The structure of the glycosaminoglycan chain of a heparan sulfate proteoglycan isolated from the conditioned medium of an endothelial cell line has been analyzed by using various degradative enzymes (heparitinase I, heparitinase II, heparinase, glycuronidase, sulfatases) from Flavobacterium heparinum. This proteoglycan inhibits the thromboplastinactivated pathway of coagulation; as a consequence, the catalytic conversion of prothrombin to thrombin is arrested. Heparitinase I (EC 4.2.2.8), an enzyme with specificity restricted to the heparan sulfate portion of the polysaccharide, releases fragments with the electrophoretic mobility and the structure of heparin. Conversely, an assessment of the size and distribution of the heparan sulfate regions has been provided by the use of heparinase (EC 4.2.2.7), which, by degrading the heparin sections of the chain, releases two segments that exhibit the structure of heparan sulfate. One of these segments is attached to the protein core. On the basis of these findings, the heparan sulfate chain can be defined as a copolymer containing heparin regions in its structure. The combined use of these enzymes has made it possible to establish the disaccharide sequence of parts of the glycosaminoglycan moiety of this proteoglycan.Heparan sulfate proteoglycans are complex macromolecules that consist of a protein backbone to which heparan sulfate chains are covalently linked (1). They are ubiquitous compounds found in a wide variety of vertebrate and invertebrate tissues (2) and are actively synthesized by cells in culture (3). These proteoglycans have been found to be present on the plasma membrane and in the extracellular matrix (4, 5) and exhibit a peculiar structural variability according to the tissue and species of origin (2,6). Despite their wide occurrence, little is known of their biological function. They have been implicated in several biological processes such as cell-cell recognition (7), tissue differentiation (8), organization of extracellular matrices (9), and cell-matrix and cell-substrate adhesion (10).The availability of two heparitinases (11, 12) and a heparinase (EC 4.2.2.7) from Flavobacterium heparinum (12, 13), which can be used in conjunction to elucidate the distribution and grouping in the polymeric chain of disaccharides with various degrees of sulfation and with different hexuronic acid moieties, has enabled us to undertake the structural study of a proteoglycan isolated from the conditioned medium of endothelial cell cultures that appears to be highly characteristic of this cell type. Using these enzymes, we have determined that the glycosaminoglycan chain of this proteoglycan contains heparin segments and have developed a strategy for the elucidation of the sequence of the disaccharide repeating units that may be applicable to the study of other structurally related compounds. MATERIALS AND METHODSSubstrates, Enzymes, and Materials. Heparin from bovine intestinal mucosa and heparan sulfate from bovine pancreas were gifts from P. Bianchini (Opocri...

A cell line from the intima of the rabbit aorta has been established. This cell line exhibits strict contact inhibition, and morphologically resembles intimal endothelial cells. B-type blood group antigens and the presence of fibrinolytic activity also distinguish these cells from smooth muscle cells and from fibroblasts of the aortic wall. Endothelial cells were assayed for changes in levels of adenosine 3': 5'-cyclic monophosphate (cAMP) and guanosine 3':5'-cyclic monophosphate (cGMP) in response to a series of vasoactive drugs. Control levels for cAMP and cGMP are 7.01 4 0.82 and 1.50 4 0.06, respectively (mean 4 SEM). Norepinephrine, acetylcholine, 5-hydroxytryptamine, and phenylephrine increased the levels of both nucleotides significantly. Propranolol (10-5 M) and phentolamine (10-5 M) inhibited, respectively, the cAMP and cGMP response to norepinephrine. Angiotensin II and histamine significantly increased cGMP levels but not cAMP levels of the endothelial cells. The cGMP increases with acetylcholine were inhibited by atropine. These results indicate that the established cell line is endothelial in nature and contains cellular receptors to a variety of vasoactive agents. The role traditionally assigned to the endothelial cell has been that of a rather passive lining of the lumen of the vessels. Recent The line of rabbit fibroblasts used for these studies has been described (6).Mixed Agglutination Test for Blood Group Antigens. Mixed agglutination tests on cells growing in plastic petri dishes were done according to the technique described by Franks and Dawson (7). Antisera were purchased from the Dade Division of the American Hospital Supply Co.Scanning and Transmission Electron Microscopy. To prepare the cultures for scanning electron microscopy, we grew the cells on cover slips (12 mm diameter). At confluence, the cells were first fixed at 370 for 1 hr with 1% glutaraldehyde buffered with 0.1 M cacodylate at pH 7.2, followed by fixing with 3% glutaraldehyde buffered with 0.1 M cacodylate at pH 7.2 for 1 hr at room temperature. After rinsing the cells with distilled water, they were dehydrated in an alcohol series (30-100%) and critical point dried (8). The cells were coated with a thin layer of palladium gold (200 A) in a vacuum evaporator and then viewed under a Cambridge S4 scanning electron microscope.The technique of preparing the cells for transmission by means of electron microscopy has been reported (6). Cultured endothelial cells were also examined by transmission electron microscopy after the second passage.

ions on either side of the membrane, although the action potential is prolonged. Changes in the concentration of H, Ca, or Mg ions may block conduction. The resting and action potentials are somewhat sensitive to Na-ion concentration. These effects are consistent with a model that emphasizes the multilayer structure of the nerve membrane and the divalent ions therein. We wish to express our deep gratitude to Prof. Katsuki of Tokyo Medical and Dental University and to Drs. Hiramoto, Kanatani and Nomura of Tokyo University Marine Station for their constant help during the present experiment.

Heparin stimulates 2-3-fold, in a concentration-dependent manner, the synthesis of heparan sulfate secreted by cultured endothelial cells. The increase in synthetic rate takes place immediately after exposure of the cells to heparin, affects only heparan sulfate, and is specific for the endothelial cell. No stimulation by other glycosaminoglycans was observed. Analysis of the disaccharide products formed by the action of heparitinases reveals a higher degree of sulfation of the uronic acid residues in the heparan sulfate of cells exposed to heparin.

The cytosol extracted from a vascular endothelial cell line binds [3H]estradiol with high affinity and a high degree of specificity. In contrast, in experiments performed with cytosol labeled in the intact cell, progesterone and, to a smaller extent, testosterone gave an apparent inhibition of estradiol binding. These data support the concept that ovarian hormones may influence the role of the endothelium in various physiological and pathophysiological conditions.