Ethanolamine metabolism in cultured bovine aortic endothelial cells

The role of extracellular ethanolamine in phospholipid synthesis was examined in cultured bovine aortic endothelial cells. Serine and ethanolamine were both readily accumulated by these cells and incorporated into phospholipid. Exposing cells to extracellular ethanolamine for 4-6 weeks had no effect on cell growth, yet increased the phosphatidylethanolamine content of these cells by 31% as compared to control cells. The intracellular content of ethanolamine was measured by high performance liquid chromatography, and results showed that the ethanolamine-treated cells contained a significantly greater amount of free ethanolamine compared to control cells (0.62 +/- 0.07 nmol/mg of protein versus 0.27 +/- 0.05 nmol/mg of protein, respectively). Ethanolamine-treated cells also had decreased accumulation and incorporation into lipid of [3H]ethanolamine throughout a 48-h incubation and increased K'm and V'max parameters of ethanolamine transport as compared to control cells. Studies were also done to examine the effect of ethanolamine on the generation of free ethanolamine from phosphatidylserine. In pulse-chase experiments with [3H]serine, a physiological concentration of ethanolamine (25 microM) decreased the amount of 3H-labeled phosphatidylethanolamine produced from 3H-labeled phosphatidylserine by 12 h as compared to the amount of 3H-labeled phosphatidyl-ethanolamine produced in the absence of ethanolamine in the chase incubation. Furthermore, ethanolamine-treated cells accumulated 20% less labeled ethanolamine in the aqueous pool from [3H]serine after 24 h of incubation than did control cells. These results can be explained by isotope dilution with the ethanolamine pool that accumulates in these cells with time when exposed to media supplemented with a physiological concentration of ethanolamine and by an effect of ethanolamine on ethanolamine generation from phosphatidylserine. The results show that an extracellular source of ethanolamine significantly influences the phospholipid metabolism of cultured bovine aortic endothelial cells.     

Bradykinin-induced potassium current in cultured bovine aortic endothelial cells

Summary Bovine aortic endothelial cells (BAECs) respond to bradykinin with an increase in cytosolic-free Ca²⁺ concentration, [Ca²⁺] _i , accompanied by an increase in surface membrane K⁺ permeability. In this study, electrophysiological measurement of K⁺ current was combined with⁸⁶Rb⁺ efflux measurements to characterize the K⁺ flux pathway in BAECs. Bradykinin- and Ca²⁺-activated K⁺ currents were identified and shown to be blocked by the alkylammonium compound, tetrabutylammonium chloride and by the scorpion toxin,noxiustoxin, but not by apamin or tetraethylammonium chloride. Whole-cell and single-channel current analysis suggest that the threshold for Ca²⁺ activation is in the range of 10 to 100nm [Ca²⁺] _i . The whole-cell current measurement show voltage sensitivity only at the membrane potentials more positive than 0 mV where significant current decay occurs during a sustained depolarizing pulse. Another K⁺ current present in control conditions, an inwardly rectifying K⁺ current, was blocked by Ba²⁺ and was not affected bynoxiustoxin or tetrabutylammonium chloride. Efflux of⁸⁶Rb^– from BAEC monolayers was stimulated by both bradykinin and ionomycin. Stimulated efflux was blocked by tetrabutyl- and tetrapentyl-ammonium chloride and bynoxiustoxin, but not by apamin or furosemide. Thus,⁸⁶Rb⁺ efflux stimulated by bradykinin and ionomycin has the same pharmacological sensitivity as the bradykinin- and Ca²⁺-activated membrane currents. The results confirm that bradykinin-stimulated⁸⁶Rb⁺ efflux occurs via Ca²⁺-activated K⁺ channels. The blocking agents identified may provide a means for interpreting the role of the Ca²⁺-activated K⁺ current in the response of BAECs to bradykinin.     

Plasminogen activator activity by cultured bovine aortic endothelial cells

This communication reports the observations that bovine aortic endothelial cells (EC) in culture during their log phase of growth secrete plasminogen activator. Hydrocortisone, dibutyryl cAMP, theophylline, colchicine and cycloheximide, dependent upon concentration, inhibit plasminogen activator activity. Several substances associated with inflammation and thrombosis, such as thrombin, serotonin,catecholomines, histamine, vasopressin, endotoxin, and indomethacin, at the concentrations tested, did not significantly alter plasminogen activator activity when compared with controls.     

Ethanolamine and choline transport in cultured bovine aortic endothelial cells

The transport of the polar head groups, ethanolamine and choline, was examined in cultured bovine aortic endothelial cells. Both ethanolamine and choline are taken up by high- and low-affinity systems. The K'm and V'max for the Na+-dependent, high-affinity ethanolamine and choline transport system are 3.0 and 3.0 microM and 5.4 and 7.3 pmol/mg protein/min, respectively. Ethanolamine and choline competitively influence one another's transport as the presence of 50 microM ethanolamine increases the K'm but not the V'max of choline uptake. Likewise, 50 microM choline increases the K'm but not the V'max of ethanolamine transport. The concentration of ethanolamine that inhibits maximal velocity of 5 microM choline by 50% is 9.7 microM, while 12 microM choline inhibits 5 microM ethanolamine maximal velocity by 50%. Uptake of both head groups is only partially Na+-dependent and is inhibited similarly by 2-methylethanolamine and 2,2-dimethylethanolamine at all concentrations examined. Hemicholinium-3, a classic inhibitor of high-affinity, Na+-dependent choline transport, reduces both ethanolamine and choline accumulation in a concentration-dependent fashion, but has a greater effect on choline transport at higher concentrations. The major portion of these data is consistent with our hypothesis that the uptake of physiological concentrations of ethanolamine and choline may occur through the same transport system. However, the results of the effect of hemicholinium-3 and the extent of Na+-dependency of choline and ethanolamine uptake could be interpreted as meaning that separate transport systems for choline and ethanolamine exist which cross react or that a single transport system exists which has separate active sites for the two compounds.     

Subcellular localization of leukotriene receptors in human endothelial cells

Leukotriene B₄ (LTB₄), a potent chemotactic and immune-modulating mediator, signals via two receptors, BLT₁ and BLT₂. Recently, we reported that BLT₁ is the predominating BLT expressed on human umbilical vein endothelial cells (HUVEC), and that BLT₁ mediated functions are enhanced by LTB₄ and lipopolysaccharide (LPS), but not by TNFα. Here, we demonstrate that BLT₁ is found on the outer cell membrane of HUVECs but also in intracellular granules, co-localized with monocyte chemotactic protein-1 and P-selectin, but not with interleukin-8 and von Willebrand factor. Upon stimulation with LTB₄ or LPS, more BLT₁ protein is found, now evenly distributed over the cytoplasm and in the cell nucleus, but less on the cell surface. An MAP kinase inhibitor prevented this enhancement and translocation, suggesting this signaling pathway to be crucial. Thus, BLT₁, a G-protein-coupled 7-transmembrane receptor, is located in various subcellular compartments in endothelial cells, which may have implications for cellular LT dependent responses and target accessibility for BLT₁ antagonists.     

Glycosaminoglycan production by bovine aortic endothelial cells cultured in sulfate-depleted medium

Bovine aortic endothelial cells were cultured in medium containing [3H]glucosamine and concentrations of [35S]sulfate ranging from 0.01 to 0.31 mM. While the amount of [3H]hexosamine incorporated into chondroitin sulfate and heparan sulfate was constant, decreasing concentrations of sulfate resulted in lower [35S]sulfate incorporation. Sulfate concentrations greater than 0.11 mM were required for maximal [35S]sulfate incorporation. Chondroitin sulfate was particularly affected so that the sulfate to hexosamine ratio in [3H]chondroitin [35S]sulfate dropped considerably more than the sulfate to hexosamine ratio in [3H] heparan [35S]sulfate. Sulfate concentration had no effect on the ratio of chondroitin 4-sulfate to chondroitin 6-sulfate. The ratios of sulfate to hexosamine in cell-associated glycosaminoglycans were essentially identical with the ratios in media glycosaminoglycans at all sulfate concentrations. DEAE-cellulose chromatography confirmed that sulfation of chondroitin sulfate was particularly sensitive to low sulfate concentrations. While cells incubated in medium containing 0.31 mM sulfate produced chondroitin sulfate which eluted later than heparan sulfate, cells incubated in medium containing less than 0.04 mM sulfate produced chondroitin sulfate which eluted before heparan sulfate and near hyaluronic acid, indicating that many chains were essentially unsulfated. At intermediate concentrations of sulfate, chondroitin sulfate was found in very broad elution patterns suggesting that most did not fit an "all or nothing" mechanism. Heparan sulfate produced at low concentrations of sulfate eluted with narrower elution patterns than chondroitin sulfate, and there was no indication of any "all or nothing" sulfation.     

Activation of cultured bovine aortic endothelial cells by extracellular pyrimidine triphosphate.

1. The addition of ATP to cultured bovine aortic endothelial cells induced the increase in intracellular free calcium concentration ([Ca2+]i) and thereby activated the sodium/proton exchanger and the prostacyclin production in a similar dose-dependent manner, as observed by the addition of ATP. 2. Other nucleoside triphosphates also activated the cells and the potency orders of the nucleotides were ATP greater than UTP greater than ITP greater than CTP greater than GTP for all the responses. 3. Pretreatment of the cells with UTP desensitized the response to ATP and the pretreatment of ATP desensitized the response to UTP. 4. The responses to ATP and UTP were inhibited by neither pertussis nor cholera toxin. 5. The receptor for UTP, however, may be a distinct pyrimidinoceptor different from the purinoceptor of the cells for ATP, because the 50% effective concentration of UDP was much larger than that of UTP, while ATP and ADP were essentially equipotent ligands to the endothelial cells.     

Substrate-dependent regulation of intracellular amino acid concentrations in cultured bovine aortic endothelial cells

Amino acid deprivation induces adaptive changes in amino acid transport and the intracellular amino acid pool in cultured cells. In this study intracellular amino acid levels were determined in cultured bovine aortic endothelial cells (EC) deprived of L-arginine or total amino acids for 1, 3, 6 and 24 h. Amino acid concentrations were analyzed by reverse phase HPLC after precolumn derivatisation. Under normal culture conditions levels of L-arginine L-citrulline, total essential and non-essential amino acids were 840 +/- 90 microM, 150 +/- 40 microM, 11.4 +/- 0.9 mM and 53.3 +/- 3.4 mM (n = 9), respectively. In EC deprived of L-arginine or all amino acids for 24 h L-arginine and L-citrulline levels were 200 microM and 50 microM, and 670 microM and 100 microM Deprivation of L-arginine or total amino acids induced rapid (1 h) decreases (30 - 50%) in the levels of other cationic (lysine, ornithine) and essential branched-chain (valine, isoleucine, leucine) and aromatic (phenylalanine, tryptophan) amino acids. L-glutamine was reduced markedly in EC deprived of total amino acids for 1 h - 6 h but actually increased 3-fold in EC deprived of L-arginine for 6 h or 24 h. Arginine deprivation resulted in a rapid decrease in the total intracellular amino acid pool, however concentrations were restored after 24 h. Increased amino acid transport and/or reduced protein synthesis may account for the restoration of amino acid levels in EC deprived of L-arginine. The sustained reduction in the free amino acid pool of EC deprived of all amino acids may reflect utilization of intracellular amino acids for protein synthesis.     

Characterization of P1,P4-diadenosine 5'-tetraphosphate binding on bovine aortic endothelial cells

In recent years it has become increasingly clear that alpha, omega-dinucleotides act as extracellular modulators of various biological processes. P1,P4-diadenosine 5'-tetraphosphate (Ap4A) is the best characterized alpha,omega-dinucleotides and acts as an extracellular signal molecule by inducing the release of nitric oxide (NO) from bovine aortic endothelial cells (BAEC) (R. H. Hilderman, and E. F. Christensen (1998) FEBS Lett. 407, 320-324). However, the characteristics of Ap4A binding to endothelial cells have not been determined. In this report we demonstrate that Ap4A binds to a heterogeneous population of receptors on BAEC. Competition ligand-binding studies using various adenosine dinucleotides, guanosine dinucleotides, adenosine/guanosine dinucleotides, and synthetic P2 purinoceptor agonists and antagonists demonstrate that Ap4A binds to a receptor on BAEC that has a high affinity for some of the adenosine dinucleotides. The apparent IC50 values for Ap4A, Ap2A, and Ap3A are between 12 and 15 microM, while the apparent IC50 values for Ap5A and Ap6A are greater than 500 microM. Evidence is also presented which suggests that this receptor can be classified as a putative P4 purinoceptor. Competition studies also demonstrate that Ap4A binds at a lower affinity to a second class of binding sites.     

Intracellular pH changes of cultured bovine aortic endothelial cells in response to ATP addition

The changes of the intracellular pH (pHi) of cultured bovine aortic endothelial cells were fluorometrically monitored using 2',7'-bis(carboxyethyl)carboxyfluorescein (BCECF). A biphasic pHi change was observed by addition of ATP: an initial acidification followed by an alkalinization of about 0.2 pH unit above the resting level of pHi 7.23. The alkalinization was dependent on [Na+]o and [H+]o, and was inhibited by 5-(N,N-hexamethylene)amiloride, indicating that the alkalinization is mediated by the Na+/H+ exchanger. The 50% effective concentration of ATP was about 1.4 microM. ADP similarly induced pHi changes, whereas AMP and adenosine were inactive. The pHi changes induced by ATP were dependent on the extracellular Ca2+, and the addition of calcium ionophore A23187 induced similar pHi changes. The results indicate that ATP activates the Na+/H+ exchanger in cultured bovine aortic endothelial cells and the activation is mediated by the P2-purinergic receptor and is dependent on the extracellular Ca2+.     

Adhesion of cultured bovine aortic endothelial cells to laminin-1 mediated by dystroglycan

Expression of dystroglycan (DG) by cultured bovine aortic endothelial (BAE) cells was confirmed by cDNA cloning from a BAE cDNA library, Northern blotting of mRNA, Western blotting of membrane proteins, and double immunostaining with antibodies against betaDG and platelet endothelial cell adhesion molecule-1. Immunocytochemical analysis revealed localization of DG in multiple plaques on the basal side of resting cells. This patchy distribution was obscured in migrating cells, in which the most prominent staining was observed in the trailing edge anchoring the cells to the substratum. Biotin-labeled laminin-1 overlay assay of dissociated BAE membrane proteins indicated the interaction of laminin-1 with alphaDG. The laminin alpha5 globular domain fragment expressed in bacteria and labeled with biotin could also bind alphaDG on the membrane blot, and the unlabeled fragment disrupted the binding of biotin-laminin-1 to alphaDG. The interaction of biotin-laminin-1 with alphaDG was inhibited by soluble alphaDG contained in the conditioned medium from DG cDNA-transfected BAE cells and by a series of glycosaminoglycans (heparin, dextran sulfate, and fucoidan). Soluble alphaDG in the conditioned medium inhibited the adhesion of BAE cells to laminin-1-coated dishes, whereas it had no effect on their adhesion to fibronectin. All three glycosaminoglycans that disrupted the biotin-laminin-1 binding to alphaDG inhibited BAE cell adhesion to laminin-1, whereas they failed to inhibit the adhesion to fibronectin. These results indicate a role of DG as a non-integrin laminin receptor involved in vascular endothelial cell adhesion to the extracellular matrix.     

Comparison of leukotriene A4 metabolism into leukotriene C4 by human platelets and endothelial cells.

Leukotriene (LT) A4 metabolism was studied in human platelets and endothelial cells, since both cells could be involved in transcellular formation of LTC4. Upon addition of exogenous LTA4, both cells produced LTC4 as a major metabolite at various incubation times, and no LTB4, LTD4, or LTE4 was detected. Kinetic studies revealed a higher apparent Km for LTA4 in endothelial cells as compared to platelets (5.8 microM for human umbilical vein endothelial cells (HUVEC) versus 1.3 microM for platelets); platelets were more efficient in this reaction with a higher Vmax (174 pmol/mg protein/min) versus 15 pmol/mg protein/min in HUVEC. The formation of LTC4 and corresponding kinetic parameters were not modified when platelets or endothelial cells were stimulated by thrombin prior to or simultaneously with the addition of LTA4. In both cells LTC4 synthase activity was not modified by repeated addition of LTA4 showing that it is not a suicide-inactivated enzyme. Furthermore, in platelets and endothelial cells, the enzyme activity was localized in the membrane fraction and was distinct from cytosolic glutathione-S-transferases. Platelet membrane fractions showed apparent Km values of 31 microM and 1.2 mM for LTA4 and GSH, respectively. Inhibition of LTC4 formation from platelets and endothelial cells preparations by S-substituted glutathione derivatives was correlated to the length of the S-alkyl chain. The same substances inhibited cytosolic glutathione-S-transferases with significantly lower IC50, confirming the distinct nature of the two enzymes. These results show that platelets and HUVEC possess similar enzymes for the production of LTC4 from LTA4; however, platelets seem to have a higher efficiency than HUVEC in performing this reaction.     

Stimulation of receptor-mediated low density lipoprotein endocytosis in neuraminidase-treated cultured bovine aortic endothelial cells

Sialic acids, occupying a terminal position in cell surface glycoconjugates, are major contributors to the net negative charge of the vascular endothelial cell surface. As integral membrane glycoproteins, LDL receptors also bear terminal sialic acid residues. Pretreatment of near-confluent, cultured bovine aortic endothelial cells (BAEC) with neuraminidase (50 mU/ml, 30 min, 37 degrees C) stimulated a significant increase in receptor-mediated 125I-LDL internalization and degradation relative to PBS-treated control cells. Binding studies at 4 degrees C revealed an increased affinity of LDL receptor sites on neuraminidase-treated cells compared to control BAEC (6.9 vs. 16.2 nM/10(6) BAEC) without a change in receptor site number. This enhanced LDL endocytosis in neuraminidase-treated cells was dependent upon the enzymatic activity of the neuraminidase and the removal of sialic acid from the cell surface. Furthermore, enhanced endocytosis due to enzymatic alteration of the 125I-LDL molecules was excluded. In contrast to BAEC, neuraminidase pretreatment of LDL receptor-upregulated cultured normal human fibroblasts resulted in an inhibition of 125I-LDL binding, internalization, and degradation. Specifically, a significant inhibition in 125I-LDL internalization was observed at 1 hr after neuraminidase treatment, which was associated with a decrease in the number of cell surface LDL receptor sites. Like BAEC, neuraminidase pretreatment of human umbilical vein endothelial cells resulted in enhanced receptor-mediated 125I-LDL endocytosis. These results indicate that sialic acid associated with either adjacent endothelial cell surface molecules or the endothelial LDL receptor itself may modulate LDL receptor-mediated endocytosis and suggest that this regulatory mechanism may be of particular importance to endothelial cells.     

Microtubules are involved in transport of macromolecules by vesicles in cultured bovine aortic endothelial cells

The macromolecular transport in bovine aortic endothelial monolayers, cultured in vitro, was studied by fluorescence microscopy, confocal laser scanning microscopy, and transmission electron microscopy. A fluid-phase endocytic tracer, fluorescein isothiocyanate dextran 70 kD (FITC-dextran 70), was found to be transported into and out of endothelial cells via vesicles arranged as chains stretching between the luminal surface and the cell interior and also from cell interior to the abluminal surface. The endocytic activity was reduced by colchicine, which disrupts microtubules, and increased during treatment with cytochalasin B, which blocks microfilament polymerization. These findings indicate that microtubules are required for fluid-phase endocytosis and that microfilaments hinder this process. © 1993 Wiley-Liss, Inc.     

Growth-dependent subcellular redistribution of protein kinase C in cultured porcine aortic endothelial cells

We have previously observed major differences in the phosphorylation of membrane proteins in sparse, proliferating versus confluent, quiescent pig aortic endothelial cells (EC) (Kazlauskas and DiCorleto, 1987). In the present study we examined whether EC growth state can influence the activity of a specific phosphorylating enzyme, protein kinase C (PKC) in cytosolic and membrane fractions of pig aortic EC. Levels of PKC were measured using two methods: 1) Ca2+ and phospholipid-dependent phosphorylation of exogenous histones using gamma-labeled [32P]ATP, and 2) [3H]phorbol-12,13-dibutyrate (PDBu) binding activity. The total amount of PKC activity in the quiescent versus proliferating cells was similar but the percentage of PKC activity in the membrane fraction correlated with the proliferative index of the cells: confluent, quiescent cultures exhibited a majority of PKC activity in the cytosolic fraction (67%), whereas sparse, proliferating cultures contained principally membrane-bound PKC (70%). We also examined the role of PKC in the mitogenic response of pig aortic EC to fetal calf serum. Following serum stimulation of sparse, serum-deprived pig aortic EC, PKC activity redistributed from the cytosolic to the membrane fraction in a rapid process that correlated with subsequent DNA synthesis. A potent activator of PKC, 12-O-tetradecanoylphorbol-13-acetate (TPA), induced a minimal mitogenic response in pig aortic EC when added alone but acted synergistically with low concentrations of fetal calf serum to greatly stimulate DNA synthesis. Furthermore, pig aortic EC treated with TPA for 24 h to down-regulate PKC exhibited only 25% of the serum-stimulated mitogenic activity of control cultures. These results suggest a role for PKC activation and translocation in the proliferation of pig aortic EC.     

Effect of glucose on Na, K-ATPase activity in cultured bovine aortic endothelial cells.

The effect of high concentrations of glucose on Na, K-ATPase activity and the polyol pathway was studied using cultured bovine aortic endothelial cells. Na, K-ATPase activity was expressed as ouabain-sensitive K+ uptake. A significant decrease in Na, K-ATPase activity with an intracellular accumulation of sorbitol was found in confluent endothelial cells incubated with 400 mg/dl glucose for 96 h. However, there was no significant change in the Na, K-ATPase activity or sorbitol content of the cells incubated with 100 mg/dl glucose plus 300 mg/dl mannitol. The decrease in Na, K-ATPase induced by the high glucose concentration was restored by the simultaneous addition of 10(-4) M ponalrestat (ICI 128,436; Statil), an aldose reductase inhibitor. The addition of this agent also significantly reduced the increase in sorbitol induced by high glucose levels. These results suggest that the decrease in Na, K-ATPase activity induced in cultured aortic endothelial cells by high concentrations of glucose may be caused in part by the accumulation of sorbitol.     

Calcium influx induced by activation of tyrosine kinase receptors in cultured bovine aortic endothelial cells

We studied the ionic currents activated by basic fibroblast growth factor (bFGF) and insulin-like growth factor-I (IGF-I) in cultured bovine aortic endothelial cells (BAE-1) by using patch-clamp and single-cell fluorimetric calcium measurements. In whole-cell, voltage-clamp experiments at V(h) = -50 mV, the addition of either bFGF (20 ng/ml) or IGF-I (50 ng/ml) induced an inward current with similar amplitude, time course, and permeation properties. The response was dependent on receptor occupancy and showed a desensitisation in the continued presence of the factors. Ionic substitutions in whole-cell experiments indicated that the current barely discriminated among Na(+), Ca(+), and K(+) ions. Accordingly, stimulation with bFGF or IGF-I induced a dose-dependent [Ca(2+)](i) elevation completely due to entry from the extracellular medium, whereas no detectable release from internal stores was observed. Calcium influx was dependent on protein tyrosine kinase (PTK) activity; it was significantly inhibited by treatment with genistein or tyrphostin 47, two PTK inhibitors, and not affected by inactive analogues, daidzein, and tyrphostin 1. Moreover, addition of 200 microM Na(3)VO(4), an inhibitor of protein tyrosine phosphatase (PTP) activity, evoked the responses to the factors both in patch-clamp and in fluorimetric measurements. Cell-attached recordings using 100 mM CaCl(2) in the pipette showed that bFGF and IGF-I activate calcium-permeable channels with similar properties. These results provide evidence for a calcium influx induced by two factors that bind to tyrosine kinase receptors (RTK) in endothelial cells.     

Agonist-dependent AT(4) receptor internalization in bovine aortic endothelial cells

Recent studies have characterized a specific binding site for the C-terminal 3-8 fragment of angiotensin II (Ang IV). In the present study we looked at the internalization process of this receptor on bovine aortic endothelial cells (BAEC). Under normal culture conditions, BAEC efficiently internalized (125)I-Ang IV as assessed by acid-resistant binding. Internalization of (125)I-Ang IV was considerably decreased after pretreatment of cells with hyperosmolar sucrose or after pretreatment of BAEC with inhibitors of endosomal acidification such as monensin or NH(4)Cl. About 50% of internalized (125)I-Ang IV recycled back to the extracellular medium during a 2 h incubation at 37 degrees C. (125)I-Ang IV remained mostly intact during the whole process of internalization and recycling as assessed by thin layer chromatography. As expected, internalization of (125)I-Ang IV was completely abolished by divalinal-Ang IV, a known AT(4) receptor antagonist. Interestingly, (125)I-divalinal-Ang IV did not internalize into BAEC. These results suggest that AT(4) receptor undergoes an agonist-dependent internalization and recycling process commonly observed upon activation of functional receptors.     

Isolation and characterization of dermatan sulfate proteoglycans synthesized by cultured bovine aortic endothelial cells

Three glucuronic acid-rich dermatan sulfate proteoglycans (DS-PGs) have been isolated by chromatographic and electrophoretic techniques from cultures of bovine aortic endothelial cells and characterized structurally. The smallest of the DS-PGs (DS-II) has an apparent Mr of approximately 100,000 and glycosaminoglycan chains of Mr approximately 29,000. Core glycoprotein samples prepared by chondroitin ABC lyase digestion run as doublets of Mr = 45,000 and 48,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A decrease in core size is apparent after N-glycanase digestion, or when DS-PG is isolated from tunicamycin-treated cultures, providing evidence that the core protein is N-glycosylated. Isolated DS-II shows evidence of self-association when subjected to liquid chromatography under conditions of reduced ionic strength, but not during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In addition, DS-II, but not other endothelial cell DS-PG subclasses, is bound by an antibody against human skin fibroblast DS-PG, indicating that this DS-PG belongs to a family of widely distributed small DS-PGs, previously isolated from various connective tissues. A slightly larger (Mr approximately 220,000) DS-PG (DS-I) can be separated from DS-II by preparative electrophoresis. Despite similarities in core size and extent of N-glycosylation between DS-I and DS-II, DS-I shows only limited ability to self-associate, and does not interact with the anti-fibroblast DS-PG antibody. DS-I glycosaminoglycan chains are also smaller (Mr approximately 18,000) than those from DS-II, similar in size to the chains borne by the DS-PG subclass of largest size (high molecular weight (HMW)-DS). HMW-DS, which predominated in cell layer extracts, runs with a Kav of 0.45 on Sepharose CL-2B and is estimated to have an Mr greater than 700,000. Reduction and alkylation of HMW-DS indicates that it forms disulfide-bonded aggregates with other matrical proteins within the cell layer. HMW-DS displayed multiple protein cores (Mr greater than 200,000) upon chondroitin ABC lyase treatment. Despite some similarity in size to the family of large, aggregating chondroitin sulfate proteoglycans and DS-PGs, immunological evidence suggests that it lacks a hyaluronic acid binding region.