MAB4140 Sigma-AldrichAnti-MDR3 Antibody, clone P3 II-26

Peroxisome proliferator-activated receptor alpha (PPARalpha) is a nuclear receptor that controls expression of genes involved in lipid metabolism and is activated by fatty acids and hypolipidaemic fibrates. Fibrates induce the hepatic expression of murine multidrug resistance 2 ( Mdr2 ), encoding the canalicular phospholipid translocator. The physiological role of PPARalpha in regulation of Mdr2 and other genes involved in bile formation is unknown. We found no differences in hepatic expression of the ATP binding cassette transporter genes Mdr2, Bsep (bile salt export pump), Mdr1a / 1b, Abca1 and Abcg5 / Abcg8 (implicated in cholesterol transport), the bile salt-uptake systems Ntcp (Na(+)-taurocholate co-transporting polypeptide gene) and Oatp1 (organic anion-transporting polypeptide 1 gene) or in bile formation between wild-type and Ppar alpha((-/-)) mice. Upon treatment of wild-type mice with ciprofibrate (0.05%, w/w, in diet for 2 weeks), the expression of Mdr2 (+3-fold), Mdr1a (+6-fold) and Mdr1b (+11-fold) mRNAs was clearly induced, while that of Oatp1 (-5-fold) was reduced. Mdr2 protein levels were increased, whereas Bsep, Ntcp and Oatp1 were drastically decreased. Exposure of cultured wild-type mouse hepatocytes to PPARalpha agonists specifically induced Mdr2 mRNA levels and did not affect expression of Mdr1a / 1b. Altered transporter expression in fibrate-treated wild-type mice was associated with a approximately 400% increase in bile flow: secretion of phospholipids and cholesterol was increased only during high-bile-salt infusions. No fibrate effects were observed in Ppar alpha((-/-)) mice. In conclusion, our results show that basal bile formation is not affected by PPARalpha deficiency in mice. The induction of Mdr2 mRNA and Mdr2 protein levels by fibrates is mediated by PPARalpha, while the induction of Mdr1a / 1b in vivo probably reflects a secondary phenomenon related to chronic PPARalpha activation.

Tumor cells may display a multidrug resistance phenotype by overexpression of ATP binding cassette transporter genes such as multidrug resistance (MDR) 1 P-glycoprotein (P-gp) or the multidrug resistance protein 1 (MRP1). MDR3 P-gp is a close homologue of MDR1 P-gp, but its role in MDR is probably minor and remains to be established. The MRP1 protein belongs to a family of at least six members. Three of these, i.e., MRP1, MRP2, and MRP3, can transport MDR drugs and could be involved in MDR. The substrate specificity of the other family members remains to be defined. Specific monoclonal antibodies are required for wide-scale studies on the putative contribution of these closely related transporter proteins to MDR. In this report, we describe the extensive characterization of a panel of monoclonal antibodies (Mabs) detecting several MDR-related transporter proteins in both human and animal tissues. The panel consists of P3II-1 and P3II-26 for MDR3 P-gp; MRPr1, MRPm6, MRPm5, and MIB6 for MRP1; M2I-4, M2II-12, M2III-5 and M2III-6 for MRP2; M3II-9 and M3II-21 for MRP3; and M5I-1 and M5II-54 for MRP5. All Mabs in the panel appeared to be fully specific for their cognate antigens, both in Western blots and cytospin preparations, as revealed by lack of cross-reactivity with any of the other family members. Indeed, all Mabs were very effective in detecting their respective antigens in cytospins of transfected cell lines, whereas in flow cytometric and immunohistochemical analyses, distinct differences in reactivity and suitability were noted. These Mabs should become valuable tools in studying the physiological functions of these transporter proteins, in screening procedures for the absence of these proteins in hereditary metabolic (liver) diseases, and in studying the possible contributions of these molecules to MDR in cancer patients.

BACKGROUND & AIMS: Biliary cholesterol secretion is coupled to that of phospholipids in a process controlled by mdr2 P-glycoprotein activity and bile salt secretion. Statins, the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors, have been shown to affect hepatobiliary lipid secretion in rats. The aim of this study was to relate the effects of statins on bile formation to the expression of mdr2 and other hepatic adenosine triphosphate-dependent transport proteins involved in bile formation in rats. METHODS: Rats received simvastatin- or pravastatin-containing chow continuously for 5 days. In one group of rats, simvastatin treatment was withdrawn 9-12 hours before the end of the experiment to induce biliary cholesterol hypersecretion (rebound). Bile and liver tissue were collected for lipid analysis, and hepatic messenger RNA (mRNA) and protein levels were studied by reverse-transcription polymerase chain reaction, immunoblotting, and immunohistochemistry. RESULTS: Simvastatin feeding did not alter biliary bile salt secretion. Secretion of phospholipids and cholesterol was stimulated by 74% and 90%, respectively, in the simvastatin-continuous group and by 72% and 235%, respectively, in the rebound group compared with controls. mdr2 mRNA levels increased only in the continuous group. mdr2 protein levels increased in both simvastatin-fed groups. Induction was most pronounced in periportal hepatocytes. mdr1b mRNA levels were moderately increased in both simvastatin-fed groups. Levels of other hepatic transport proteins did not change. Similar results were obtained in pravastatin-fed rats. CONCLUSIONS: Statins increase expression of mdr2 and mdr1b in rats, revealing a novel effect of these commonly used drugs.