Multiple Alterations of Canalicular Membrane Transport Activities in Rats with CCl₄-induced Hepatic Injury

Abstract

This article is available online at http://dmd.aspetjournals.org ABSTRACT:The influence of CCl 4 -induced experimental hepatic injury (CCl 4 -EHI) on the expression and transport activities of primary active transporters on the canalicular membrane, including P-glycoprotein (P-gp), a bile salt export pump (Bsep) and a multidrug resistance associated protein2 (Mrp2), was assessed. CCl 4 -EHI was induced by an intraperitoneal injection of CCl 4 to rats at a dose of 1 ml/kg 24 h prior to the preparation of canalicular liver plasma membrane (cLPM) vesicles and pharmacokinetic studies. The expression of each transporter was measured for the vesicles via Western blot analysis at 6, 12, 24, 36, and 48 h after the injection of and Mrp2, respectively, was determined following an i.v. infusion to rats. The uptake of each substrate into cLPM vesicles in the presence of ATP was also measured by a rapid filtration technique. As the result of the CCl 4 -EHI, the protein level of transporters was altered as a function of time in multiple manners; it was increased by 3.6-fold for P-gp, unchanged for Bsep, and decreased by 73% for Mrp2 at 24 h. The in vivo CL exc and the intrinsic uptake clearance into cLPM vesicles (CL int ) at 24 h after the CCl 4 injection (CCl 4 -EHI 24 h ) were also influenced by the EHI in a similar manner; they were increased by 1.8-and 1.9-fold for daunomycin, unchanged for taurocholate, and decreased by 41 and 39% for E 2 17␤G, respectively, consistent with multiple alterations in the expression of the relevant transporters.Primary active transporters in the liver canalicular membrane, which contain the ATP binding cassette (ABC 1 ), play an important role as efflux pumps in the excretion of endogenous bile constituents or xenobiotics into the bile canaliculi (Hooiveld et al., 2001). As might be expected, certain types of liver diseases have an influence on this hepatobiliary excretion. Experimental hepatic injury (EHI) induced by a single administration of carbon tetrachloride (CCl 4 ) has been widely used as a pathological model for liver diseases, since it is known that CCl 4 produces acute hepatocellular injury with centrilobular necrosis and steatosis (Recknagel, 1967). The effects of CCl 4 -EHI on biochemical characteristics such as increased lipid peroxidation (Recknagel, 1967) and the activities (Mourelle et al., 1987;Romero et al., 1994) of glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) and hepatobiliary excretion of xenobiotics (for example, sulfobromophthalein and leukotriene; Iga et al., 1977;Wettstern et al., 1990) have been widely studied. Our earlier study revealed that transport systems for some organic cations on the sinusoidal membrane are prone to damage as the result of CCl 4 -EHI, as demonstrated by a decrease in the in vitro maximal rates of hepatic uptake and efflux without any influence on in vivo canalicular excretion clearance (Hong et al., 2000). In addition, a dose-dependent inhibition of Na ϩ /taurocholate cotransport (Ntcp) into sinusoidal m...

Altered Pharmacokinetics of Daunorubicin in Rats with CCl4-Induced Hepatic Injury

“…The expression of multidrug-resistance related protein (Mrp2) is not affected for hepatic microsomes (14), whereas a significant decrease is observed for the canalicular membrane (15). In addition, our previous studies has revealed that the in vitro transport of organic cations across the sinusoidal membrane is decreased as a result of CCl 4 -EHI (16), while the transport activity of Pglycoprotein (P-gp) in the canalicular membrane is increased by CCl 4 -EHI (15).…”

“…The concentrations of daunorubicin in plasma, bile and urine, as well as those of daunorubicinol in plasma, were quantified by HPLC with a fluorescence detector, as previously described (15). Briefly, a 100-µL aliquot of the biological samples (i.e., plasma, bile, and urine) was deproteinized by adding 250 µL of methanol and 1 mL ethyl acetate and centrifuging at 10,000 rpm for 5 min.…”

“…Canalicular liver plasma membrane (cLPM) vesicles were prepared from control (n=3) and CCl 4 -EHI rats (n=3) according to the method of Inoue et al (21) and characterized as described previously (15). Uptake was initiated by adding 40 µL of membrane suspension buffer (MSB, 250 mM sucrose, 10 mM Hepes, 10 mM Tris, 10 mM MgCl 2 , and 0.2 mM CaCl 2 , pH 7.4) containing 0.2 µM daunorubicin hydrochloride (0.035 µCi) with an ATP-regenerating system (1.2 mM ATP, 3 mM phosphocreatine, and 3.6 µg/100 µL creatine phosphokinase) to the preincubated vesicle suspension.…”

“…Recently, multiple alterations in the expression of organic anion transporters by EHI have been reported. For example, the expression of Na + /taurocholate cotransporting polypeptide (ntcp), organic anion transporting polypeptide 1 (Oatp1), and Oatp2 decreases, whereas .that .Oatp4 .and .bile .salt .export .pump ________________________________________ (Bsep) remains unchanged (14,15). The expression of multidrug-resistance related protein (Mrp2) is not affected for hepatic microsomes (14), whereas a significant decrease is observed for the canalicular membrane (15).…”

“…For example, the expression of Na + /taurocholate cotransporting polypeptide (ntcp), organic anion transporting polypeptide 1 (Oatp1), and Oatp2 decreases, whereas .that .Oatp4 .and .bile .salt .export .pump ________________________________________ (Bsep) remains unchanged (14,15). The expression of multidrug-resistance related protein (Mrp2) is not affected for hepatic microsomes (14), whereas a significant decrease is observed for the canalicular membrane (15). In addition, our previous studies has revealed that the in vitro transport of organic cations across the sinusoidal membrane is decreased as a result of CCl 4 -EHI (16), while the transport activity of Pglycoprotein (P-gp) in the canalicular membrane is increased by CCl 4 -EHI (15).…”