Acidification of rat liver lysosomes: quantitation and comparison with endosomes

Abstract

Both lysosomes and endosomes are acidified by an electrogenic proton pump, although studies in intact cells indicate that the steady-state internal pH (pHi) of lysosomes is more acid than that of endosomes. We undertook the present study to examine in detail the acidification mechanism of purified rat liver secondary lysosomes and to compare it with that of a population of early endosomes. Both endosomes and lysosomes exhibited ATP-dependent acidification, but proton influx rates were 2.4- to 2.7-fold greater for endosomes than for lysosomes because of differences in both buffering capacity and acidification rates, suggesting that endosomes exhibited greater numbers or rates of proton pumps. Lysosomes, however, exhibited a more acidic steady-state pHi due in part to a slower proton leak rate. Changes in medium Cl- increased acidification rates of endosomes more than lysosomes, and the lysosome ATP-dependent interior-positive membrane potential was only partially eliminated by high-Cl- medium. Permeability studies suggested that lysosomes were less permeable to Na+, Li+, and Cl- and more permeable to K+ and PO4(2-) than endosomes. Na-K-adenosine-triphosphatase did not appear to regulate acidification of either vesicle type. Endosome and lysosome acidification displayed similar inhibition profiles to N-ethylmaleimide, dicyclohexyl-carbodiimide, and vanadate, although lysosomes were somewhat more sensitive [concentration producing 50% maximal inhibition (IC50) 1 nM] to bafilomycin A1 than endosomes (IC50 7.6 nM). Oligomycin (1.5-3 microM) stimulated lysosome acidification due to shunting of membrane potential. Overall, acidification of endosomes and lysosomes was qualitatively similar but quantitatively somewhat different, possibly related to differences in the density or rate of proton pumps as well as vesicle permeability to protons, anions, and other cations.

Saturable Uptake of Lipophilic Amine Drugs into Isolated Hepatocytes: Mechanisms and Consequences for Quantitative Clearance Prediction

“…The saturable uptake of quinine, fluvoxamine, and fluoxetine was substantially, but incompletely, reduced after pretreatment of hepatocytes with the ATP inhibitor, FCCP, providing evidence of involvement of an active process. Partial inhibition of saturable uptake does not necessarily indicate involvement of an additional, nonactive, process; partial inhibition by several ATP inhibitors has been observed in other studies (D'Souza et al, 1987;Dell'Antone, 1988;Van Dyke, 1993). Also, there is evidence to support the specificity of the FCCP cellular effect in reducing ATP (Yamazaki et al, 1993(Yamazaki et al, , 1996.…”

Acute Renal Effects of Intravenous Bisphosphonates in the Rat

“…Differences in bisphosphonate-related morphological changes may be a result of their different molecular mechanisms of action. Non-nitrogen-containing bisphosphonates, such as clodronate, form cytotoxic non-hydrolysable analogues of adenosine triphosphate (ATP) ultimately leading to apoptosis; the depletion of intracellular ATP may affect the ATP-dependent acidification of lysosomes resulting in changes of membrane permeability and/or inhibition of lysosomal function (Schneider 1983;van Dyke 1993;Piqueras et al 1994;Zatta et al 2000). However, any causal relationship between ATP-depletion and increased mitoses is currently unclear.…”

Effect of Cholera Toxin on Rat Liver Lysosome Acidification

“…Lysosomes were purified from livers of overnight-fasted rats as described (10,11) 18 h after intraperitoneal (IP) injection of a sterile mixture of 50 mg 70,000 mw FITC-dextran and either cholera toxin (120 g/100 gm body weight) or an equivalent volume of solvent (4,10). Purified lysosomes were suspended in buffers containing 140 mM Kgluconate or N-methyl-D-glucamine (NMDG) gluconate with 70 mM sucrose (to improve osmotic stability (10)) and 30 mM Bis-Tris (pH 7.0), kept on ice and used for acidification assays within 3-6 h (10). Samples of lysosomes, postnuclear supernatants (PNS) and homogenates were frozen for other assays.…”

“…Vesicle pH i was assessed in fresh lysosomes or Percoll fractions from the fluorescence of intravesicular fluorescein as described (4,5,10) in buffers containing 70 mM sucrose, 30 mM Bis-Tris (pH 7.0) and 140 mM of the indicated salts. When Cl -concentration was altered, gluconate was used to maintain electroneutrality.…”