reversible inhibition of
bile flow in the rat together with endocytic retrieval of Mrp2 from the canalicular
membrane to intracellular structures. We analyzed the effect of pretreatment (100
min) with the microtubule inhibitor, colchicine, or lumicholchicine, its inactive
isomer (1 µmol/kg, i.v.), on changes in bile flow and localization and function of
Mrp2 induced by E2-17G (15 µmol/kg, i.v.). Bile flow and biliary excretion of
bilirubin, an endogenous Mrp2 substrate, were measured throughout, while Mrp2
localization was examined at 20 and 120 min after E2-17G by confocal
immunofluorescence microscopy and Western analysis. Colchicine pretreatment
alone did not affect bile flow or Mrp2 localization and activity over the short time
scale examined (3-4 hr). Administration of E2-17G to colchicine-pretreated rats
induced a marked decrease (85%) in bile flow and biliary excretion of bilirubin as
well as internalization of Mrp2 at 20 min. These alterations were of a similar
magnitude as in rats pretreated with lumicolchicine followed by E2-17G. Bile flow
and Mrp2 localization and activity were restored to control levels within 120
minutes of E2-17G in animals pretreated with lumicolchicine. In contrast, in
colchicine-pretreated rats followed by E2-17G, bile flow and Mrp2 activity remained
significantly inhibited by 60%, and confocal and Western studies revealed
sustained internalization of Mrp2 120 min after E2-17G. We conclude that recovery
from E2-17G cholestasis, associated with exocytic insertion of Mrp2 in the
canalicular membrane, but not its initial E2-17G-induced endocytosis, is a
microtubule-dependent process.
