Chloroquine reduces the number of asialo-glycoprotein receptors in the hepatocyte plasma membrane

doi:10.1016/0006-2952(79)90586-0

Biochemical Pharmacology

Volume 28, Issue 19, 1 October 1979, Pages 2919-2922

https://doi.org/10.1016/0006-2952(79)90586-0 Get rights and content

Abstract

Rat hepatocytes which had been incubated with chloroquine in concentrations ranging from 0.2 to 1.0 mM showed markedly reduced ability to take up asialo-glycoproteins. Chloroquine had no effect on the actual binding of asialo-orosomucoid to the receptor on hepatocytes. Chloroquine caused a progressive reduction in the binding capacity of the plasma membrane for asialo-glycoproteins; in hepatocytes that had been exposed to 1.0 mM chloroquine for 30 min at 37°C, the binding capacity of the plasma membrane was reduced to 15 per cent of controls. The results support the idea that the asialo-glycoprotein receptors of hepatocytes are internalized regardless of binding of ligand.

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Acidification and protein traffic
2003, International Review of Cytology
Citation Excerpt :
However, in some cases, pH perturbation also has a profound effect on the trafficking and distribution of the receptors themselves. In the case of the ASGPR, the primary effect of ammonium chloride, chloroquine, or primaquine was a rapid and profound loss of cell surface receptors and the intracellular accumulation of receptor and ligand in an endocytic compartment (Harford et al., 1983a; Schwartz et al., 1984; Zijderhand-Bleekemolen et al., 1987; Tolleshaug and Berg, 1979). The rate of internalization of asialoglycoprotein was unaffected, suggesting an effect on receptor recycling (Harford et al., 1983a; Schwartz et al., 1984).
Acidification of some organelles, including the Golgi complex, lysosomes, secretory granules, and synaptic vesicles, is important for many of their biochemical functions. In addition, acidic pH in some compartments is also required for the efficient sorting and trafficking of proteins and lipids along the biosynthetic and endocytic pathways. Despite considerable study, however, our understanding of how pH modulates membrane traffic remains limited. In large part, this is due to the diversity of methods to perturb and monitor pH, as well as to the difficulties in isolating individual transport steps within the complex pathways of membrane traffic. This review summarizes old and recent evidence for the role of acidification at various steps of biosynthetic and endocytic transport in mammalian cells. We describe the mechanisms by which organelle pH is regulated and maintained, as well as how organelle pH is monitored and quantitated. General principles that emerge from these studies as well as future directions of interest are discussed.
Effects of inhibitors of the vacuolar proton pump on hepatic heterophagy and autophagy
2001, Biochimica et Biophysica Acta - Biomembranes
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Acidotropic agents and/or specific inhibitors of the vacuolar proton pump (BAF, ConcA) have been shown to interfere both with retroendocytosis [10,11], transport to lysosomes [12–16], and transport to the TGN [17]. It was suggested several years ago that chloroquine, ammonium chloride and monensin inhibited degradation of endocytosed probes indirectly, by inhibiting the transport from endosomes to lysosomes, rather than inhibiting degradation in the lysosomes [18–20]. Furthermore, it was shown that high concentrations of chloroquine inhibit the recycling of the asialoglycoprotein receptor (ASGPR) from endosomes back to the plasma membrane.
Bafilomycin A₁ (BAF) and concanamycin A (ConcA) are selective inhibitors of the H⁺-ATPases of the vacuolar system. We have examined the effects of these inhibitors on different steps in endocytic pathways in rat hepatocytes, using [¹²⁵I]tyramine-cellobiose-labeled asialoorosomucoid ([¹²⁵I]TC-AOM) and [¹²⁵I]tyramine-cellobiose-labeled bovine serum albumin ([¹²⁵I]TC-BSA) as probes for respectively receptor-mediated endocytosis and pinocytosis (here defined as fluid phase endocytosis). The effects of BAF and ConcA were in principle identical, although ConcA was more effective than BAF. The main findings were as follows. (1) BAF/ConcA reduced the rate of uptake of both [¹²⁵I]TC-AOM and [¹²⁵I]TC-BSA. The reduced uptake of [¹²⁵I]TC-AOM was partly due to a redistribution of the asialoglycoprotein receptors (ASGPR) such that the number of surface receptors was reduced ≈40% without a change in the total number of receptors. (2) BAF/ConcA at the same time increased retroendocytosis (recycling) of both probes. The increased recycling of the ligand ([¹²⁵I]TC-AOM) is partly a consequence of the enhanced pH in endosomes, which prevents dissociation of ligand. (3) It was furthermore found that the ligand remained bound to the receptor in presence of BAF/ConcA and that the total amount of ligand molecules internalized in BAF/ConcA-treated cells was only slightly in excess of the total number of receptors. These data indicate that reduced pH in endosomes is the prime cause of receptor inactivation and release of ligand in early endosomes. (4) Subcellular fractionation experiments showed that [¹²⁵I]TC-AOM remained in early endosomes, well separated from lysosomes in sucrose gradients. The fluid phase marker, [¹²⁵I]TC-BSA, on the other hand, seemed to reach a later endosome in the BAF/ConcA-treated cells. This organelle coincided with lysosomes in the gradient, but hypotonic medium was found to selectively release a lysosomal enzyme (β-acetylglucosaminidase), indicating that even [¹²⁵I]TC-BSA remained in a prelysosomal compartment in the BAF/ConcA-treated cells. (5) Electron microscopy using horseradish peroxidase (HRP) as a fluid phase marker verified that BAF/ConcA inhibited transfer of material from late endosomes (‘multivesicular bodies’). (6) BAF/ConcA led to accumulation of lactate dehydrogenase (LDH) in autophagic vacuoles, but although the drugs partly inhibited fusion between autophagosomes and lysosomes a number of autolysosomes was formed in the presence of BAF/ConcA. This observation explains the reduced buoyant density of lysosomes (revealed in sucrose density gradients). In conclusion, BAF/ConcA inhibit transfer of endocytosed material from late endosomes to lysosomes, but do not at the same time prevent fusion between autophagosomes and lysosomes.
Intracellular traffic of asialoglycoproteins depends on vacuolar pH in polarized isolated rat hepatocyte couplets
1999, Hepatology Research
Asialoglycoproteins are internalized via specific receptors localized on the hepatic parenchymal cells. The role of vacuolar type H⁺-ATPase (v-ATPase) and vacuolar pH in intracellular transport of asialoglycoproteins was investigated in isolated rat hepatocyte couplets. Two specific inhibitors of v-ATPase, bafilomycin A1 and concanamycin B, were used to inhibit acidification of the vacuolar system. Intracellular transport of asialoglycoproteins was investigated using gold-conjugated asialofetuin with electron microscopy. Fluorescent staining by acridine orange showed that bafilomycin A1 and concanamycin B each inhibited acidification of endocytic compartments. Electron microscopy demonstrated that the number of autophagic vacuoles was increased, and the uptake of gold-conjugated asialofetuin was significantly inhibited by these agents. Vesicle budding and membrane fusion between endocytic structures in the peripheral area and intracellular transport of endocytosed asialofetuin to lysosomes were inhibited by these agents. These results suggest that the intracellular transport of endocytosed asialoglycoproteins to lysosomes depends on acidification of the vacuolar system (vacuolar pH) in hepatocytes.
Ligand-induced endocytosis of the asialoglycoprotein receptor: Evidence for heterogeneity in subunit oligomerization
1998, FEBS Letters
The hepatic asialoglycoprotein receptor, a non-covalent hetero-oligomer of two subunits, is a constitutively cycling endocytic receptor. However, the ligand asialoorosomucoid caused downregulation of up to 40% of surface binding sites and a twofold increase in internalization rate. This was not the result of receptor crosslinking, since monovalent ligands had the same effect. Ligand binding thus appears to transmit a signal to the cytosolic portion of the receptor not unlike in signaling receptors. The two subunits were endocytosed at different average rates lower than that of ligand, indicating heterogeneity in oligomer formation and potentially in ligand specificity.
In vitro and in vivo ultrastructural changes induced by macrolide antibiotic LY281389
1996, Fundamental and Applied Toxicology
High doses of LY281389 (9-N-(n-propyl)-erythromycylamine) cause cytoplasmic vacuolar changes in striated and smooth muscle characteristic of drug-induced phospholipidosis. This study characterized phospholipidosis in striated and smooth muscle of rats and dogs, comparedin vivoobservations with those in a cultured rat myoblast model, and attempted to confirm the lysosomal origin of the drug-induced vacuoles. Standard transmission electron microscopy and acid phosphatase cytochemistry techniques were used to evaluate ultrastructural changesin vivoandin vitro.Rats and dogs exposed to LY281389 had a time- and dose-related increase in number and size of vacuoles containing concentric lamellar figures in cardiac and skeletal muscle. Cytochemical staining of dog stomach smooth muscle for acid phosphatase, a lysosomal enzyme, stained the periphery of vacuoles that contained concentric lamellar figures. Cultured rat L6 myoblast cells were exposed to 0.25 mg LY281389/ml for 2.5, 5, 10, 20, 30, or 90 min and 2, 6, 12, 24, or 48 hr. Cell cultures exposed for 2 hr had several predominantly large, clear, membrane-bound vacuoles, and at 6 and 12 hr there were greater numbers of large vacuoles that contained increased amounts of membranous figures. Following 24- or 48-hr exposures, vacuoles occupied most of the cytoplasmic volume, and were engorged predominantly with amorphous or granular material. These findings indicate that LY281389 can induce similar phospholipidosis-like vacuolar changes in rat and dog muscle and in a cultured rat muscle cell line. Further, positive acid phosphatase staining of drug-induced vacuolar structures, in conjunction with standard transmission electron microscopy techniques, strongly suggests that vacuoles seenin vitroandin vivoare lysosomal in origin.
Bafilomycin A1, a specific inhibitor of vacuolar-type H<sup>+</sup>-ATPases, inhibits the receptor-mediated endocytosis of asialoglycoproteins in isolated rat hepatocytes
1996, Journal of Hepatology
Background/Methods: The role of vacuolar type H⁺-ATPases (v-ATPases) and pH gradient between the endocytic compartments and cytoplasm in the endocytosis of asialoglycoproteins was morphologically investigated in isolated rat hepatocytes using bafilomycin A1, a specific inhibitor of v-ATPases.
Results: Fluorescent staining by acridine orange showed that bafilomycin A1 inhibited the acidification of the endocytic compartments. Uptake of gold-conjugated asialofetuin was significantly inhibited by bafilomycin A1. However, bafilomycin A1 did not significantly inhibit uptake of a fluid phase marker, horseradish peroxidase. The number of autophagic vacuoles increased after the bafilomycin A1 treatment. However, materials in the autophagic vacuoles were rapidly degraded after the removal of bafilomycin A1.
Conclusions: Results suggest that: (a) v-ATPases are necessary for acidification of the endocytic compartments; (b) the pH gradient between the endocytic compartments and the cytoplasm which is generated by v-ATPases is necessary for the receptor-mediated endocytosis of asialoglycoproteins, and (c) v-ATPases may contribute to the degradation of the materials in autophagic vacuoles.

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Chloroquine reduces the number of asialo-glycoprotein receptors in the hepatocyte plasma membrane

Abstract

Biochim. biophys. Acta

Biochem. Pharmac.

Biochem. Pharmac.

Biochem. biophys. Res. Commun.

J. biol. Chem.

J. biol. Chem.

Biochem. Pharmac.

Biochim. biophys. Acta

Adv. Enzymol.

Acta biol. med. germ.

Acta biol. med. germ.