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      Transendothelial Transport of Low-Density Lipoprotein and Albumin Across the Rat Peritoneum in vivo: Effects of the Transcytosis Inhibitors NEM and Filipin

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          This study was performed to investigate the mechanisms responsible for the transport of albumin and low-density lipoprotein (LDL) across capillary walls in vivo. To separate transcytosis from passive, ‘porous’ transport, we tested the effects of the transcytosis inhibitors N-ethylmaleimide (NEM) and filipin given intraperitoneally on the peritoneal capillary clearance of LDL and albumin in anesthetized rats undergoing peritoneal dialysis. Radiolabeled human albumin or LDL was given intra-arterially, and <sup>51</sup>Cr-EDTA was infused intravenously. A 2-hour peritoneal dialysis dwell was performed using 16 ml of conventional 1.36% glucose-based dialysis fluid. The clearance of LDL and albumin to the dialysate and the peritoneal mass transfer coefficient for <sup>51</sup>Cr-EDTA were assessed. Following intraperitoneal NEM incubations (0.5–5 m M), there were marked increases in the peritoneal transport of albumin and LDL for NEM doses exceeding 1 m M. For lower NEM doses, there were no reductions in clearance. Filipin incubations (0.2–4 µg/ml) did not affect the clearance of either macromolecule. In conclusion, neither NEM nor filipin caused reductions in albumin or LDL clearance across the peritoneal capillaries. The present data clearly show that NEM and filipin are unsuitable as transcytosis inhibitors in vivo.

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          Most cited references 6

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          Filipin-sensitive caveolae-mediated transport in endothelium: reduced transcytosis, scavenger endocytosis, and capillary permeability of select macromolecules

          Caveolae or noncoated plasmalemmal vesicles found in a variety of cells have been implicated in a number of important cellular functions including endocytosis, transcytosis, and potocytosis. Their function in transport across endothelium has been especially controversial, at least in part because there has not been any way to selectively inhibit this putative pathway. We now show that the ability of sterol binding agents such as filipin to disassemble endothelial noncoated but not coated plasmalemmal vesicles selectively inhibits caveolae-mediated intracellular and transcellular transport of select macromolecules in endothelium. Filipin significantly reduces the transcellular transport of insulin and albumin across cultured endothelial cell monolayers. Rat lung microvascular permeability to albumin in situ is significantly decreased after filipin perfusion. Conversely, paracellular transport of the small solute inulin is not inhibited in vitro or in situ. In addition, we show that caveolae mediate the scavenger endocytosis of conformationally modified albumins for delivery to endosomes and lysosomes for degradation. This intracellular transport is inhibited by filipin both in vitro and in situ. Other sterol binding agents including nystatin and digitonin also inhibit this degradative process. Conversely, the endocytosis and degradation of activated alpha 2- macroglobulin, a known ligand of the clathrin-dependent pathway, is not affected. Interestingly, filipin appears to inhibit insulin uptake by endothelium for transcytosis, a caveolae-mediated process, but not endocytosis for degradation, apparently mediated by the clathrin-coated pathway. Such selective inhibition of caveolae not only provides critical evidence for the role of caveolae in the intracellular and transcellular transport of select macromolecules in endothelium but also may be useful for distinguishing transport mediated by coated versus noncoated vesicles.
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            Endothelial caveolae have the molecular transport machinery for vesicle budding, docking, and fusion including VAMP, NSF, SNAP, annexins, and GTPases.

            Transport by discrete vesicular carriers is well established at least in part because of recent discoveries identifying key protein mediators of vesicle formation, docking, and fusion. A general mechanism sensitive to N-ethylmaleimide (NEM) is required for the transport of a divergent group of vesicular carriers in all eukaryotes. Many endothelia have an abundant population of non-coated plasmalemmal vesicles or caveolae, which have been reported with considerable controversy to function in transport. We recently have shown that like other vesicular transport systems, caveolae-mediated endocytosis and transcytosis are inhibited by NEM (Schnitzer, J. E., Allard, J., and Oh, P. (1995) Am. J. Physiol. 268, H48-H55). Here, we continue this work by utilizing our recently developed method for purifying endothelial caveolae from rat lung tissue (Schnitzer, J. E., Oh, P., Jacobson, B. S., and Dvorak, A. M. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 1759-1763) to show that these caveolae contain key proteins known to mediate different aspects of vesicle formation, docking, and/or fusion including the vSNARE VAMP-2, monomeric and trimeric GTPases, annexins II and VI, and the NEM-sensitive fusion factor NSF along with its attachment protein SNAP. Like neuronal VAMPs, this endothelial VAMP is sensitive to cleavage by botulinum B and tetanus neurotoxins. Caveolae in endothelium are indeed like other carrier vesicles and contain similar NEM-sensitive molecular machinery for transport.
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              Endocytosis and exocytosis events regulate vesicle traffic in endothelial cells.

              We used water-soluble styryl pyridinium dyes that fluoresce at the membrane-water interface to study vesicle traffic in endothelial cells. Cultured endothelial cells derived from bovine and human pulmonary microvessels were incubated in styryl probes, washed to remove dye from the plasmalemmal outer face, and observed by digital fluorescence microscopy. Vesicles that derived from plasmalemma by endocytosis were filled with the styryl dye. These vesicles were distributed throughout the cytosol as numerous particles of heterogeneous diameter and brightness. Vesicle formation was activated 2-fold following addition of extracellular albumin whereas a control protein, immunoglobulin G, had no effect. Dye uptake was abrogated by labeling at low temperatures and inhibitors of phosphoinositide-3-kinase (PI 3-kinase). Tyrosine kinase inhibitors (genistein and herbimycin A) prevented the albumin-induced vesicle formation. Cytochalasin B prevented vesicle redistribution indicating involvement of actin filaments in translocation of endosomes away from sites of vesicle formation. Styryl dye was lost from cells by exocytosis as evident by the disappearance of discrete fluorescent particles. N-ethylmaleimide and botulinum toxin types A and B caused cells to accumulate increased number of vesicles suggesting that exocytosis was regulated by NSF-dependent SNARE mechanism. The results suggest that phosphoinositide metabolism regulates endocytosis in endothelial cells and that extracellular albumin activates endocytosis by a mechanism involving tyrosine phosphorylation, whereas exocytosis is a distinct process regulated by the SNARE machinery. The results support the hypothesis that albumin regulates its internalization and release in vascular endothelial cells via activation of specific endocytic and exocytic pathways.

                Author and article information

                J Vasc Res
                Journal of Vascular Research
                S. Karger AG
                June 2002
                22 August 2008
                : 39
                : 3
                : 230-237
                Departments of Physiological Sciences and Nephrology, University of Lund, Sweden
                63688 J Vasc Res 2002;39:230–237
                © 2002 S. Karger AG, Basel

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                Page count
                Figures: 6, References: 33, Pages: 8
                Research Paper


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