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

Abstract

Abstract. 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, d. 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 ~40% by 10 mM NaF and 30%, or sometimes less in the case of long-lived proteins, by 100 #M chloroquine. When the cells were supplied with BSA, a mixture of cell proteins, or even inert endocytosible materims, 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 extracellulady, 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 (~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 longlived proteins.

Selective upregulation of TNFα expression in classically-activated human monocyte-derived macrophages (M1) through pharmacological interference with V-ATPase

“…Elevation of the lysosomal pH by archazolid was pronounced at 100 nM but only slightly affected at 10 nM. In agreement with the literature [43,44], chloroquine (100 µM) and NH4Cl (50 mM), used as control tools, elevated the lysosomal pH in MLPS to a comparable degree as 10 nM archazolid (Fig. 3A).…”

“…Therefore, it appeared possible that lysosomal pH elevation is causative for increased TNFα expression. However, the control tool compounds chloroquine and NH4Cl that are known to increase lysosomal pH in macrophages [43,44], also elevated the pH in lysosomes of MLPS comparable as archazolid, but TNFα release was not increased but rather impaired. These data suggest that simply elevation of lysosomal pH is probably not the cause for increased TNFα expression by targeting V-ATPase.…”

Phosphorylation of paracellin-1 at Ser217 by protein kinase A is essential for localization in tight junctions

“…These results suggest that dephosphorylated and non-phosphorylated PCLN-1 might be degraded by proteases. The decrease of PCLN-1 expression was inhibited by chloroquine, a specific lysosome inhibitor (Ohkuma et al, 1986), but not by lactacystin, a specific proteasome inhibitor (Dick et al, 1996). Furthermore, a new band was detected at about 38 kDa by the treatment with chloroquine.…”

Mechanisms of TNF induction by heat-killed Staphylococcus aureus differ upon the origin of mononuclear phagocytes

“…However, this drug inhibits actin polymerization and, therefore, may also impact other actin-dependent processes. To confirm the involvement of phagocytosis, we used chloroquine, a drug that increases phagosome pH and blocks phagosome maturation and its fusion with lysosomes (30). Cells were isolated and incubated with various doses of chloroquine (10,20, and 50 M) and then stimulated with 10 g/ml of HKSA for 24 h. As shown in Fig.…”