scholarly journals Cytoplasmic vacuolation of mouse peritoneal macrophages and the uptake into lysosomes of weakly basic substances.

1981 ◽  
Vol 90 (3) ◽  
pp. 656-664 ◽  
Author(s):  
S Ohkuma ◽  
B Poole
Keyword(s):  
Peritoneal Macrophages ◽  
Weak Base ◽  
Neutral Form ◽  
Osmotic Swelling ◽  
Cytoplasmic Vacuolation ◽  
Weak Bases ◽  
Protonated Forms ◽  
Basic Compounds ◽  
Mouse Peritoneal Macrophages ◽  
Basic Substances

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 than 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 greater than approximately 30 microM cause less uptake and less vacuolation than do lower concentrations.

scholarly journals Effect of weak bases on the intralysosomal pH in mouse peritoneal macrophages.

1981 ◽  
Vol 90 (3) ◽  
pp. 665-669 ◽  
Author(s):  
B Poole ◽  
S Ohkuma
Keyword(s):  
Fluorescence Probe ◽  
Spectral Characteristics ◽  
Active Form ◽  
Peritoneal Macrophages ◽  
Weak Bases ◽  
Wide Range ◽  
A Minor ◽  
Energy Dependent ◽  
Mouse Peritoneal Macrophages ◽  
In Cells

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 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.

scholarly journals The effects of basic substances and acidic ionophores on the digestion of exogenous and endogenous proteins in mouse peritoneal macrophages.

1986 ◽  
Vol 102 (3) ◽  
pp. 959-966 ◽  
Author(s):  
S Ohkuma ◽  
J Chudzik ◽  
B Poole
Keyword(s):  
Bovine Serum Albumin ◽  
Enzyme Activities ◽  
Inhibitory Effect ◽  
Peritoneal Macrophages ◽  
The Other ◽  
Selective Effect ◽  
Lysosomal Ph ◽  
Endogenous Proteins ◽  
Mouse Peritoneal Macrophages ◽  
Basic Substances

Basic substances and acidic ionophores that increase the lysosomal pH in cultured macrophages (Ohkuma, S., and B. Poole, 1978, Proc. Natl. Acad. Sci. USA., 75:3327-3331; Poole, B., and S. Ohkuma, 1981, J. Cell Biol., 90:665-669) inhibited the digestion of heat-denatured acetylated bovine serum albumin (BSA) taken up by the cells. For several substances, the shift in pH sufficed to explain the inhibition of proteolysis. Additional effects, presumably on enzyme activities, have to be postulated for tributylamine, amantadine, and chloroquine. Sodium fluoride (10 mM) had no significant effect on the breakdown of BSA by macrophages. The breakdown of endogenous macrophage proteins, whether short lived or long lived, was inhibited approximately 40% by 10 mM NaF and 30%, or sometimes less in the case of long-lived proteins, by 100 microM chloroquine. When the cells were supplied with BSA, a mixture of cell proteins, or even inert endocytosible materials, the breakdown of endogenous long-lived proteins and the inhibitory effect of chloroquine on this process were selectively reduced. Inhibition of endocytosis by cytochalasins B or D did not affect the chloroquine-sensitive breakdown of endogenous proteins, indicating that the proteins degraded by this process were truly endogenous and not taken in from the outside by cellular cannibalism. On the other hand, when macrophage proteins were supplied extracellularly, their breakdown occurred at the same rate for short-lived and long-lived proteins, and it was strongly inhibited by chloroquine and not by NaF. It is concluded from these results that the breakdown of endogenous proteins, both short-lived and long-lived, probably takes place partly (approximately 30%) in lysosomes and partly through one or more nonlysosomal mechanism(s) unaffected by chloroquine and presumably susceptible to inhibition by fluoride. A difference must exist between short-lived and long-lived proteins in the manner in which they reach lysosomes or are handled by these organelles; this difference would account for the selective effect of the supply of endocytosible materials on the lysosomal processing of long-lived proteins.

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