Characteristics and functional relevance of apolipoprotein-A1 and cholesterol binding in mammary gland tissues and epithelial cells.
Cholesterol in milk is derived from the circulating blood through a complex transport process involving the mammary alveolar epithelium. Details of the mechanisms involved in this transfer are unclear. Apolipoprotein-AI (apoA-I) is an acceptor of cellular cholesterol effluxed by the ATP-binding cassette (ABC) transporter A1 (ABCA1). We aimed to 1) determine the binding characteristics of (125)I-apoA-I and (3)H-cholesterol to enriched plasma membrane vesicles (EPM) isolated from lactating and non-lactating bovine mammary glands (MG), 2) optimize the components of an in vitro model describing cellular (3)H-cholesterol efflux in primary bovine mammary epithelial cells (MeBo), and 3) assess the vectorial cholesterol transport in MeBo using Transwell(®) plates. The amounts of isolated EPM and the maximal binding capacity of (125)I-apoA-I to EPM differed depending on the MG's physiological state, while the kinetics of (3)H-cholesterol and (125)I-apoA-I binding were similar. (3)H-cholesterol incorporated maximally to EPM after 25±9 min. The time to achieve the half-maximum binding of (125)I-apoA-I at equilibrium was 3.3±0.6 min. The dissociation constant (KD) of (125)I-apoA-I ranged between 40-74 nmol/L. Cholesterol loading to EPM increased both cholesterol content and (125)I-apoA-I binding. The ABCA1 inhibitor Probucol displaced (125)I-apoA-I binding to EPM and reduced (3)H-cholesterol efflux in MeBo. Time-dependent (3)H-cholesterol uptake and efflux showed inverse patterns. The defined binding characteristics of cholesterol and apoA-I served to establish an efficient and significantly shorter cholesterol efflux protocol that had been used in MeBo. The application of this protocol in Transwell(®) plates with the upper chamber mimicking the apical (milk-facing) and the bottom chamber corresponding to the basolateral (blood-facing) side of cells showed that the degree of (3)H-cholesterol efflux in MeBo differed significantly between the apical and basolateral aspects. Our findings support the importance of the apoA-I/ABCA1 pathway in MG cholesterol transport and suggest its role in influencing milk composition and directing cholesterol back into the bloodstream.
Institute of Biochemistry and Molecular Medicine, Faculty of Medicine, University of Bern, Bern, Switzerland.
Edgar Corneille
EC
Ontsouka
Xiao
X
Huang
Bruno
B
Stieger
Christiane
C
Albrecht
D064286:ATP Binding Cassette Transporter 1 / Q000037:antagonists & inhibitors / Q000378:metabolism; D000818:Animals; D000924:Anticholesteremic Agents / Q000378:metabolism / Q000494:pharmacology; D016632:Apolipoprotein A-I / Q000378:metabolism*; D001692:Biological Transport / Q000187:drug effects; D002417:Cattle; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D002478:Cells, Cultured; D002784:Cholesterol / Q000378:metabolism*; D004847:Epithelial Cells / Q000378:metabolism*; D005260:Female; D006801:Humans; D007457:Iodine Radioisotopes / Q000378:metabolism; D007700:Kinetics; D008321:Mammary Glands, Animal / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure; D046529:Microscopy, Electron, Transmission; D011341:Probucol / Q000378:metabolism / Q000494:pharmacology; D011485:Protein Binding; D022161:Transport Vesicles / Q000378:metabolism / Q000648:ultrastructure; D014316:Tritium / Q000378:metabolism
D064286:ATP Binding Cassette Transporter 1; D000924:Anticholesteremic Agents; D016632:Apolipoprotein A-I; D007457:Iodine Radioisotopes; D014316:Tritium; D002784:Cholesterol; D011341:Probucol