Endocytosis of hyaluronic acid by rat liver endothelial cells. Evidence for receptor recycling.

Hyaluronic acid (HA) is cleared from the blood by liver endothelial cells through receptor-mediated endocytosis [Eriksson, Fraser, Laurent, Pertoft & Smedsrod (1983) Exp. Cell Res. 144, 223-238]. We have measured the capacity of cultured rat liver endothelial cells to endocytose and degrade 125I-HA (Mr approximately 44,000) at 37 degrees C. Endocytosis was linear for 3 h and then reached a plateau. The rate of endocytosis was concentration-dependent and reached a maximum of 250 molecules/s per cell. Endocytosis of 125I-HA was inhibited more than 92% by a 150-fold excess of non-radiolabelled HA. HA, chondroitin sulphate and heparin effectively competed for endocytosis of 125I-HA, whereas glucuronic acid, N-acetylglucosamine, DNA, RNA, polygalacturonic acid and dextran did not compete. In the absence of cycloheximide, endothelial cells processed 13 times more 125I-HA in 6 h than their total (cell-surface and intracellular) specific HA-binding capacity. This result was not due to degradation and rapid replacement of receptors, because, even in the presence of cycloheximide, these cells processed 6 times more HA than their total receptor content in 6 h. Also, in the presence of cycloheximide, no decrease in 125I-HA-binding capacity was seen in cells processing or not processing HA for 6 h, indicating that receptors are not degraded after the endocytosis of HA. During endocytosis of HA at 37 degrees C, at least 65% of the intracellular HA receptors became occupied with HA within 30 min. This indicates that the intracellular HA receptors (75% of the total) function during continuous endocytosis. Hyperosmolarity inhibits endocytosis and receptor recycling in the asialoglycoprotein and low-density-lipoprotein receptor systems by disrupting the coated-pit pathway [Heuser & Anderson (1987) J. Cell Biol. 105, 230a; Oka & Weigel (1988) J. Cell. Biochem. 36, 169-183]. Hyperosmolarity inhibited 125I-HA endocytosis in liver endothelial cells by more than 90%, suggesting use of a coated-pit pathway by this HA receptor. We conclude that liver endothelial cell HA receptors are recycled during the continuous endocytosis and processing of HA.     

Characterization of an intracellular hyaluronic acid binding site in isolated rat hepatocytes.

125I-HA, prepared by chemical modification at the reducing sugar, specifically binds to rat hepatocytes in suspension or culture. Intact hepatocytes have relatively few surface 125I-HA binding sites and show low specific binding. However, permeabilization of hepatocytes with the nonionic detergent digitonin results in increased specific 125I-HA binding (45-65%) and a very large increase in the number of specific 125I-HA binding sites. Scatchard analysis of equilibrium 125I-HA binding to permeabilized hepatocytes in suspension at 4 degrees C indicates a Kd = 1.8 x 10(-7) M and 1.3 x 10(6) molecules of HA (Mr approximately 30,000) bound per cell at saturation. Hepatocytes in primary culture for 24 h show the same affinity but the total number of HA molecules bound per cell at saturation decreases to approximately 6.2 x 10(5). Increasing the ionic strength above physiologic concentrations decreases 125I-HA binding to permeable cells, whereas decreasing the ionic strength above causes an approximately 4-fold increase. The divalent cation chelator EGTA does not prevent binding nor does it release 125I-HA bound in the presence of 2 mM CaCl2, although higher divalent cation concentrations stimulate 125I-HA binding. Ten millimolar CaCl2 or MnCl2 increases HA binding 3-6-fold compared to EGTA-treated cells. Ten millimolar MgCl2, SrCl2, or BaCl2 increased HA binding by 2-fold. The specific binding of 125I-HA to digitonin-treated hepatocytes at 4 degrees C increased greater than 10-fold at pH 5.0 as compared to pH 7.(ABSTRACT TRUNCATED AT 250 WORDS)     

The endocytic hyaluronan receptor in rat liver sinusoidal endothelial cells is Ca(+2)-independent and distinct from a Ca(+2)-dependent hyaluronan binding activity.

Isolated and cultured rat liver sinusoidal endothelial cells (LECs) retain the ability to specifically bind 125I-hyaluronan (HA) and internalize it using a coated pit pathway [Biochem J, 257:875-884, 1989]. Here we have determined the effect of Ca+2 on the binding and endocytosis of HA by LECs. 125I-HA binding to intact LECs at 4 degrees C occurred both in the absence (10 mM EGTA) or the presence of physiologic concentrations of Ca+2 (1.8 mM). However, the specific binding of 125I-HA to LECs increased linearly with increasing Ca+2 concentrations. After permeabilization with the nonionic detergent digitonin, the Ca(+2)-independent HA binding activity increased approximately 743%, while the Ca(+2)-dependent binding activity was enhanced only approximately 46%. Therefore, the Ca(+2)-dependent HA binding activity appears not to be intracellular, whereas the Ca(+2)-independent HA receptor is found both inside LECs and on the cell surface. When LECs were allowed to endocytose 125I-HA at 37 degrees C in 10 mM EGTA or in 1.8 mM Ca+2, no differences were seen in the extent or rate of endocytosis. When LECs were allowed to endocytose 125I-HA in the presence of 10 mM Ca+2, the amount of cell-associated radioactivity increased approximately 20-50-fold. However, this additional cell-associated 125I-HA was not sensitive to hyperosmolarity and was removed by washing the cells in 10 mM EGTA at 4 degrees C. Therefore, the Ca(+2)-dependent cell-associated 125I-HA had accumulated on the cell surface and had not been internalized. From these studies we conclude that LECs have at least two types of specific HA binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endocytic function, glycosaminoglycan specificity, and antibody sensitivity of the recombinant human 190-kDa hyaluronan receptor for endocytosis (HARE)

The human hyaluronan receptor for endocytosis (hHARE) mediates the endocytic clearance of hyaluronan (HA) and chondroitin sulfate from lymph fluid and blood. Two hHARE isoforms (190 and 315 kDa) are present in sinusoidal endothelial cells of liver, spleen, and lymph nodes (Zhou, B., McGary, C. T., Weigel, J. A., Saxena, A., and Weigel, P. H. (2003) Glycobiology 13, 339-349). Here we report the specificity and function of the 190-kDa HARE, expressed without the larger isoform, in Flp-In 293 cell lines (190hHARE cells). Like the native protein, recombinant hHARE contains approximately 25 kDa of N-linked oligosaccharides, binds HA in a ligand blot assay, cross-reacts with three anti-rat HARE monoclonal antibodies, and is inactivated by reduction. The 190hHARE cell lines mediated rapid, continuous (125)I-HA endocytosis and degradation for >1 day. About 30-50% of the total cellular receptors were on the cell surface, and their recycling time for reutilization was approximately 8.5 min. The average K(d) for the binding of HA to the 190-kDa hHARE at 4 degrees C was 7 nm with 118,000 total HA binding sites per cell. Competition studies at 37 degrees C indicated that the 190-kDa hHARE binds HA and chondroitin better than dermatan sulfate and chondroitin sulfates A, C, D, and E, but it does not bind to heparin, heparan sulfate, or keratan sulfate. Although competition was observed at 37 degrees C, none of the glycosaminoglycans tested, except HA, competed for (125)I-HA binding by 190hHARE cells at 4 degrees C. Anti-HARE monoclonal antibodies #30 and #154, which do not inhibit (125)I-HA uptake mediated by the 175-kDa rat HARE, partially blocked HA endocytosis by the 190-kDa hHARE. We conclude that the 190-kDa hHARE can function independently of other hHARE isoforms to mediate the endocytosis of multiple glycosaminoglycans. Furthermore, the rat and human small HARE isoforms have different glycosaminoglycan specificities and sensitivities to inhibition by cross-reacting antibodies.     

Expression, processing, and glycosaminoglycan binding activity of the recombinant human 315-kDa hyaluronic acid receptor for endocytosis (HARE)

The hyaluronic acid (HA) receptor for endocytosis (HARE; also designated stabilin-2 and FEEL-2) mediates systemic clearance of glycosaminoglycans from the circulatory and lymphatic systems via coated pit-mediated uptake. HARE is primarily found as two isoforms (315- and 190-kDa) in sinusoidal endothelial cells of the liver, lymph node, and spleen. Here we characterize the ligand specificity and function of the large stably expressed 315-HARE isoform in Flp-In 293 cell lines. Like human spleen sinusoidal endothelial cells, Flp-In 293 cell lines transfected with a single cDNA encoding the full-length 315-HARE express both the 315-kDa and the proteolytically truncated 190-kDa isoforms in a ratio of approximately 3-4:1. The 190-kDa HARE isoform generated from the 315-kDa HARE and the 315-kDa HARE specifically bound 125I-HA. Like the 190-kDa HARE expressed alone (Harris, E. N., Weigel, J. A., and Weigel, P. H. (2004) J. Biol. Chem. 279, 36201-36209), the 190- and 315-kDa HARE isoforms expressed in 315-HARE cell lines were recognized by anti-HARE monoclonal antibodies 30, 154, and 159. All 315-HARE cell lines could endocytose and degrade 125I-HA. Competition studies with live cells indicate that 190-HARE and 315-HARE bind HA with higher apparent affinity (Kd approximately 10-20 nM) than chondroitin sulfate (CS) types A, C, D, or E. Only slight competition of HA endocytosis was observed with CS-B (dermatan sulfate) and chondroitin. Direct binding assays with the 315-HARE ectodomain revealed high affinity HA binding, and lower binding affinities for CS-C, CS-D, and CS-E. A majority of each HARE isoform was intracellular, within the endocytic system, suggesting transient surface residency typical of an active endocytic recycling receptor.     

Insulin-like growth factor-II receptors in cultured rat hepatocytes: regulation by cell density

Insulin-like growth factor-II (IGF-II) receptors in primary cultures of adult rat hepatocytes were characterized and their regulation by cell density examined. In hepatocytes cultured at 5 X 10(5) cells per 3.8 cm2 plate [125I]IGF-II bound to specific, high affinity receptors (Ka = 4.4 +/- 0.5 X 10(9) l/mol). Less than 1% cross-reactivity by IGF-I and no cross-reactivity by insulin were observed. IGF-II binding increased when cells were permeabilized with 0.01% digitonin, suggesting the presence of an intracellular receptor pool. Determined by Scatchard analysis and by polyacrylamide gel electrophoresis after affinity labeling, the higher binding was due solely to an increase in binding sites present on 220 kDa type II IGF receptors. In hepatocytes cultured at low densities, the number of cell surface receptors increased markedly, from 10-20,000 receptors per cell at a culture density of 6 X 10(5) cells/well to 70-80,000 receptors per cell at 0.38 X 10(5) cells/well. The increase was not due simply to the exposure of receptors from the intracellular pool, as a density-related increase in receptors was also seen in cells permeabilized with digitonin. There was no evidence that IGF binding proteins, either secreted by hepatocytes or present in fetal calf serum, had any effect on the measurement of receptor concentration or affinity. We conclude that rat hepatocytes in primary culture contain specific IGF-II receptors and that both cell surface and intracellular receptors are regulated by cell density.     

Characterization of the recombinant rat 175-kDa hyaluronan receptor for endocytosis (HARE)

Hyaluronan (HA) and chondroitin sulfate (CS) clearance from lymph and blood in mammals is mediated by the HA receptor for endocytosis (HARE), which is present as two isoforms in rat and human (175/300 kDa and 190/315 kDa, respectively) in the sinusoidal endothelial cells of liver, spleen, and lymph nodes (Zhou, B., McGary, C. T., Weigel, J. A., Saxena, A., and Weigel, P. H. (2003) Glycobiology 13, 339-349). The small rat and human HARE proteins are not encoded directly by mRNA but are derived from larger precursors. Here we characterize the specificity and function of the 175-kDa HARE, expressed in the absence of the 300-kDa species, in stably transfected SK-Hep-1 cells. The HARE cDNA was fused with a leader sequence to allow correct orientation of the membrane protein. The recombinant rHARE contained approximately 25 kDa of N-linked oligosaccharides and, like the native protein, was able to bind HA in a ligand blot assay, even after de-N-glycosylation. SK-HARE cell lines demonstrated specific 125I-HA endocytosis, receptor recycling, and delivery of HA to lysosomes for degradation. The Kd for the binding of HA (number-average molecular mass approximately 133 kDa) to the 175-kDa HARE at 4 degrees C was 4.1 nm with 160,000 to 220,000 HA-binding sites per cell. The 175-kDa rHARE binds HA, dermatan sulfate, and chondroitin sulfates A, C, D, and E, but not chondroitin, heparin, heparan sulfate, or keratan sulfate. Surprisingly, recognition of glycosaminoglycans (GAGs) other than HA by native or recombinant HARE was temperature-dependent. Although competition was observed at 37 degrees C, none of the other GAGs competed for 125I-HA binding to SK-HARE cells at 4 degrees C. Anti-HARE monoclonal antibody-174 showed a similar temperature-dependence in its ability to block HA endocytosis. These data suggest that temperature-induced conformational changes may alter the GAG specificity of HARE. The results confirm that the 175-kDa rHARE does not require the larger HARE isoform to mediate endocytosis of multiple GAGs.     

Identification of the hyaluronan receptor for endocytosis (HARE)

Rat liver sinusoidal endothelial cells (LECs) express two hyaluronan (HA) receptors, of 175 and 300 kDa, responsible for the endocytic clearance of HA. We have characterized eight monoclonal antibodies (mAbs) raised against the 175-kDa HA receptor partially purified from rat LECs. These mAbs also cross-react with the 300-kDa HA receptor. The 175-kDa HA receptor is a single protein, whereas the 300-kDa species contains three subunits, alpha, beta, and gamma at 260, 230, and 97 kDa, respectively (Zhou, B., Oka, J. A., and Weigel, P. H. (1999) J. Biol. Chem. 274, 33831-33834). The 97-kDa subunit was not recognized by any of the mAbs in Western blots. Based on their cross-reactivity with these mAbs, the 175-, 230-, and 260-kDa proteins appear to be related. Two of the mAbs inhibit (125)I-HA binding and endocytosis by LECs at 37 degrees C. All of these results confirm that the mAbs recognize the bone fide LEC HA receptor. Indirect immunofluoresence shows high protein expression in liver sinusoids, the venous sinuses of the red pulp in spleen, and the medullary sinuses of lymph nodes. Because the tissue distribution for this endocytic HA receptor is not unique to liver, we propose the name HARE (HA receptor for endocytosis).     

A blocking antibody to the hyaluronan receptor for endocytosis (HARE) inhibits hyaluronan clearance by perfused liver

Hyaluronan (HA) and chondroitin sulfate clearance from lymph and blood is mediated by the hyaluronan receptor for endocytosis (HARE). The purification and molecular cloning (Zhou, B., Weigel, J. A., Saxena, A., and Weigel, P. H. (2002) Mol. Biol. Cell 13, 2853-2868) of this cell surface receptor were finally achieved after we developed monoclonal antibodies (mAbs) against HARE. There are actually two independent isoreceptors for HA, which in rat are designated the 175-kDa HARE and 300-kDa HARE. Only one mAb (number 174) effectively and completely blocked the specific uptake of 125I-HA at 37 degrees C by rat liver sinusoidal endothelial cells. 125I-HA binding to both the 175-kDa and 300-kDa HARE proteins in a ligand blot assay was almost completely inhibited by <1 microg/ml mAb-174, whereas mouse IgG had little or no effect. MAb-174 also performed very well in Western analysis, indirect fluorescence microscopy, and a variety of immuno-procedures. Immunohistochemistry using mAb-174 localized HARE to the sinusoidal cells of rat liver, spleen, and lymph node. Western analysis using mAb-174 revealed that the sizes of both HARE glycoproteins were the same in these three tissues. 125I-HA was taken up and degraded by excised rat livers that were continuously perfused ex vivo with a recirculating medium. This HA clearance and metabolism by liver, which is a physiological function of HARE, was very effectively blocked by mAb-174 but not by mouse IgG. The results indicate that mAb-174 will be a useful tool to study the functions of HARE and the physiological significance of HA clearance.     

Specific intracellular hyaluronic acid binding to isolated rat hepatocytes is membrane-associated

Intact isolated rat hepatocytes show a small amount of specific 125I-labeled hyaluronic acid (HA) binding. However, in the presence of digitonin, a very large increase in the specific binding of 125I-HA is observed. Chondroitin sulfate, heparin and dextran sulfate were as effective as unlabeled HA in competing for 125I-HA binding to permeabilized hepatocytes, indicating that the binding sites may have a general specificity for glycosaminoglycans. After rat hepatocytes had been homogenized in a hypotonic buffer, more than 98% of the 125I-HA binding activity could be pelleted by centrifugation at 100,000 x g for 1 h. Mild alkaline treatment of hepatocyte membranes did not release 125I-HA binding activity, suggesting that the HA binding site is an integral membrane molecule. Furthermore, trypsin treatment of deoxycholate-extracted membranes destroyed the binding activity, as assessed by a dot-blot assay. This suggests that a protein component in the membrane is necessary for 125I-HA binding activity. Rat fibrinogen could be a possible candidate for the HA binding activity because HA binds specifically to human fibrinogen (LeBoeuf et al. (1986) J. Biol. Chem. 261, 12 586). Also, fibrinogen can be found in a quasi-crystalline form in rat hepatocytes and could be pelleted with the membranes. Rat fibrinogen was not responsible for the 125I-HA binding activity, since (1) purified rat fibrinogen did not bind to 125I-HA, and (2) immunoprecipitation of rat fibrinogen from hepatocyte extracts did not decrease the 125I-HA binding of these extracts. We conclude that the internal HA binding sites are membrane- or cytoskeleton-associated proteins and are neither cytosolic proteins nor fibrinogen.     

Distinct receptors for IGF-I, IGF-II, and insulin are present on bovine capillary endothelial cells and large vessel endothelial cells

Endothelial cells were cultured from bovine fat capillaries, aortae and pulmonary arteries and their interactions with 125I-IGF-I, 125I-MSA (an IGF-II), 125I-insulin and the corresponding unlabeled hormones were evaluated. Each endothelial culture showed similar binding parameters. With 125I-insulin, unlabeled insulin competed with high affinity while IGF-I and MSA were approximately 1% as potent. With 125I-MSA, MSA was greater than or equal to IGF-I in potency and insulin did not compete for binding. Using 125I-IGF-I, IGF-I was greater than or equal to MSA whereas insulin decreased 125I-IGF-I binding by up to 72%. Exposing cells to anti-insulin receptor antibodies inhibited 125I-insulin binding by greater than 90%, did not change 125I-MSA binding, while 125I-IGF-I binding was decreased by 30-44%, suggesting overlapping antigenic determinants between IGF-I and insulin receptors that were not present on MSA receptors. We conclude that cultured capillary and large vessel endothelial cells have distinct receptors for insulin, IGF-I and MSA (IGF-II).     

Identification and characterization of a divalent cation-dependent glycosaminoglycan-binding protein from rat liver endothelium

Rat liver endothelial cells (LECs) express a membrane-associatedCa²⁺-dependent hyaluronan-binding activity (CaHA-BP) which isdistinct from the Ca²⁺-dependent, endocytic LEC HA receptor(Yannariello-Brown et al., J. Cell Biochem., 48, 73–80,1992). The CaHA-BP is specific for a subset of glycosaminoglycans,since Ca²⁺-dependent binding of ¹²⁵I-HA ({small tilde}80kDa)to LECs was competed with a 100-fold excess (w/w) of HA, chondroitinsulfate, and heparin, but not with chondroitin. The CaHA-BPactivity on intact LECs was pH-dependent. Optimal binding occurredat pH 6.0; no binding was detected at pH values 5 or 9. ¹²⁵I-HA,pre-bound in the presence of Ca²⁺ could also be dissociatedwith an acidic buffer (pH 5.0), as well as the divalent cationchelators EDTA and EGTA. ¹²⁵I-HA binding was stimulated by divalentcations other than Ca²⁺ such as Mg²⁺, Mn²⁺ and Ba²⁺; with theexception of Zn²⁺. A photoaffinity crosslinking reagent (¹²⁵I-ASD-HA)was used to identify specifically crosslinked polypeptides onLECs. In the absence of Ca²⁺ and in the presence of EGTA, onlybands at 175/166 kDa were consistently crosslinked. These bandshave been previously identified as the LEC Ca²⁺-independentendocytic HA receptor (Yannariello-Brown et al., J. Biol. Chem.,267, 20451/20455, 1992). In the presence of Ca²⁺, crosslinkingwas consistently seen to a 68 kDa polypeptide. Crosslinkingwas competed with a 100-fold excess (w/w) of HA. These and otherdata suggest that a 68 kDa protein is the most likely candidatefor the CaHA-BP in LECs. photoaffinity crosslinking hyaluronan calcium lectin     

Identification of the Ca(2+)-independent endocytic hyaluronan receptor in rat liver sinusoidal endothelial cells using a photoaffinity cross-linking reagent.

The Ca(2+)-independent endocytic hyaluronan (HA) receptor in rat liver sinusoidal endothelial cells (LECs) was identified using a novel cross-linking derivative of HA. The heterobifunctional, photoactivatable, reducible reagent sulfosuccinimidyl 2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate (SASD) was coupled to the terminal amino group of uniquely modified HA-amine oligosaccharides (M(r) approximately 60,000) and subsequently iodinated. 125I-ASD-HA bound to cultured LECs with similar specificity and affinity as a previously characterized 125I-HA-amine/Bolton-Hunter adduct. Permeabilized LECs were incubated with 125I-ASD-HA with 10 mM EGTA and photolysed with UV light. Detergent extracts were reduced to release the HA oligosaccharides and radiolabeled proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Two polypeptides were consistently and equally labeled at M(r) = 175,000 and 166,000. Photoaffinity labeling of these two proteins was virtually identical in cultured LECs or membranes and was competed greater than 90% with a 100-fold excess of HA. As with the previously characterized bona fide LEC HA receptor, cross-linking was also competed by chondroitin sulfate and heparin, but less efficiently by chondroitin and not with galacturonan. We conclude that the Ca(2+)-independent LEC HA receptor is composed of at least two polypeptides of M(r) approximately 175,000 and 166,000 and may exist as a heterodimer of M(r) approximately 340,000. We also conclude that the LEC HA receptor is distinct from the CD44 family of HA-binding proteins.     

Internalization, recycling, and redistribution of vasopressin receptors in rat hepatocytes

Three hours after isolation, cultured hepatocytes have approximately 150,000 surface vasopressin receptors/cell, and these exhibit a Kd for 125I-vasopressin of 6 nM based on calculation of Koff/Kon, or a Kd of 9.5 nM based on Scatchard plot analysis. After the binding of 125I-vasopressin to its receptor on the hepatocyte surface, this complex is internalized with a t1/2 of 3-6 min. Following this internalization, the number of vasopressin receptors on the cell surface is restored both in vitro and in the isolated perfused liver with a t1/2 of 8-10 min. This restoration is blocked in vitro by incubation of the hepatocytes at 18 degrees C, but not by cycloheximide, suggesting that internalized vasopressin receptors recycle back to the cell surface. Prolonged incubation of hepatocytes with vasopressin results in the loss of greater than 75% of the vasopressin surface binding at concentrations of vasopressin approximately equivalent to its Kd. The binding of vasopressin to cultured hepatocytes 3-5 h after isolation resembles binding to the isolated perfused whole liver with respect to receptor dynamics. During culture for 48 h, however, we observe a progressive loss of hepatocyte surface vasopressin receptors. Concomitant with this reduction in surface receptors with time in culture, there appears to be a marked elevation in intracellular receptors.     

Insulin receptor recycling in vascular endothelial cells. Regulation by insulin and phorbol ester

Endothelial cell insulin receptors mediate the transcytosis of insulin from luminal to abluminal cell surface. We have investigated the kinetics of insulin receptor translocation by immunoprecipitation of radiolabeled receptors at various times before and after trypsin treatment of intact endothelial cells. Insulin receptors were constitutively internalized with t1/2 = 18 +/- 2 min and were recycled to the cell surface. Insulin stimulated receptor internalization and externalization rates 2.6- and 2.4-fold, respectively. Changes in cell-surface binding of 125I-insulin were consistent with the receptor translocation rates observed in surface-labeling experiments. Phorbol myristate acetate (PMA) treatment increased the rate of insulin-stimulated receptor externalization 1.7-fold. PMA treatment increased the constitutive externalization rate 3.5-fold without affecting the constitutive internalization rate, suggesting that recycling might occur via a mobilization of receptors from intracellular sites in a manner independent of internalization rate. Analysis of the intracellular distribution of receptors by 125I-insulin binding and immunogold electron microscopy revealed that less than one-third of the total insulin receptor pool resided on the cell surface. In summary, endothelial cell insulin receptors are constitutively recycled, and internalization and externalization rates are increased by receptor occupancy and PMA treatment.     

Nonionic detergents increase the stoichiometry of ligand binding to the rat hepatic galactosyl receptor

When digitonin is used to expose intracellular galactosyl (Gal) receptors in isolated rat hepatocytes, only about half of the binding activity for 125I-asialoorosomucoid (ASOR) is found as compared to cells solubilized with Triton X-100. The increased ligand binding in the presence of detergent is not due to a decrease in Kd but could be due either to an increase in the number of ASORs bound per receptor or to exposure of additional receptors. Several experiments support the former explanation. No additional activity is exposed even when 80% of the total cell protein is solubilized with 0.4% digitonin. It is, therefore, unlikely that receptors are in intracellular compartments not permeabilized by digitonin and inaccessible to 125I-ASOR. Digitonin-treated cells are not solubilized by Triton X-100 if they are first treated with glutaraldehyde under conditions that retain specific binding activity. 125I-ASOR binding to these permeabilized/fixed cells increases about 2-fold in the presence of Triton X-100 and a variety of other detergents (e.g., Triton X-114, Nonidet P-40, Brij-58, and octyl glucoside) but not with the Tween series, saponin, or other detergents. When these fixed cells are washed to remove detergent, 125I-ASOR binding decreases almost to the initial level. Affinity-purified Gal receptor linked to Sepharose 4B binds approximately twice as much 125I-ASOR in the presence of Triton X-100 as in its absence. The results suggest that the increase in Gal receptor activity in the presence of nonionic detergents is due to an increase in the valency of the receptor rather than to exposure of additional receptors.     

Different fate in vivo of oxidatively modified low density lipoprotein and acetylated low density lipoprotein in rats. Recognition by various scavenger receptors on Kupffer and endothelial liver cells

Human low density lipoprotein was oxidized (Ox-LDL) by exposure to 5 microM Cu2+ and its fate in vivo was compared to acetylated low density lipoprotein (Ac-LDL). Ox-LDL, when injected into rats, is rapidly removed from the blood circulation by the liver, similarly as Ac-LDL. A separation of rat liver cells into parenchymal, endothelial, and Kupffer cells at 10 min after injection of Ox-LDL or Ac-LDL indicated that the Kupffer cell uptake of Ox-LDL is 6.8-fold higher than for Ac-LDL, leading to Kupffer cells as the main liver site for Ox-LDL uptake. In vitro studies with isolated liver cells indicated that saturable high affinity sites for Ox-LDL were present on both endothelial and Kupffer cells, whereby the capacity of Kupffer cells to degrade Ox-LDL is 6-fold higher than for endothelial cells. Competition studies showed that unlabeled Ox-LDL competed as efficiently (90%) as unlabeled Ac-LDL with the cell association and degradation of 125I-labeled Ac-LDL by endothelial and Kupffer cells. However, unlabeled Ac-LDL competed only partially (20-30%) with the cell association and degradation of 125I-labeled Ox-LDL by Kupffer cells, while unlabeled Ox-LDL or polyinosinic acid competed for 70-80%. It is concluded that the liver contains, in addition to the scavenger (Ac-LDL) receptor which interacts efficiently with both Ac-LDL and Ox-LDL and which is concentrated on endothelial cells, an additional specific Ox-LDL receptor which is highly concentrated on Kupffer cells. In vivo the specific Ox-LDL recognition site on Kupffer cells will form the major protection system against the occurrence of the atherogenic Ox-LDL particles in the blood.     

Cyclic AMP increases bradykinin receptor binding affinity in human endothelial cells

We demonstrated bradykinin receptors in human endothelial cells and studied whether bradykinin receptors might be regulated by cyclic AMP. Messenger RNA for bradykinin B(1) and B(2) receptors was detected with real-time PCR and B(2) receptor protein was confirmed by immunoblotting. Saturation binding experiments with increasing concentrations of (125)I-[Tyr(8)]-bradykinin (25-700 pM) were made to determine maximal binding capacity and dissociation constant. However, saturation binding experiments suggested one class of binding sites, maximal binding capacity of 39.3 +/- 1.3 fmol/mg protein and dissociation constant of 352 +/- 27 pM. Competition studies with bradykinin B(1) and B(2) receptor antagonists showed that binding was competed by a B(1) antagonist, and when internalization was inhibited with hypertonic buffer, by both B(1) and B(2) antagonists. Stimulating cells with dibutyryl-cAMP, cholera toxin and forskolin for 24 h increased (125)I-[Tyr(8)]-bradykinin (90 pM) binding with approximately 50%. Saturation binding experiments with dibutyryl-cAMP stimulated cells showed, that the dissociation constant was altered from 352 +/- 27 pM in non-stimulated cells, to 203 +/- 18 pM (P < 0.001) in stimulated cells, while maximal binding capacity remained unchanged. Binding was competed similarly by the B(1) antagonist in stimulated and control cells. These results suggest, that the dibutyryl-cAMP stimulated increase in (125)I-[Tyr(8)]-bradykinin binding is probably due to increased B(1) receptor affinity with no change in receptor capacity. In conclusion, bradykinin B(1) and B(2) receptor mRNA was shown in human endothelial cells. Binding studies suggest that bradykinin receptors are competable with bradykinin antagonists. Adenylate cyclase activators probably increase bradykinin B(1) receptor affinity, without changing capacity, and thus increase bradykinin binding.     

Purification and molecular identification of the human hyaluronan receptor for endocytosis

The clearance of hyaluronan (HA) and chondroitin sulfates from the circulating blood and lymph in the body is mediated by the membrane-bound HA receptor for endocytosis (HARE). Previously, we found that two HARE species of approximately 175 kDa and approximately 300 kDa are abundant in the sinusoidal endothelial cells in rat liver, spleen, and lymph nodes (Zhou et al. [2000], J. Biol. Chem., 275, 37733-37741). In the present study, immunocytochemical analysis of human tissues showed a similar pattern with abundant expression of HARE in the sinusoidal endothelial cells of human liver, spleen, and lymph nodes. The two human HARE proteins were immunoaffinity-purified from human spleen. Each protein was recognized in western blots using several anti-rat HARE monoclonal antibodies and was able to bind 125I-HA specifically. In nonreducing SDS-PAGE, these two human HARE species migrated at approximately 190 kDa and approximately 315 kDa; both proteins are approximately 15 kDa larger than the corresponding rat HAREs, although the de-N-glycosylated core proteins are essentially the same mass. After reduction, the human 190-kDa HARE gave a single 196-kDa species, which was not seen in the approximately 315-kDa HARE after reduction. The reduced approximately 315-kDa HARE yielded two major proteins at approximately 250 kDa and approximately 220 kDa. We determined the sequence of the human 190-kDa HARE cDNA based on analysis of internal tryptic peptides, as well as RT-PCR and 5' RACE analyses using human spleen and lymph node cDNA libraries. The human gene that encodes HARE is on chromosome 12.     

Studies on the interaction between hyaluronan and a rat colon cancer cell line

Binding studies with 125I-Tyr labelled hyaluronan (HA) on a cultured rat colon cancer cell line were performed to characterize the association of HA to tumour cells in vitro. Results show a specific and saturable binding (Kd= 1.36nM) which indicates the presence of an HA binding receptor on the tumour cells. There is a specific constant increase of cell-associated HA over time, which indicates that HA is specifically taken up by the cells through endocytosis. The binding of 125I-Tyr labelled HA was more effectively inhibited by unlabelled HA of high MW in relation to low MW species of the polysaccharide indicating that the receptor binds HA of high MW with greater affinity than low MW species. In competition experiments, the HA-binding could not be inhibited by other polysaccharides such as chondroitin sulphate and heparin. Nor could ligands for scavenger receptors and antibodies directed towards ICAM-1, CD 44 and RHAMM (Receptor for HA Mediated Motility) significantly inhibit the association of HA to tumour cells.