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.

Distribution of colon cancer cells permanently labeled by lectin-mediated endocytosis of a trap label.

A method was elaborated for high-yield 125I-trap labeling of rat colon carcinoma cells using conjugates of dichlorotriazine aminofluorescein and bovine serum albumin substituted with either N-acetylgalactosamine or N-acetylglucosamine as vehicles. Fluorescence microscopy revealed that the ligands accumulated in perinuclear vesicles that were probably lysosomes. Monensin inhibited accumulation by 40%, signifying receptor-mediated endocytosis. Competition experiments revealed that the same receptor(s) mediated endocytosis of the two neoglycoproteins. Accumulation of label was greatly enhanced in the absence of serum, resulting in a labeling efficiency of at least 15 cpm/cell, with no sign of toxic effects. At least 75% of the initially accumulated radioactivity resided in the cells 4 days after labeling. After that the loss of radioactivity was linear with time and stabilized at 1.1%/day for at least 2 weeks. Injection of labeled carcinoma cells i.v. into syngeneic rats revealed a very rapid clearance from the circulation. Isolation of the liver cells 24 h later revealed that a great proportion of the administered cells or their remnants had been engulfed by sinusoidal Kupffer and endothelial cells; the parenchymal cells contained a smaller proportion of label. In conclusion, we have developed a technique of labeling colon carcinoma cells with 125I and fluorescein utilizing specific lectin-like receptors for endocytosis. Since the label is trapped intralysosomally, it will also label Kupffer cells and other members of the reticuloendothelial system after internalization. These features make the procedure well suited for studies on the fate of the colon carcinoma cells after administration in vivo. Since the label is trapped intralysosomally for an extended length of time, parameters such as the formation of metastasis and elimination by phagocytosis can readily be determined.

High-affinity von Willebrand factor binding does not affect the anatomical or hepatocellular distribution of factor VIII in rats.

UNLABELLED: Essentials Von Willebrand factor (VWF) stabilizes factor VIII (FVIII) and prevents its premature clearance. Rat anatomical and hepatocellular distribution studies assessed the VWF effect on FVIII clearance. Hepatocytes and liver sinusoidal endothelial cells play a key role in FVIII clearance. Anatomical and hepatocellular distribution of FVIII is independent of high-affinity VWF binding. ABSTRACT: Background Von Willebrand factor (VWF) stabilizes factor VIII in the circulation and prevents its premature clearance. Objective To study the effects of VWF on FVIII clearance in rats with endogenous VWF. Methods Anatomical and hepatocellular distribution studies were performed in rats following intravenous administration of glycoiodinated recombinant FVIII (rFVIII) and a FVIII variant, FVIII-Y1680F, lacking high-affinity VWF binding. Radioactivity was quantified in organs, and in distinct liver cell populations. The role of VWF binding was also studied by immunohistochemical staining of rat livers perfused ex vivo with rFVIII alone or with a FVIII-binding VWF fragment. Results The liver was the predominant organ of rFVIII distribution, and a radioactivity peak was also observed in the intestines, suggesting FVIII secretion to the bile by hepatocytes. In the liver, ~60% of recovered radioactivity was associated with hepatocytes, 32% with liver sinusoidal endothelial cells (LSECs), and 9% with Kupffer cells (KCs). When calculated per cell, 1.5-fold to 3-fold more radioactivity was associated with LSECs than with hepatocytes. The importance of hepatocytes and LSECs was confirmed by immunohistochemical staining; strong staining was seen in LSECs, and less intense, punctate staining in hepatocytes. Minor staining in KCs was observed. Comparable anatomical and hepatocellular distributions were observed with rFVIII and FVIII-Y1680F, and the presence of the VWF fragment, D'D3A1, did not change the FVIII staining pattern in intact livers. Conclusions The present data support FVIII clearance via the liver, with hepatocytes and LSECs playing a key role. High-affinity VWF binding did not alter the anatomical or hepatocellular distribution of FVIII.

A novel immunomodulator soluble aminated beta-1,3-D-glucan: binding characteristics to mouse peritoneal macrophages.

We have previously reported that soluble aminated beta-1,3-D-glucan (AG), a potent immunomodulator, specifically inhibited binding and internalization of AG-coated microbeads (GDM) in mouse peritoneal macrophages. The present study was undertaken to determine parameters of AG binding to macrophages. For this purpose, AG was conjugated with tyraminyl cellobiose (TC), which can be radioiodinated. With this method the immunomodulator was labelled with a very high specific radioactivity, allowing sensitive measurements of binding. Maximal binding capacity was 0.33 micrograms [125I]TC-AG/10(6) cells. Binding was inhibited by TC-AG and AG, but not by mannose and mannan, showing that the receptor different from the mannose receptor was involved. Binding was reversible, with an initial association rate of 120 cpm/min, and a much faster initial dissociation rate of 680 cpm/min. Bound [125I]TC-AG was internalized. These findings suggest that both AG and GDM are bound and internalized via the same beta-glucan receptor in mouse peritoneal macrophages.

Clearance of circulating gamma-glutamyltransferase by the hepatic galactose receptor. Variability in clearance rate due to carbohydrate heterogeneity of the enzyme.

The clearance and organ uptake of gamma-glutamyltransferase was studied by injecting the purified human liver enzyme intravenously in the rat. The enzyme was almost exclusively taken up by liver hepatocytes with a rapid initial uptake. The clearance was significantly inhibited by asialofetuin as well as by galactose and fucose. The uptake of neuraminidase-treated enzyme was much more rapid than that of the native enzyme. Subfractions of gamma-glutamyltransferase obtained by lectin affinity chromatography revealed significant differences in clearance rates. The data strongly indicates that the uptake of circulating gamma-glutamyltransferase involves the galactose (asialo-glycoprotein) receptor of the parenchymal cells, and that the heterogeneity of gamma-glutamyltransferase results in varying clearance rates.

Preparation of pure hepatocytes and reticuloendothelial cells in high yield from a single rat liver by means of Percoll centrifugation and selective adherence.

A rapid method for mass isolation of functionally intact hepatocytes and reticuloendothelial cells from a single rat liver is described. The technique is based on collagenase perfusion of the liver, isopycnic sedimentation in Percoll, and selective adherence of the cells. The Kupffer cells (KC) attach and spread on glass or plastic in serum-free medium 15 min following seeding. Cultures of KC are 90%-95% pure with about 5% liver endothelial cells (LEC), less than 1% parenchymal cells (PC) and a maximum of 5% stellate cells (SC). The LEC adhere and spread on fibronectin 60-120 min following seeding, forming cultures that are contaminated with 5-10% SC and less than 1% KC and PC. The yield of plated LEC is 50-60 X 10(6) per 200-g rat. Ultrastructural analysis shows that Percoll does not associate with the cells during the separation procedure.

Serglycin secreted by leukocytes is efficiently eliminated from the circulation by sinusoidal scavenger endothelial cells in the liver.

This study was undertaken to determine the fate of the circulating chondroitin sulfate proteoglycan serglycin. The human monocytic cell line THP-1 was cultured under serum-free conditions in the presence of [35S]sulfate. The conditioned medium was harvested and 35S-macromolecules were purified by Q-Sepharose anion-exchange chromatography and Superose 6 gel chromatography. After labeling with 125I, the purified material was treated with chondroitinase ABC and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. A major band with mr of approximately 14 kDa appeared, consistent with the core protein of serglycin. The identity of the proteoglycan was confirmed by amino-terminal amino acid sequencing. Purified serglycin, labeled either with [35S]sulfate or 125I and fluorescein isothiocyanate, was injected intravenously into rats. The blood content of radiolabeled serglycin fell by 50% from 1 to 2.4 min after injection, indicating an initial t1/2 of 1.4 min or shorter. Approximately 90% of the recovered radioactivity was localized in the liver, 5% in the blood, and 5% altogether in urine, kidneys, and spleen about 30 min after injection. Isolation of liver cells at the same time point showed that 70% of the radioactivity was taken up by the sinusoidal scavenger endothelial cells, and 23 and 7% by the hepatocytes and Kupffer cells, respectively. When excess amounts of unlabeled hyaluronan was coinjected with radiolabeled serglycin, the elimination of serglycin was significantly inhibited, indicating that the hyaluronan receptor on the sinusoidal scavenger endothelial cells is responsible for the elimination of serglycin.

Clearance of tissue plasminogen activator by mannose and galactose receptors in the liver.

The mechanism of uptake of radio-iodinated tissue plasminogen activator (125I-t-PA) was studied in rats. When trace amounts of 125I-t-PA were injected alone, the clearance followed a biphasic pattern in which 65% and 35% were cleared with alpha- and beta-kinetics (t1/2 (alpha) = 0.6 min, and t1/2 (beta) = 6.4 min), respectively. Co-injection with excess unlabelled t-PA or mannan changed the uptake kinetics to the monophasic beta-elimination pattern. Mannosylated albumin and ovalbumin, both of which bind to the hepatic mannose receptor, reduced the proportion of t-PA cleared with t1/2 (alpha) to 48% and 21%, respectively. A corresponding increase in the beta-elimination of t-PA was observed. The t1/2 (alpha) and t1/2 (beta) were unchanged. Studies on the clearance of 125I-ovalbumin also showed a biphasic elimination with an initial rapid phase, t1/2 (alpha), accounting for only 39% of the clearance of ovalbumin, as compared to 65% in the case of t-PA. Macromolecules with affinity for the galactose-receptor only, such as asialofetuin, or galactosylated albumin, did not significantly affect the clearance kinetics at the concentrations used. Asialoorosomucoid, which also carries galactosyl residues in the terminal position, reduced somewhat (from 65% to 48%) the proportion cleared with alpha-kinetics. Very high concentrations of galactose and N-acetyl-galactosamine, which are also known to compete for binding to the galactose receptor, lowered the proportion of t-PA cleared in the late beta-phase (reduced from 35% to 26% with galactose and to 19% with N-acetyl-galactosamine).(ABSTRACT TRUNCATED AT 250 WORDS)

Uptake and degradation of tissue plasminogen activator in rat liver.

The mechanism of uptake of tissue plasminogen activator (tPA) in rat liver was studied. Radio-iodinated tPA was removed from the circulation after intravenous administration in a biphasic mode. The initial half life, t1/2(alpha), and the terminal phase, t1/2 (beta), were determined to be 0.5 min and 7.5 min, resp. Separation of the liver cells by collagenase perfusion and density centrifugation, revealed that the uptake per cell was two to three times higher in the non-parenchymal cells than in the parenchymal cells. Endocytosis of fluorescein isothiocyanate-labelled or 125I-labelled tPA was studied in pure cultures of liver cells in vitro. Liver endothelial cells and parenchymal cells took up and degraded tPA. Endocytosis was more efficient in liver endothelial cells than in parenchymal cells, and was almost absent in Kupffer cells. Competitive inhibition experiments showing that excess unlabelled tPA could compete with the uptake and degradation of 125I-tPA, suggested that liver endothelial cells and parenchymal cells interact with the activator in a specific manner. Endocytosis of trace amounts of 125I-tPA in cultures of liver endothelial cells and parenchymal cells was inhibited by 50% in the presence of 19 nM unlabelled tPA. Agents that interfere with one or several steps of the endocytic machinery inhibited uptake and degradation of 125I-tPA in both cell types. These findings suggest that 1) liver endothelial cells and parenchymal cells are responsible for the rapid hepatic clearance of intravenously administered tPA; 2) the activator is taken up in these cells by specific endocytosis, and 3) endocytosed tPA is transported to the lysosomes where it is degraded.

Tissue plasminogen activator is endocytosed by mannose and galactose receptors of rat liver cells.

Experiments were carried out to characterize the specificity of uptake of tPA in rat liver cells. Endocytosis in liver endothelial cells of the native carbohydrate variants of tissue plasminogen activator (tPA), and tPA inactivated by diisopropyl fluorophosphate was found to be competitive, suggesting that the determinant being recognized by these cells is different from the active site. Fibronectin and urokinase, which show partial homology with tPA, did not compete with tPA for uptake in liver endothelial cells. Hyaluronic acid, collagen, or IgG, which are endocytosed by specific receptors in liver endothelial cells, did not interfere with the uptake. Reduced endocytosis by liver endothelial cells was observed with tPA modified in the carbohydrate side chains, suggesting that these structures are important for uptake. Ovalbumin, mannan, mannose, fructose, and EDTA, but not galactose, effectively inhibited uptake in liver endothelial cells of both native and diisopropyl fluorophosphate-inhibited tPA, but had very little effect on the uptake of tPA modified in the carbohydrate side chains. Endocytosis of native tPA by parenchymal cells could be inhibited by galactose, ovalbumin, and EDTA, but not by mannose. These results suggest that endocytosis of tPA by liver endothelial cells and parenchymal cells is mediated by the mannose and galactose receptors, respectively.

The effect of cyclooxygenase inhibitor diclofenac on experimental murine colon carcinoma.

BACKGROUND: Cyclooxygenase-2 (Cox-2) has been found to be overexpressed in several types of human cancers and its role in tumorigenesis has been proposed. The aim of this study was to investigate the effects of the cyclooxygenase inhibitor diclofenac on the growth of murine C-26 colon carcinoma cells. MATERIALS AND METHODS: Expression of Cox-2 mRNA and protein was examined by RT-PCR analysis and immunohistochemistry, respectively. By using MTT-assay, we examined the effects of diclofenac at various concentrations on the growth of C-26 cells in vitro. The effect of diclofenac on the growth of the C-26 tumor in syngeneic mice was also investigated. RESULTS: By RT-PCR, Cox-2 mRNA was detected in C-26 cells. Cox-2 protein was localized to C-26 cells and treatment with diclofenac resulted in apoptotic cell death in a dose-dependent manner. Diclofenac administered in drinking water resulted in growth inhibition of C-26 tumor in mice and correlated with plasma levels of both PGE2 and TXB2. CONCLUSION: Our data show that diclofenac may be a potential agent for the prevention and treatment of human colon cancer.

Interferon-gamma modulates TRAIL-mediated apoptosis in human colon carcinoma cells.

BACKGROUND: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is expressed by immune cells and has been shown to play an important role in tumor surveillance due to its ability to induce apoptosis in various transformed cells. Interferon gamma (IFN-gamma), a multipotent cytokine with broad stimulatory effects on anti-tumoral immune reactions, may exert its cytotoxic activity directly on tumor cells or indirectly via stimulation of effector cells. This study was designed to determine the effect of IFN-gamma on TRAIL-mediated apoptosis in human colon carcinoma cell lines. MATERIALS AND METHODS: Cytotoxicity was assessed by MTT-assay. Expression of death receptors was measured by reverse transcriptase polymerase chain reaction. Apoptosis was assessed by caspase-8 immunoblot, DNA fragmentation and morphological studies. RESULTS: Treatment with TRAIL resulted in detectable cytotoxicity within 5 hours and was enhanced in a dose-dependent manner. When cells were pretreated with IFN-gamma, the cytotoxic effect of TRAIL increased significantly. Treated cells showed a typical apoptotic morphology that was accompanied by internucleosomal cleavage of DNA. Up-regulation of caspase-8 expression and activation were detected as a result of pretreatment with IFN-gamma and subsequent apoptosis mediated by TRAIL. CONCLUSION: Our results demonstrated that IFN-gamma sensitises human colon carcinoma cells to TRAIL-mediated apoptosis, partly by elevated caspase-8 expression.

Studies on uptake and intracellular processing of infectious pancreatic necrosis virus by Atlantic cod scavenger endothelial cells.

Previous work in our group has identified the scavenger endothelial cells (SECs) of heart endocardium in cod, Gadus morhua L., as the major site for elimination of both physiological and foreign macromolecular waste from the circulation. The present study was undertaken to establish the role of cod SECs in the clearance of virus. We focused on infectious pancreatic necrosis virus (IPNV) as it is a well-known virus with a broad host range, and causes significant economic losses in the salmon industry. Our results showed that cod SEC cultures infected by the IPNV produce high titres of new virus. Ligand-receptor inhibition experiments suggested that the virus did not enter the cells through any of the major endocytosis receptors of cod SECs. Yet, the infection lowered the capacity of the cells to endocytose ligands via the scavenger receptor. Inhibitors of receptor recycling and vesicle acidification did not affect virus infectivity. The finding that SEC cultures prepared from 25% of the cod produced high titres of IPNV without being infected in the laboratory, suggests that SECs of cod may serve as reservoirs for IPNV in persistently infected cod.

Non-invasive means to study the functional status of sinusoidal liver endothelial cells.

Circulating connective tissue macromolecules are efficiently eliminated by receptor-mediated endocytosis in liver endothelial cells (LEC). In patients with a seriously diseased liver, for example in liver cirrhosis or when a transplanted liver is about to be rejected, dysfunctional LEC or altered blood perfusion through the liver results in significantly increased serum levels of connective tissue macromolecules and other substances that are normally taken up by LEC. With the aid of high affinity antibodies or other kinds of binding proteins it has been possible to measure a number of connective tissue macromolecules in the blood, and some of these substances have been used to diagnose serious liver disease. However, the metabolic patterns of these molecules are such that it is difficult to extrapolate directly on the basis of increased serum levels to conclude that the main underlying cause of increased serum levels is dysfunctional LEC. Elevated serum levels of many connective tissue proteins may reflect either increased synthesis, increased release from cell or tissue depots, and/or decreased removal by LEC. Therefore, to use measurement of serum connective tissue molecule levels as indicators of LEC function, it is imperative to know the catabolic routes of these substances in health and disease. Due to the fact that we presently know more about the total metabolic pattern of hyaluronan (HYA) than of other connective tissue macromolecules that are cleared solely by LEC, serum HYA should be preferentially used to monitor the function of LEC, in particular in liver transplantation and cirrhosis.

Aminoterminal propeptide of type III procollagen is cleared from the circulation by receptor-mediated endocytosis in liver endothelial cells.

Intravenously administered [125I]-labelled bovine aminoterminal propeptide of type III procollagen ([125I]-BPIIINP) had a half-life in blood of about 2 minutes. Low molecular weight degradation products appeared in the circulation about 5 minutes after injection. BPIIINP coupled to [125I]-labelled tyramine cellobiose ([125I]-TC-BPIIINP) was administered intravenously to determine the cellular site of uptake. TC is non-degradable and is therefore accumulated intralysosomally. With this ligand I could show that PIIINP is taken up mainly by the liver endothelial cells (LEC), with very low uptake in other types of liver cells and at extrahepatic sites. Studies on binding and endocytosis of labelled PIIINP in cultures of purified populations of liver cells can be summarized as follows: 1) Uptake and degradation were observed mainly in LEC; 2) PIIINP associated with Kupffer and parenchymal cells, but degradation was very low; 3) Serum was not required for binding of PIIINP to LEC; 4) Binding was specific, that is, other ligands, such as collagen type III, hyaluronan, chondroitin sulfate, formaldehyde-treated albumin, and mannose, that are recognized by distinct receptors on LEC, did not compete with PIIINP for binding; 5) BPIIINP, TC-BPIIINP, and rat PIIINP (RPIIINP) were recognized with the same specificity by LEC; 6) BPIIINP bound to LEC with high affinity (dissociation constant = 1 nM), and about 4.2 x 10(5), 3.2 x 10(5), and 1.6 x 10(5) molecules of BPIIINP, TC-PIIINP, and R-PIIINP, respectively were bound per cell; 7) PIIINP could not be degraded by conditioned medium from cultured Kupffer cells; 8) Leupeptin, which is a strong inhibitor of lysosomal collagenolysis, only weakly inhibited degradation of PIIINP; 9) Binding and endocytosis of PIIINP was not Ca++-dependent; 10) Agents that inhibit the endocytic machinery inhibited uptake and degradation of PIIINP. In conclusion, the present results suggest that PIIINP is rapidly eliminated from the circulation by receptor-mediated endocytosis in LEC.

Fate of intravenously injected aminated beta(1----3) polyglucose derivatized with 125I-tyraminyl cellobiose.

Aminated beta(1----3)glucan (polyglucose, AG), a potent soluble immunomodulator, was radio-iodinated and traced after intravenous administration to rats. Since the glucose polymer cannot be 125I-labelled directly by conventional methods, the polysaccharide had to be substituted with an adduct which binds the radiolabel. To this end, tyraminyl cellobiose (TC) was coupled to amino groups of AG by means of cyanuric chloride. This procedure resulted in a degree of substitution corresponding to 3.6% (or 1 molecule of tyraminyl cellobiose being incorporated per 28 molecules of glucose). AG substituted with TC (TC-AG) could be labelled with 125I by conventional procedures. After intravenous administration of 125I-TC-AG the serum concentration dropped about 50% from 1 min to about 15 min after injection, while a further drop from 50% to about 25% was observed during the next 15-60 min. The finding that 60 min after injection most of the radioactivity was recovered in the kidneys and urine, together with the results from gel chromatography showing that the low Mw fraction of the injected material disappeared first from the circulation, suggests that the initial rapid phase of elimination is due mainly to glomerular filtration. The molecules that are too large for kidney excretion are taken up mainly by the liver (about 10% of injected dose) at a slower speed. This notion was supported by the finding that a preparation of high Mw glucan obtained by gel chromatography survived for a long period in the circulation, and was eliminated mainly by accumulation in liver, whereas a preparation of low Mw glucan was rapidly eliminated by glomerular filtration. Several days after injection the liver contained nearly 90% of the recovered radioactivity, whereas the kidneys and other organs contained only insignificant amounts. This indicates that radioactivity associated with the kidneys after 60 min reflects glomerular filtration, whereas radioactivity in liver results from uptake leading to lysosomal accumulation. Isolation of liver cells after injection disclosed that the radioactivity per cell was the same in Kupffer cells (KC) and liver endothelial cells (LEC), whereas the uptake per parenchymal cell (PC) was about 30% of the uptake per KC and LEC. It could be calculated that in the intact liver, the population of PC was responsible for 50% of the uptake, whereas the populations of LEC and KC contained 35% and 15%, respectively, of the total liver radioactivity. These findings raise the question whether not only KC, but also LEC and PC may be mediators of the immune responses caused by beta(1----3) polyglucose.

Identification of a plasma gelatinase in preparations of fibronectin.

Preparations of fibronectin purified from human plasma according to conventional methods was found to contain a latent gelatinolytic activity. The protease was activated by exposure to trypsin or electrophoresis in sodium dodecyl sulfate. Zymography of the enzyme under nonreducing conditions gave an estimated Mr of 72,000. Reducing agents destroyed the activity of the enzyme. The gelatinase co-purified with fibronectin in chromatography on Sepharoses conjugated with gelatin, arginine, and heparin but could be separated from fibronectin by gel filtration in a physiological buffer. This protease was found to be a normal constituent of plasma and was probably not derived from the blood cells since the 72-kDa protease was not detected in lysates of these cells.

Use of 75Se-labeled methionine to study the sequestration of senescent red blood cells.

Labeling red blood cells with Na251CrO4 enabled us to study certain aspects of red cell survival and sequestration from the circulation. As a random labeling procedure, however, the 51Cr method has certain limitations. Therefore, we developed a cohort labeling method using 75Se-methionine as a two-rat procedure. This gives us a clear pulse-labeled population of rat red cells to study the dynamics of sequestration. With this labeling procedure, it was possible to demonstrate that 1) there is an increase in the density of red cells with age, 2) a significant sequestration of red cells from the circulation is apparent at the end of 48 days and essentially is complete at the end of 60 days, 3) there is a corresponding uptake of senescent red cells in the spleen, which peaks at 55 days, and 4) the 60-day end point is sharper and is more definitive when the "specific activity" (cpm per red blood cell) of the labeled red cells in the spleen is compared to that of the red cells still in the circulation. Asialo red cells, obtained by removal of sialic acid with sialidase, frequently have been used as a model for the study of sequestration of senescent red cells. With the technique herein described, it was possible to show that while asialo red cells will inhibit the uptake of labeled asialo red cells, they have no effect on the sequestration of senescent red cells. Presumably, different sites and mechanisms of sequestration are involved.

Receptor mediated endocytosis of formaldehyde treated albumin, yeast invertase and chondroitin sulfate in suspensions of rat liver endothelial cells.

Isolated rat liver endothelial cells take up and degrade formaldehyde serum albumin (FSA), invertase and chondroitin sulfate (CS) efficiently. Degradation products start to appear in the medium after 5-30 min. Calcium was necessary for binding of invertase to the cells, but not for the two other ligands. Ammonia and monensin inhibited uptake as well as degradation of all three ligands, whereas leupeptin only inhibited the degradation of FSA and invertase. Uptake of CS was strongly inhibited in the presence of 1 microM FSA. The possibility that these two ligands bind to a common receptor is discussed.