Dissection of hepatic receptor-mediated endocytic pathways using self-generated gradients of iodixanol (Optiprep)

Iodixanol is a new, nonionic, iodinated density gradient medium which has an advantage over other similar media in that it rapidly forms self-generated gradients in vertical or near-vertical rotors. Endocytosis of Tc-99m-labeled neogalactosyl albumin (Tc-99m-NGA), a synthetic ligand for the asialoglycoprotein receptor, was studied by administering the ligand as a short pulse to perfused rat livers operating under single-pass conditions. Intracellular processing was arrested at various times after the pulse and the resultant homogenate cleared of nuclei and heavy mitochondria by centrifugation at 3000g for 10 min. After adjustment to 12.5% (w/v) iodixanol, the 3000g supernatants were centrifuged at 350,000g for 60 min to form the gradients in which early, clathrin-containing vesicles, low-density endosomes, and lysosomes were well-resolved. Tc-99m-NGA bound to the sinusoidal membrane could be partially resolved from clathrin-containing vesicles by inclusion of 1 mM CaCl2 in the homogenization and gradient buffers. Two populations of early clathrin-containing vesicles could be resolved by rate-zonal centrifugation in preformed iodixanol gradients. Thus, iodixanol is an excellent density gradient medium for the rapid and efficient resolution of endo some compartments. (C) 1998 Academic Press.