Regulation and translocation of ATP-dependent apical membrane proteins in rat liver

Abstract

The bile canalicular membrane contains four specific ATP-dependent transport processes that are involved in secretion of bile acids, non-bile acid organic anions (mrp1), phospholipids (mdr2), and organic cations (mdr3). The aim of this study was to determine how the canalicular presence of these transport proteins is regulated. Canalicular membrane vesicles (CMV) were prepared from livers of rats treated with taurocholate (TC) and/or dibutyryl-adenosine 3',5'-cycle monophosphate (DBcAMP) with and without pretreatment with colchicine. Transport studies were performed with radiolabeled substrates. Changes in the relative amounts of transport proteins were determined by Western blots. Compared with controls, the specific activity of each of the transport processes was enhanced 1.5- and 3-fold with TC and DBcAMP treatments, respectively. Western blots revealed the same increases with mdr2 and mdr3. Pretreatment of rats with colchicine prevented these responses fully with TC treatment, whereas only partial prevention was obtained with DBcAMP treatment. Besides the ATP-dependent transporters, the relative specific activities of the canalicular membrane ectoenzyme markers, leucine aminopeptidase and gamma-glutamyltranspeptidase, were also affected the same way. These results suggest that TC and DBcAMP increase the specific activity of the canalicular ATP-dependent transport proteins and some canalicular membrane ectoenzymes by stimulating an increase in the relative amounts of these proteins in the membrane.

Hepatic sequestration and modulation of the canalicular transport of the organic cation, daunorubicin, in the rat

“…51,52 TC infusions in the intact rat also increase the specific activity and relative amounts of several ATP-dependent canalicular membrane transporters in isolated membrane vesicles. 22 Together, these findings support the conclusion that TC stimulates transcytosis and fusion of pericanalicular vesicles with the canalicular membrane, and that these vesicles contain transporters for the biliary excretion of a variety of substrates including DNR.…”

“…17,18 In the present study, we have used the fluorescent properties of this mdr1 substrate to assess the hepatobiliary excretion and intracellular compartmentalization of this compound, and to determine if these transport processes are regulated, as has been observed for the biliary excretion of organic anions. [19][20][21][22] The question is of general interest, because nearly 50% of therapeutic agents are organic cations or have cationic properties, and various amounts of these compounds are often eliminated into bile. 18 We found that DNR excretion in bile was extremely limited (Ϸ10%), in contrast to equivalent molar concentrations of a fluorescent bile acid derivative, CGamF, which is a substrate for the canalicular bsep, 12 and a fluorescent substrate for mrp2, GSMF, where approximately 100% and 60% of the administered dose was excreted in bile, respectively.…”

“…18 Previous studies have established that some of these excretory transport systems are highly regulated rather than constitutive functions of the hepatocyte, and that compounds such as N 6 ,2Ј-O-dibutyryl-adenosine 3Ј,5Јcyclic monophosphate (DBcAMP) and bile salts are capable of up-regulating their transport capacity. [19][20][21][22] In contrast, inhibitors of microtubule function down-regulate these transport processes. 19,21,23 This type of regulation has been demonstrated for the fluorescent bsep substrate, cholyglycylamidofluorescein (CGamF), and for the mrp2 substrate, glutathione-Smethylfluorescein.…”

“…20,21 These findings have led to the conclusion that the plasma membrane transport proteins may also reside on intracellular pericanalicular vesicles, and that apical transport capacity may be regulated by the insertion and/or retrieval of these vesicles to or from the apical canalicular membrane. 21,22,24,25 In the present study, we examined whether the canalicular excretion of the mdr1 transport system can be regulated using the fluorescent mdr1 substrate, daunorubicin (DNR). DNR is a naturally fluorescent organic cation that is widely used as an antineoplastic agent.…”

The bile salt export pump

“…This stimulation of bile salt secretion is independent from protein synthesis but sensitive to the microtubule disrupting agent colchicine, suggesting the presence of an intracellular regulatory pool of Bsep transporters. Treatment of rats with intravenous taurochcholate or dibutyryl cAMP resulted in a fast increase in functional Bsep insertion into canalicular plasma membranes [31]. Inhibition of this process by colchicine confirmed the postulated presence of an intracellular vesicular pool of Bsep, which upon stimulation is incorporated into the canalicular plasma membrane in a microtubule-dependent process.…”