Measurement of total hepatic low density lipoprotein receptor levels in the hamster.
The ability to measure the total concentration of low density lipoprotein (LDL) receptors in hepatic tissues is of crucial importance to understanding changes in hepatic cholesterol metabolism. Such measurements can be made in conjunction with estimates of LDL receptor transcriptional activity, cell surface LDL receptor number, and rates of hepatic LDL uptake to evaluate the mechanisms controlling cellular LDL receptor expression. Current methods for assessing hepatic LDL receptor levels use microsomes as a source of LDL receptor, and thus rely on consistent contamination of the microsomal preparation with LDL receptor-containing plasma membranes, endocytic vesicles, and/or secretory vesicles. Because this contamination is variable, and may vary with alterations in either the distribution of LDL receptors among the various cellular membrane fractions or in the composition of the intracellular membranes, measurement of LDL receptor concentration in microsomal fractions may not accurately reflect the total compliment of LDL receptors within the cell. We have developed the methodology for isolating the full complement of hepatic LDL receptor containing membranes by discontinuous sucrose density gradient centrifugation, and for quantitating LDL receptor concentration using a Western immunoblotting procedure that uses an anti-C-terminal LDL receptor peptide polyclonal antiserum and assesses the intensity of color formation by reflectance densitometry. Using this methodology, we observed a 126 kDa immunoreactive band for the bovine adrenal cortex LDL receptor that also exhibited LDL binding activity as visualized by biotinylated LDL-ligand blotting, and a doublet of 140 kDa for the hamster liver LDL receptor. These bands were not observed when ligand blotting was conducted in the presence of either 10 mM EDTA or a 5-fold excess of unlabeled LDL, or when immunoblotting was conducted using either preimmune serum or antiserum that had been preabsorbed with LDL receptor peptide. The intensity of color formation was a linear function of the amount of membrane extract separated by electrophoresis. Intra-assay variation averaged 7%, and inter-animal variation averaged 20%. Cholestyramine, tiqueside, CP-88488, 17 alpha-ethinyl estradiol, mevinolin, and the combination of cholestyramine plus mevinolin, pharmacological interventions known to increase LDL receptor activity in experimental animals, produced the predicted increases in hamster total hepatic LDL receptor concentration that were highly correlated with concomitant increases in HMG-CoA reductase activity and reductions in serum cholesterol.(ABSTRACT TRUNCATED AT 400 WORDS)
Cosgrove
P G
PG
Department of Metabolic Diseases, Pfizer Central Research, Pfizer, Inc., Groton, CT 06340.
Gaynor
B J
BJ
Harwood
H J
HJ
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D001710:Biotin; D015153:Blotting, Western; D002499:Centrifugation, Density Gradient; D002784:Cholesterol / Q000097:blood; D006224:Cricetinae; D006801:Humans; D006903:Hydroxymethylglutaryl CoA Reductases / Q000378:metabolism; D007106:Immune Sera / Q000276:immunology; D015151:Immunoblotting; D008099:Liver / Q000737:chemistry*; D008297:Male; D008647:Mesocricetus; D008862:Microsomes, Liver / Q000737:chemistry; D008969:Molecular Sequence Data; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D011973:Receptors, LDL / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D015203:Reproducibility of Results
D007106:Immune Sera; D011480:Protease Inhibitors; D011973:Receptors, LDL; D001710:Biotin; D002784:Cholesterol; D006903:Hydroxymethylglutaryl CoA Reductases