| Abstract: | We have previously described the preparation of hepatocytes from which the plasma membrane was removed by digitonin treatment. Such "nude" cells were found to be very stable in sucrose media containing above 50 mM NaCl or KCl, but they disintegrate near instantly in salt-free media, liberating nuclei, mitochondria, and other organelles. We show here that disintegration occurs at a physiologic pH and in the presence of oxygen. Disintegration was blocked by rotenone, oligomycin, KCN, and carboxyatractyloside, establishing that oxidative phosphorylation and ATP generation is essential for disintegration to occur. The addition of ATP, GTP, ITP, or ADP (but not AMP) in the presence of the inhibitors, induced breakdown. Taxol, an inhibitor of tubulin depolymerization and phalloidin, a drug that stabilizes actin fibers, prevented disintegration in salt-free media. The effect of these drugs was counteracted by the addition of ATP. Our results show that two conditions are essential to induce the disintegration of the nude cell: media of low ionic strength, and ATP generation. The ATP effect is likely to be of physiological significance, suggesting role of ATP generation in affecting polymerization of cytoskeletal elements. |
