Uptake of liposomes containing the photoprotein obelin by rat isolated adipocytes. Adhesion, endocytosis or fusion?
1. The uptake of liposomes containing the photoprotein obelin by rat isolated adipocytes was investigated with the aim of producing liposome-cell fusion, enabling obelin to be introduced into the cytoplasm of intact cells. 2. Incubation of liposomes containing obelin with rat isolated adipocytes resulted in a time-dependent uptake of entrapped obelin by the adipocytes. The uptake by adipocytes (at 2h) of liposomes prepared from phosphatidylcholine, phosphatidylcholine+phosphatidylserine (molar ratio 4:1) and phosphatidylcholine+N-octadecylamine (molar ratio 4:1) was approx. 6, 10 and 10% of original entrapped obelin per g dry wt. of adipocytes respectively. 3. During incubation with adipocytes some of the liposomes became permeable to Ca(2+) ions, resulting in stimulation of obelin luminescence from within the liposomes. This increase in permeability to Ca(2+) seemed to be the result of the interaction of liposomes with the cell membrane. 4. Approx. 50% of liposome uptake could be inhibited by cytochalasin B (500mum). This was consistent with this uptake being the result of endocytosis. The remaining uptake was probably the result of adhesion of liposomes to the cell membrane. 5. In an attempt to detect the presence of cytoplasmic obelin, after incubation of adipocytes with liposomes, a method of causing a rapid rise in cell-membrane permeability to Ca(2+) was developed in which an anti-cell anti-body-complement reaction occurred at the cell membrane. There was no detectable transfer of active obelin into the cell cytoplasm. 6. After incubation of liposomes with adipocytes in the absence of bovine serum albumin, obelin luminescence from a small proportion of liposomes increased (approx. 1.5%) in response to anti-(5'-nucleotidase) antibody plus complement. 7. It was concluded that under the conditions of these experiments, (a) no detectable transfer (<0.1%) of liposome-entrapped obelin to the adipocyte cytoplasm had occurred, (b) an increase in liposome permeability to Ca(2+) occurred during incubation with adipocytes, (c) at least 50% of liposome uptake by adipocytes was the result of endocytosis, presumably into secondary lysosomes, and the remaining uptake was apparently the result of loose attachment of liposomes to the cell surface, and (d) in the absence of bovine serum albumin, a portion of at least one surface antigen, the ectoenzyme 5'-nucleotidase, was transferred from the adipocyte membrane to the liposome membrane.
D000273:Adipose Tissue / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D000818:Animals; D000954:Antigens, Surface / Q000032:analysis; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000276:immunology / Q000378:metabolism; D003571:Cytochalasin B / Q000494:pharmacology; D004705:Endocytosis / Q000187:drug effects; D066298:In Vitro Techniques; D008081:Liposomes / Q000276:immunology / Q000378:metabolism*; D008164:Luminescent Proteins / Q000378:metabolism*; D010539:Permeability; D051381:Rats
D000954:Antigens, Surface; D008081:Liposomes; D008164:Luminescent Proteins; C010497:obelin; D003571:Cytochalasin B; D002118:Calcium