Permeability properties of rat renal lysosomes

Abstract

Although lysosomes maintain large pH gradients and may be subjected to significant osmotic gradients in vivo, little is known about their passive permeability properties. In recent studies, vacuolar H(+)-adenosine-triphosphatases (ATPases), such as those found in lysosomes, have been suggested to act as water channels. In addition, the erythrocyte and proximal tubule water channel CHIP28 is present on the plasma membrane of proximal tubule cells and may undergo endocytosis so that it is incorporated in lysosomes. We therefore examined water, proton, and small nonelectrolyte permeabilities in freshly purified lysosomes from rat renal proximal tubule. Lysosomes were purified by differential and Percoll gradient centrifugation. The preparation contained only lysosomes when examined by electron microscopy. Moreover, analysis by flow cytometry showed virtually all particles to be positive for acid phosphatase and cathepsin B activities. Permeabilities were measured on a stopped-flow fluorimeter by monitoring the self-quenching or pH-sensitive quenching of entrapped fluorescein derivatives. Osmotic water permeability (Pf) averaged 0.011 +/- 0.003 cm/s (n = 6), a value similar to that of biological membranes containing water channels. However, Pf was insensitive to the organic mercurial reagent p-chloromercuribenzene-sulfonate and to HgCl2 and exhibited an activation energy of 10.8 +/- 0.8 kcal/mol. These results indicate that water flux in lysosomes occurred via the lipid bilayer, and not via water channels. Addition of ATP led to lysosomal acidification (proton flux = 4.6 +/- 0.8 x 10(-11) mmol H+.s-1.cm-2), which was completely inhibited by 0.1 microM bafilomycin. Pf was insensitive to this agent as was the passive proton permeability (0.36 +/- 0.18 cm/s, n = 4). Permeabilities to small nonelectrolytes varied in proportion to the oil-water partition coefficient, confirming the applicability of Overton's rule to lysosomes. We conclude that proximal tubular lysosomes exhibit high Pf, which occurs via the lipid bilayer and not via vacuolar H(+)-ATPase.