Clearance of NH2-terminal propeptides of types I and III procollagen is a physiological function of the scavenger receptor in liver endothelial cells.

This study was undertaken to determine the fate of circulating NH2-terminal propeptide of type I procollagen (PINP) in rats. Radiolabeled PINP showed a biphasic serum decay curve after intravenous injection. 79% of the material disappeared from the blood during the initial alpha-phase (t1/2 alpha = 0.6 min), while the remaining 21% was eliminated with a t1/2 beta of 3.3 min. The major site of uptake was the liver, 78, 1, and 21% of its radioactivity being recovered in isolated liver endothelial cells (LEC), Kupffer cells, and parenchymal cells, respectively. In LEC, fluorescently labeled PINP accumulated in small (0.1 microns) peripheral and larger (> 0.1 microns) perinuclear vesicles within 10 min at 37 degrees C after a binding pulse at 4 degrees C. These grew in size with increasing chasing time, reaching a maximum diameter of 1 microns or more after 30 min, and taking the shape of rings that were stained only along their periphery. At chase intervals exceeding 30 min, the size of the vesicles decreased, and after 60 min the stain appeared in smaller, densely stained perinuclearly located vesicles. Degradation of 125I-PINP to free smaller fragments and 125I- was significant after 30 min. Only formaldehyde-treated albumin, acetylated LDL, polyinosinic acid and NH2-terminal propeptide of type III procollagen (PIIINP) competed with PINP for uptake. These findings indicate that clearance of PINP and PIIINP, which are normal waste products generated in large quantities, is a physiological function of the scavenger receptor in LEC.

Synaptotagmin VII regulates Ca(2+)-dependent exocytosis of lysosomes in fibroblasts.

Synaptotagmins (Syts) are transmembrane proteins with two Ca(2+)-binding C(2) domains in their cytosolic region. Syt I, the most widely studied isoform, has been proposed to function as a Ca(2+) sensor in synaptic vesicle exocytosis. Several of the twelve known Syts are expressed primarily in brain, while a few are ubiquitous (Sudhof, T.C., and J. Rizo. 1996. Neuron. 17: 379-388; Butz, S., R. Fernandez-Chacon, F. Schmitz, R. Jahn, and T.C. Sudhof. 1999. J. Biol. Chem. 274:18290-18296). The ubiquitously expressed Syt VII binds syntaxin at free Ca(2+) concentrations ([Ca(2+)]) below 10 microM, whereas other isoforms require 200-500 microM [Ca(2+)] or show no Ca(2+)-dependent syntaxin binding (Li, C., B. Ullrich, Z. Zhang, R.G.W. Anderson, N. Brose, and T.C. Sudhof. 1995. Nature. 375:594-599). We investigated the involvement of Syt VII in the exocytosis of lysosomes, which is triggered in several cell types at 1-5 microM [Ca(2+)] (Rodríguez, A., P. Webster, J. Ortego, and N.W. Andrews. 1997. J. Cell Biol. 137:93-104). Here, we show that Syt VII is localized on dense lysosomes in normal rat kidney (NRK) fibroblasts, and that GFP-tagged Syt VII is targeted to lysosomes after transfection. Recombinant fragments containing the C(2)A domain of Syt VII inhibit Ca(2+)-triggered secretion of beta-hexosaminidase and surface translocation of Lgp120, whereas the C(2)A domain of the neuronal- specific isoform, Syt I, has no effect. Antibodies against the Syt VII C(2)A domain are also inhibitory in both assays, indicating that Syt VII plays a key role in the regulation of Ca(2+)-dependent lysosome exocytosis.

Intracellular fate of endocytosed collagen in rat liver endothelial cells.

The fate of endocytosed collagen (COL) in sinusoidal liver endothelial cells was studied using COL labeled with FITC (F-COL) or iodine (125I-COL) or both (125I-FCOL). In pulse-chase experiments in vitro, F-COL localized after 10 min along the limiting membrane of vesicles, taking the appearance of rings. After 20 min chase the probe appeared more concentrated in fewer but larger ring structures, and after 60 min the probe was observed mainly in the interior of smaller vesicles. Gel filtration of solubilized cultures after pulse-chase experiments using 125I-FCOL or 125I-COL revealed that degradation was initiated and largely completed in the small, filled vesicles, judged as a pre- or early lysosomal compartment. In the presence of monensin, or by incubation at 20 degrees C, the probe was arrested at the level of the larger ring structures, and degradation could not be observed. Lysosomal preloading by iv injection of TRITC-COL (T-COL) 24 h prior to pulse-chase experiments with culture cells using F-COL disclosed colocalization of the two dyes only after 8 h in perinuclear vesicles of a size larger than the early lysosomal vesicles observed after 60 min, suggesting a slow, unidirectional transport of F-COL to the T-COL labeled lysosomal compartment. Esterase reaction product was observed mainly in vesicles resembling the double-stained lysosomes. We conclude that (1) the early endosomes, (2) the vesicles appearing after 60 min are prelysosomes mediating degradation, and (3) the lysosomes accumulating in the probe after 8 h are responsible for final degradation and storage of residual stain.

Lymphatic transport and organ uptake of gelatin and hyaluronan injected into the rat mesentery.

The metabolic pathways of denatured collagen (gelatin) and hyaluronan were studied by injecting labelled macromolecules into the mesentery of rats. The label, [125]tyramine-cellobiose is trapped intracellularly after endocytosis, allowing localization of the site of uptake. Mesenteric and thoracic lymph was sampled for 6 h in anaesthetized rats. Separate rats were investigated after an awake period of 6 or 24 h. About 30% of the gelatin remained at the site of injection and of the remaining activity 1.7% was recovered in lymph, 11% in the liver and 15% in the kidneys, whereas 3 h after an intravenous injection of gelatin > 70% was recovered in the liver. The change in preferable site of uptake from the liver to the kidney was attributed to local degradation in the mesentery as confirmed by chromatography of tissue extracts and lymph. Following hyaluronan injection and 6 h lymph sampling approximately 30% was left at the site of injection and of the remaining activity 5.7% was recovered in lymph. After an awake period of 6 or 24 h, 30% was regained in the liver. The recoveries in other organs were negligible and mesenteric lymph nodes seem quantitatively unimportant in the uptake of hyaluronan or gelatin from lymph or blood. The liver has a central role in intestinal hyaluronan metabolism, while denatured collagen is more prone to local degradation with remote uptake shared between the liver and the kidney.

Transport of residual endocytosed products into terminal lysosomes occurs slowly in rat liver endothelial cells.

Receptor-mediated endocytosis of circulating collagen is a major physiological scavenger function of the liver endothelial cell and an important catabolic event in the complete turnover of this abundant connective tissue protein. In the present study, transport of collagen through the endocytic pathway was investigated in cultured liver endothelial cells. Collagen conjugated to fluorescein isothiocyanate, to allow detection of the ligand by fluorescence and immunoelectron microscopy, was found sequentially in three different organelles that compose the basic degradative endocytic pathway of eukaryotic cells: early endosomes, late endosomes, and terminal lysosomes. Early endosomes were identified as vesicles positive for early endosome antigen 1 (EEA1). Late endosomes were distinguished as structures positive for the late endosomal/lysosomal marker rat lysosomal membrane glycoprotein 120, but negative for EEA1 and lysosomally targeted BSA-gold. Lysosomes were defined by their content of BSA-gold, injected 24 hours before isolation of cells. Coated pits and coated vesicles mediated an extremely rapid internalization. Shortly after internalization and during the first 20 minutes, ligand was found in early endosomes. From 20 minutes on, ligand started to appear in late endosomes (23%), and by 2 hours the transfer was largely complete (82.5%). Only 2.5% of ligand was transferred to the lysosomes after 2 hours, and this number slowly increased to 21% and 53% after 6 and 16 hours, respectively. We conclude that 1) EEA1 is a useful marker for tracing early events of endocytosis in liver endothelial cells; 2) in contrast to the rapid internalization, transit of internalized ligand through early sorting endosomes generally takes from 20 minutes to 2 hours; and 3) exit from the late endosomes is very slow, requiring several hours.