Up–Regulation of the Multidrug Resistance Genes, Mrp1 And Mdr1b , and Down–Regulation of the Organic Anion Transporter, Mrp2, and the Bile Salt Transporter, Spgp, in Endotoxemic Rat Liver

Abstract

Endotoxin-induced cholestasis is mainly caused by an impaired canalicular secretion. Mrp2, the canalicular multispecific organic anion transporter, is strongly downregulated in this situation, and canalicular bile salt secretion is also reduced. We hypothesized that other adenosine triphosphate-binding cassette (ABC) transporters may compensate for the decreased transport activity to protect the cell from cytokine-induced oxidative damage. Therefore, we examined the expression of ABC-transport proteins in membrane fractions of whole liver and of isolated hepatocytes of endotoxin-treated rats and performed reversetranscriptase polymerase chain reaction (RT-PCR) on mRNA isolated from these livers. In addition, the localization of these transporters was examined using confocal scanning laser microscopy. By 6 hours after endotoxin administration, we found a clear increase of mrp1 mRNA and protein, whereas mrp2 mRNA and protein were decreased. This was confirmed in isolated hepatocytes. In addition, mdr1b mRNA was strongly increased, whereas mdr1a and mdr2 mRNA did not change significantly. Both the mRNA and protein levels of the sister of P-glycoprotein (spgp), the recently cloned bile salt transporter, decreased. After endotoxin treatment, the normally sharply delineated canalicular staining of mrp2 and spgp had changed to a fuzzy pattern, suggesting localization in a subapical compartment. We conclude that endotoxin-induced cholestasis is caused by decreased mrp2 and spgp levels, as well as an abnormal localization of these proteins. The simultaneous up-regulation of mrp1 and mdr1b may confer resistance to hepatocytes against cytokine-induced metabolic stress. (HEPATOLOGY 1998;28:1637-1644.)Excretion of a large variety of endogenous and exogenous compounds from hepatocytes into bile is an adenosine triphosphate (ATP)-dependent process, predominantly performed by members of the P-glycoprotein (Pgp) subfamily and the multidrug-resistance protein (MRP) subfamily of the ATP-binding cassette (ABC) protein superfamily. 1,2 At least four members of the Pgp subfamily are located at the canalicular membrane of rodent liver: mdr1a, mdr1b, mdr2, and spgp. In normal rodent liver, mdr1a and mdr1b are present at low levels. Overexpression of mouse mdr1a/mdr1b confers multidrug resistance against a broad variety of natural product drugs. 3,4 The main physiological function of these multidrug-resistance proteins is presumably the transport of bulky amphiphilic compounds, such as cationic drugs, hydrophobic peptides, steroids, and atypical glycolipids, across the canalicular membrane. 1,4,5 The expression of mdr2 in normal rodent liver is high. This transporter functions as a flippase that translocates phosphatidylcholine across the membrane. 6 From pig liver, Childs et al. cloned part of another member of the Pgp subfamily: the sister gene of Pgp (spgp). 7 Most recently, Gerloff et al. cloned the full-length cDNA of spgp from rat liver. 8 They provided evidence that spgp, which is exclusively present in the liver, most likely is ...

Down-Regulation of Hepatic Transporters for BSP in Rats with Indomethacin-Induced Intestinal Injury

“…Western Blot Analysis Hepatic plasma membrane fractions were prepared as described previously 18) with modification. Briefly, the liver was homogenized with glass/glass homogenizer, and the liver homogenate was centrifuged at 1500 g for 15 min.…”

Activation of the Raf-1/MEK/ERK cascade by bile acids occurs via the epidermal growth factor receptor in primary rat hepatocytes

“…Membranes were prepared as described by Vos et al 21 Hepatocytes were treated and washed with cold Krebs buffer. They were then permeabilized in 1 mmol/L NaHCO 3 with phosphatase inhibitors for 1 hour at 4°C with shaking.…”

Regulation of the dynamic localization of the rat Bsep gene-encoded bile salt export pump by anisoosmolarity

“…These transport ATPases have been cloned and characterized at the molecular level. [5][6][7][8][9][10][11] Recent evidence suggests that expression of these pumps is regulated by lipopolysaccharide, [12][13][14] dexamethasone, 15 drugs, 16 and ambient osmolarity, 15,17 and gene defects of these pumps may underlie cholestatic liver disease, such as the Dubin Johnson syndrome 18 or subgroups of progressive familial intrahepatic cholestasis. [19][20][21][22] Short-term regulation of canalicular bile acid excretion in perfused rat liver involves effects of TUDCA 23 and liver cell hydration.…”