The role of lipid peroxidation and antioxidants in oxidative modification of LDL.

The study of endocytosis has traditionally focused on the contents of endocytic vacuoles, i.e., extracellular fluid, dissolved solutes, and macromolecules or particles which specifically or nonspecifically bind to the plasma membrane (PM). Substances that are endocytosed include important nutrients, toxins, effector molecules (growth factors, hormones, antibodies), enzymes, and pathogens. This article centers on the properties and dynamics of the endocytic vacuole membrane. In many instances we will stress observations on cultured mouse macrophages with which we are most familier. We will emphasize four points: (a) Movement of vesicles is rapid such that endocytosed membrane and contents move from one cellular compartment to another in seconds to minutes. (b) Vesicular movement requires the interiorization and flow of large amounts of PM) (c) In many instances, internalized PM must recycle or return intact to the cell surface. (d) During recycling, contents and membrane components can be sorted from one another; e.g., endocytosed contents can accumulate within the cell while the container (membrane) can move into and out of the cell after one or more fusion events with other endocytic vacuoles, lysosomes (Ly), or Golgi apparatus. While it has been difficult to obtain direct evidence, the literature is replete with examples in which rapid membrane flow and recycling readily explains the data. Two of the more striking examples derive from studies of pinocytosis in cultured ceils. Fibroblasts, for instance, interiorize the equivalent of 50% of their surface area and 5-10% of their cell volume during each hour of pinocytic activity. Yet, the overall dimensions of the cells and the vacuolar system remain constant throughout hours, even days, of endocytic activity. Since it is unlikely that internalized PM is rapidly degraded, it was proposed that

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Endocytosis and the recycling of plasma membrane.

For a long time, lysosomes were considered merely to be cellular 'incinerators' involved in the degradation and recycling of cellular waste. However, now there is compelling evidence indicating that lysosomes have a much broader function and that they are involved in fundamental processes such as secretion, plasma membrane repair, signalling and energy metabolism. Furthermore, the essential role of lysosomes in autophagic pathways puts these organelles at the crossroads of several cellular processes, with significant implications for health and disease. The identification of a master regulator, transcription factor EB (TFEB), that regulates lysosomal biogenesis and autophagy has revealed how the lysosome adapts to environmental cues, such as starvation, and targeting TFEB may provide a novel therapeutic strategy for modulating lysosomal function in human disease.

Signals from the lysosome: a control centre for cellular clearance and energy metabolism.

Epidermal-growth-factor receptor status as predictor of early recurrence of and death from breast cancer

Bone resorption depends on the formation, by osteoclasts, of an acidic extracellular compartment wherein matrix is degraded. The mechanism by which osteoclasts transport protons into that resorptive microenvironment was identified by means of adenosine triphosphate-dependent weak base accumulation in isolated osteoclast membrane vesicles, which exhibited substrate and inhibition properties characteristic of the vacuolar, electrogenic H+-transporting adenosine triphosphatase (H+-ATPase). Identify of the proton pump was confirmed by immunoblot of osteoclast membrane proteins probed with antibody to vacuolar H+-ATPase isolated from bovine kidney. The osteoclast's H+-ATPase was immunocytochemically localized to the cell-bone attachment site. Immunoelectron microscopy showed that the H+-ATPase was present in the ruffled membrane, the resorptive organ of the cell.

https://science.sciencemag.org/content/sci/245/4920/855.full.pdf

Osteoclastic bone resorption by a polarized vacuolar proton pump

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 approximately 5 and gradually increased in KCl (to 7.0) at 25 degrees C. In the presence of Mg2+, ATP caused acidification of lysosomes 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.

https://www.pnas.org/content/pnas/79/9/2758.full.pdf

Identification and characterization of a proton pump on lysosomes by fluorescein-isothiocyanate-dextran fluorescence.

Transcellular transport of fluorescein dextran (FD) of various molecular weights (4K, 10K, 20K, 70K and 150K daltons) through porcine arterial endothelial cells cultured on a type I collagen gel supported by a dacron sheet was studied and compared with the transport of low density lipoprotein labeled with rhodamine B (RB-LDL) described previously (Hashida et al., Cell Struct. Funct. 11, 31-42, 1986). The rate of FD transport through the monolayer depended on the size of the FD. FD transport was not temperature-dependent and was not a saturable process. Our findings show that FD transport differs from RB-LDL transport which is temperature- and dose-dependent. The mechanism of the transport of FD is compared with that of RB-LDL.

https://www.jstage.jst.go.jp/article/csf1975/11/4/11_4_343/_pdf

Transcellular transport of fluorescein dextran through an arterial endothelial cell monolayer.

Monoclonal DLR1a/104G antibody recognizing peroxidized lipoproteins in atherosclerotic lesions

To study the mechanism of lipoprotein transport through arterial endothelial cells, porcine endothelial cells were cultured on gelated type I collagen supported by a dacron sheet, and the transport of low density lipoprotein (LDL) labeled with rhodamine B isothiocyanate (RB-LDL) through the cells was measured. Light and scanning electron microscopy showed that the cells on the gel were confluent. There was little RB-LDL transport through the endothelial monolayer at 0 degrees C. RB-LDL transport through the monolayer at 37 degrees C was dose-dependent saturable at 0.4 mg protein/ml. The transport was energy-dependent, since its rate was affected by temperature and was inhibited by a combination of 2-deoxyglucose (50 mM) and NaN3 (10 mM). RB-LDL was shown not to be degraded during transport.

Shoji Ohkuma

Papers

Identification and characterization of a proton pump on lysosomes by fluorescein-isothiocyanate-dextran fluorescence.

Transcellular transport of fluorescein dextran through an arterial endothelial cell monolayer.

Monoclonal DLR1a/104G antibody recognizing peroxidized lipoproteins in atherosclerotic lesions

Transcellular transport of lipoprotein through arterial endothelial cells in monolayer culture.

Effect of PGI2 on transcellular transport of fluorescein dextran through an arterial endothelial monolayer