Permeabilization of rat hepatocytes with Staphylococcus aureus alpha-toxin.
Pathogenic staphylococci secrete a number of exotoxins, including alpha-toxin. alpha-Toxin induces lysis of erythrocytes and liposomes when its 3S protein monomers associate with the lipid bilayer and form a hexomeric transmembrane channel 3 nm in diameter. We have used alpha-toxin to render rat hepatocytes 93-100% permeable to trypan blue with a lactate dehydrogenase leakage less than or equal to 22%. Treatment conditions included incubation for 5-10 min at 37 degrees C and pH 7.0 with an alpha-toxin concentration of 4-35 human hemolytic U/ml and a cell concentration of 13-21 mg dry wt/ml. Scanning electron microscopy revealed signs of swelling in the treated hepatocytes, but there were no large lesions or gross damage to the cell surface. Transmission electron microscopy indicated that the nucleus, mitochondria, and cytoplasm were similar in control and treated cells and both had large regions of well-defined lamellar rough endoplasmic reticulum. Comparisons of the mannose-6-phosphatase and glucose-6-phosphatase activities demonstrated that 5-10 U/ml alpha-toxin rendered cells freely permeable to glucose-6-phosphate, while substantially preserving the selective permeability of the membranes of the endoplasmic reticulum and the functionality of the glucose-6-phosphatase system. Thus, alpha-toxin appears to have significant potential as a means to induce selective permeability to small ions. It should make possible the study of a variety of cellular functions in situ.
McEwen
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BF
Arion
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WJ
D000818:Animals; D001427:Bacterial Toxins / Q000494:pharmacology*; D002463:Cell Membrane Permeability / Q000187:drug effects*; D002467:Cell Nucleus / Q000648:ultrastructure; D003600:Cytosol / Q000648:ultrastructure; D005372:Filipin / Q000494:pharmacology; D005952:Glucose-6-Phosphatase / Q000378:metabolism; D006460:Hemolysin Proteins; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008099:Liver / Q000201:enzymology / Q000648:ultrastructure*; D008297:Male; D008854:Microscopy, Electron; D008855:Microscopy, Electron, Scanning; D008933:Mitochondrial Swelling / Q000187:drug effects; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D012503:Saponins / Q000494:pharmacology
D001427:Bacterial Toxins; D006460:Hemolysin Proteins; D012503:Saponins; C021091:staphylococcal alpha-toxin; D005372:Filipin; D007770:L-Lactate Dehydrogenase; C023553:mannose-6-phosphatase; D010744:Phosphoric Monoester Hydrolases; D005952:Glucose-6-Phosphatase