ATP-DEPENDENT LYSIS OF ISOLATED LYSOSOMES BY BASIC SUBSTANCES AND ACIDIC IONOPHORES

We established an in vitro cell-free system with which to evaluate the effects of basic substances and acidic ionophores on the internal pH and integrity of FITC-dextran (FD)-loaded lysosomes isolated from the rat liver. In this system, basic substances and acidic ionophores not only increased the internal pH dose-dependently, but also disrupted th e lysosomes in the presence of Mg-ATP, which was detected as the relea se of FD from lysosomes. All of the vacuoligenic bases and acidic iono phores, but none of the non-vacuoligenic bases or neutral ionophores d isrupted the lysosomes, suggesting that this phenomenon is an in vitro manifestation of vacuole formation induced in vivo by basic substance s and acidic ionophores. Lysosome disruption required a functional pro ton pump as well as permeant anions. It was inhibited by inhibitors of the lysosomal proton pump, including bafilomycin A(1), N-ethylmaleimi de (NEM), and N,N'-dicyclohexylcarbodiimide (DCCD), or when permeant a nions were replaced with impermeant anions. It was also suppressed by increasing the osmotic pressure of the surrounding medium, suggesting that it was caused by osmotic swelling of lysosomes induced by protona ted bases or cations characteristic of particular ionophores that accu mulated within lysosomes driven by the proton pump. Furthermore, this lysosomal disruption was inhibited by cytosolic factors. This phenomen on will provide an in vitro system for studies on osmoregulation and t he intracellular dynamics of the lysosomal system, including membrane fusion.