A quantitative method is described for the measurement of intralysosomal pH in living cells. Fluorescein isothiocyanate-labeled dextran (FD) is endocytized and accumulates in lysosomes where it remains without apparent degradation. The fluorescence spectrum of this compound changes with pH in the range 4-7 and is not seriously affected by FD concentration, ionic strength, or protein concentration. Living cells on coverslips are mounted in a spectrofluorometer cell and can be perfused with various media. The normal pH inside macrophage lysosomes seems to be 4.74.8, although it can drop transiently as low as 4.5. Exposure of the cells to various weak bases and to acidic potassium ionophores causes the pH to increase. The changes in pH are much more rapid than is the intralysosomal accumulation of the weak bases. Inhibitors of glycolysis (-deoxyglucose) and of oxidative phosphorylation (cyanide or azide) added together, but not separately, cause the intralysosomal pH to increase. These results provide evidence for the existence of an active proton accumulation mechanism in the lysosomal membrane and support the theory of lysosomal accumulation of weak bases by proton trapping. There have been conflicting reports and theories about the pH inside lysosomes. This controversy has been the subject of a review by Tager and Reijngoud (1). Isolated lysosomes have an internal pH about 1 unit lower than that of the medium, apparently as a consequence of a Donnan equilibrium (2-4). However, there is some evidence of an energy-dependent mechanism that is capable of lowering the pH (5, 6). In living cells, various weakly basic substances are concentrated in lysosomes as a consequence of lysosomal acidity (7). When the intralysosomal concentration of these substances becomes sufficiently high, the lysosomes swell osmotically to form large vacuoles (8, 9). Vacuoles of similar appearance have been observed in cells exposed to the acidic ionophore X537A (10).Attempts have been made to estimate the pH inside lysosomes by visual inspection of color changes in pH indicator dyes (see ref. 1). Here we describe a quantitative method for the measurement of intralysosomal pH based on the pH-dependent fluorescence signals from fluorescein isothiocyanate-labeled dextran (FD) in the lysosomes of living cells. Control experiments indicate that these signals should provide an accurate measure of pH. We report the results of some measurements of pH in the lysosomes of living cells under various conditions that support the existence of an active process of pH maintenance and provide some confirmation of the theory of pHdependent concentration of certain substances in lysosomes (7). MATERIALS AND METHODSMouse peritoneal macrophages isolated by the method of Cohn and Benson (11) were cultured in modified Eagle's medium (12) The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fa...

The spectral characteristics of dextran, labeled with fluorescein, depend upon pH . We have loaded the lysosomes of mouse peritoneal macrophages with this fluorescence probe and used it to measure the intralysosomal pH under various conditions . The pH of the medium has no effect on the intralysosomal pH . Weakly basic substances in the medium cause a concentration-dependent increase in the intralysosomal pH . However, the concentration of base necessary to produce a significant change in the intralysosomal pH varies over a wide range for different bases. The active form of the base is the neutral, unprotonated form . Although most of these weak bases cause an increase in the volume of the lysosomes, increase in lysosomal volume itself causes only a minor perturbation of the intralysosomal pH . This was demonstrated in cells whose lysosomes were loaded with sucrose, and in cells vacuolated as a consequence of exposure to concanavalin A.The results of these studies are interpreted in terms of energy-dependent lysosomal acidification and leakage of protons out of the lysosomes in the form of protonated weak bases.In a previous paper (6) we reported the results of experiments showing that exposure of macrophages to medium containing any one of a large number of weak bases results in the uptake of the bases into lysosomes, and, when the concentration in lysosomes becomes sufficiently high, osmotic swelling of these particles to form large phase-lucent vacuoles occurs. We showed that the concentration of the free base in the medium is the determining factor for uptake and vacuolation, and that the protonated base in the medium has no effect. Widely different concentrations are required to elicit vacuolation by different bases.We have described previously a fluorescence probe technique for the measurement of intralysosomal pH (5) . In this study we use this technique to explore, in some detail, the pH changes caused in the lysosomes by some of these weak bases, and we discuss the results in terms of the leakage of protons out of lysosomes in the form of protonated base. We conclude that the high acidity in lysosomes is the consequence of an active process of proton secretion into lysosomes . MATERIALS AND METHODS Cell CultureOur culture procedures for mouse peritoneal macrophages, adapted from This paper is appearing without fmal revision due to the untimely death of Dr . Brian Poole.THE JOURNAL OF CELL BIOLOGY " VOLUME 90 SEPTEMBER 1981 665-669 © The Rockefeller University Press " 0021-9525/81/09/0665/05 $1 .00Cohn and Benson (2), have been described fully in the previous paper in this series (6). Lysosomal pH MeasurementThis technique has been described in more detail elsewhere (5) . Briefly, cells were exposed to medium containing 1 mg/ml fluorescein-labeled dextran for 24 h. They were then washed free of medium and the cover slips were mounted in a special holder in a regular fluorescence cell that was perfused with various media . The fluorescence of the cells was measured with a Hitachi Perkin-Elmer MPF...

With few exceptions, weakly basic compounds that are sufficiently lipophilic in their neutral forms and sufficiently hydrophilic in their protonated forms accumulate in lysosomes. When the concentration within the lysosomes becomes sufficiently high, osmotic swelling occurs . The cells then take on a vacuolated appearance . The concentrations at which different weak bases cause lysosomal vacuolation vary over almost three orders of magnitude. For any particular weak base, it is the concentration of the neutral form that determines the extent of uptake and the degree of vacuolation. Chloroquine is anomalous in that concentrations > -30 [LM cause less uptake and less vacuolation than do lower concentrations .It has been found that the treatment of cells with a variety of chemical compounds leads to the formation, in the cytoplasm, ofmanylarge vacuoles (3,20,22,23). Many ofthese substances, the best know being neutral red, are weak bases. De Duve et al. (7) have proposed a quantitative theory to account for the accumulation, in lysosomes, of weakly basic substances and the formation of vacuoles. This theory is the logical consequence of three assumptions. First, that the plasma and lysosomal membranes are highly permeable to the neutral forms of weak bases. Second, that these same membranes are impermeable, or very slightly permeable, to the protonated forms ofthe weak bases. And third, that the pH inside the lysosomes is considerably lower than it is outside the lysosomes . These assumptions have the following consequences. First, that weak bases will be trapped by protonation inside lysosomes and accumulate there. And, second, that when the concentration of the base inside the lysosomes approaches isotonicity, water will enter osmotically and the lysosomes will swell to form large vacuoles.At the time this theory was proposed we had no quantitative measure of the pH in lysosomes. Recently we have devised a technique to measure this parameter in mouse peritoneal macrophages (13) and have discovered that the pH rises in the presence of weak bases, a possibility not considered in the original theory (7) .In this paper we will examine, in some detail, the ability of This paper is appearing without final revision due to the untimely death of Dr . Brian Poole .a number of weak bases to induce vacuolation in mouse peritoneal macrophages and the kinetics of uptake of these bases into the cells. In subsequent papers we will explore, in more detail, the pH changes that occur in the lysosomes of mouse peritoneal macrophages exposed to weak bases, and the effect of these compounds on lysosomal protein degradation (18). MATERIALS AND METHODS Cell CultureMouse peritoneal macrophages were isolated by the method of Cohn and Benson (6) from NCS strain mice . They were cultured in Dulbecco's modified Eagle's minimum essential medium (l2) at pH 7.6 (unless otherwise indicated), containing 20% fetal calf serum, 25 Pg/ml Gentamicin, and 2.5 jug/ml Fungizone in glass Leighton tubes, either on the tube surface or on cover slip...

Fluorescein isothiocyanate-conjugated dextran was introduced preferentially into hepatic lysosomes by intraperitoneal injection into rats. The pH in isolated lysosomes, measured by fluorescein fluorescence, was =5 and gradually increased in KCI (to 7.0) at 250C. In the presence of Mg+, ATP caused acidification oflysosomes that was reversed by the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Mn2+, Co2+, and Fe2+ could replace Mg2+ but Ca2+ could not. Cu2 , Zn2 , and Cd2+ were inhibitory. A membrane-permeant anion, in practice chloride, was required for this acidification. ATP analogues, including 5'-adenylyl imidodiphosphate, could not be substituted for ATP. ATP-driven acidification was sensitive to N-ethylmaleimide and quercetin but insensitive to oligomycin, ouabain, and vanadate. There were some differences between '"normal" lysosomes and tritosomes; the acidification was resistant to azide and N,N'-dicyclohexylcarbodiimide in normal lysosomes but sensitive to these reagents in tritosomes. These results provide evidence for the presence of an electrogenic proton pump driven by MgATP (H+-ATPase) on lysosomes.Lysosomes are acidic inside (1-3). The mechanism whereby this low pH is maintained is unknown, but two different mechanisms have been proposed. (i) A Donnan-type equilibrium or a modification ofthis equilibrium has been proposed from studies of the permeability properties of the lysosomal membrane (4-6).(ii) The existence of an ATP-driven proton pump on lysosomes has been proposed from the finding that proteolysis is stimulated by addition ofATP to intact lysosomes in vitro (7). The latter hypothesis is supported by observations and theoretical considerations of massive uptake of basic substances by lysosomes in living cells (8). Schneider found relatively high ATPase activity on lysosomal membranes (9) and observed that MgATP stimulates the uptake of methyl amine by tritosomes 1.5-to 2-fold (10). In the mitochondria/lysosomes fraction, similar enhancement has been reported for the uptake of weakly basic dyes (11) and amino acid methyl esters (12). Recently, Schneider found evidence for the presence of a MgATP-driven proton pump on lysosomes by using the methyl amine uptake method and suggested that this pump may be electroneutral (13).We measured the intralysosomal pH of living macrophages 'by'determining the fluorescence. spectrum of fluorescein isothiocyanate-conjugated dextran (FITC-dex) that had been introduced preferentially into lysosomes and found a value of pH 4.7-4.8, which was 2 to 3 pH units lower than that of the medium and provided evidence for the presence of an active proton accumulation mechanism(s) in lysosomes (2).In this work, we obtained direct evidence for the presence of a MgATP-driven proton pump (H+-ATPase) on lysosomes and also evidence that this pump is electrogenic in nature.MATERIALS AND METHODS Materials. Male Wistar rats (150-250 g) were purchased from Sankyo (Tokyo). FITC-dex (average Mr, 67,000; 0.007 mol of fluorescein per mol of glucose residue...

AcKNOwledgemeNTSWe are indebted to Prof. Mori M. (Kumamoto University, Japan) for generously providing pCAGGS-pOTC-GFP. We thank Ms. Nagami Yamashita for her supports. Prof. Ohkuma died on November 5, 2006 by myocardial infarction at his home. We greatly miss him as a scientist and a friend. We offer sincere thanks to all the friends, colleagues and former collaborators of Prof. Ohkuma who showed him kindness during his lifetime. AbSTrAcTAutophagy is the bulk degradation of cytoplasmic constituents in response to starvation and other environmental or intracellular cues. During this process, most of the cytoplasm is sequestered into autophagosomes, which then fuse with lysosomes where the degradation of the sequestered material proceeds. We investigated the relationship between autophagosome-lysosome fusion and the pH in acidic compartments by visualizing the fusion process using fluorescence in CHO cells. In this experiment, mitochondria were labeled with GFP by transfecting CHO cells with the presequence of ornithine transcarbamylase, and lysosomes were labeled with Texas Red Dextran; any fusion was identified by the colocalization of mitochondria (in autophagosomes) and lysosomes using fluorescence microscopy. When CHO cells were treated with rapamycin or starvation medium to induce autophagy, the colocalization of fluorescence was observed. Whereas when they were treated with 3-MA, an inhibitor of autophagy, the colocalization disappeared. We conclude that the colocalization reflects the fusion of autophagosomes and lysosomes. Moreover, when the CHO cells were treated with drugs that increase the pH of acidic compartments, the colocalization disappeared. This suggests that the autophagosome-lysosome fusion is inhibited by increasing pH in acidic compartments independently of V-ATPase activity in CHO cells.