Permeability properties of isolated enterocytes from rat small intestine

Metabolic and permeability properties of enterocytes isolated by treatment of rat small intestine with hyaluronidase or EDTA were compared. No significant difference was observed in the ability of the two types of cell to produce lactate from glucose. However, while cells obtained with hyaluronidase accumulate alpha-methylglucoside, cells obtained with EDTA were unable to accumulate the sugar above the medium concentrations. When resuspended in a medium designed to resemble the intracellular medium, potentiometric measurements showed that cells obtained with hyaluronidase released Ca2+ to the medium while cells obtained with EDTA accumulated it. Using 45Ca transport assays, this was shown to be an ATP-dependent process, the accumulated 45Ca being totally released by the addition of the ionophore A23187. When cells obtained with EDTA were resuspended in a medium containing concentrations of free Ca2+ higher that 10 microM, the uptake was partially inhibited by sodium orthovanadate and also by oligomycin and antimycin. At free Ca2+ concentrations lower than 1 microM, the accumulation was inhibited up to 87% by sodium orthovanadate while mitochondrial inhibitors inhibited only 5%. Thus, it appears that during their preparation cells obtained with hyaluronidase retain their integrity while cells obtained with EDTA become permeable to Ca2+ and other ions. The usefulness of both types of preparation in metabolic and transport studies is discussed.     

THE ENZYMATIC PREPARATION OF ISOLATED INTACT PARENCHYMAL CELLS FROM RAT LIVER

Suspensions of isolated parenchymal cells were prepared from rat liver by incubation with collagenase and hyaluronidase followed by mechanical treatment. Utilization of 0.15% collagenase together with 0.15% hyaluronidase yielded adequate numbers of cells for experimental purposes. As shown by light and electron microscopy, approximately 75% of the isolated cells retain their structural integrity. The cell suspensions are capable of maintaining endogenous respiration in the presence of 1% albumin for periods of time up to 8 hr. These cell preparations consist almost entirely of parenchymal cells and offer a unique tissue preparation for the study of hepatic metabolism.     

Improved isolation of villus and crypt cells from rat small intestinal mucosa.

An enzymic method is described which allows the isolation under comparable conditions of crypt and villus cells from rat jejunum with normal morphologic appearance and high metabolic activity when compared with previous preparations. The method is based on a differential scraping of short lengths of everted small intestine to yield two villus cell fractions and a gut wall residue. The scrapings and the gut tube are incubated for the same length of time in a HEPES-buffered modified Hanks' balanced salt solution containing hyaluronidase, DNase, and soybean trypsin inhibitor. The cells of the crypt region are recovered by a further scraping of the digested gut wall. Cells from all fractions are dispersed by gentle agitation, washed, and harvested by centrifugation. The final crypt and villus cells are 95--99% viable by dye exclusion and exhibit 5--20% cross-contamination on the basis of differential marker enzymes. The isolated crypt and villus cells prepared by the new procedure are suitable for comparative studies of metabolic activity in the absence of chelation-induced structural and metabolic abnormalities.     

Morphological study of chicken cecal and jejunal mucosa during epithelial cell isolation

Epithelial cells from jejunum and proximal cecum of the chicken were isolated by using a sodium citrate containing medium. Cell dissociation during the isolation process was studied by scanning electron microscopy. Results after 5, 15 and 30 min incubation show a sequential cell detachment from the tip to the lower regions of the villi. Inspection of the cell suspension by scanning and transmission electron microscopy reveals that most cells are enterocytes that retain their characteristic cylindrical shape. The brush border of isolated cells maintains its architecture, while organelles remain intact.     

Citrate oscillates in liver and pancreatic beta cell mitochondria and in INS-1 insulinoma cells

Oscillations in citric acid cycle intermediates have never been previously reported in any type of cell. Here we show that adding pyruvate to isolated mitochondria from liver, pancreatic islets, and INS-1 insulinoma cells or adding glucose to intact INS-1 cells causes sustained oscillations in citrate levels. Other citric acid cycle intermediates measured either did not oscillate or possibly oscillated with a low amplitude. In INS-1 mitochondria citrate oscillations are in phase with NAD(P) oscillations, and in intact INS-1 cells citrate oscillations parallel oscillations in ATP, suggesting that these processes are co-regulated. Oscillations have been extensively studied in the pancreatic beta cell where oscillations in glycolysis, NAD(P)/NAD(P)H and ATP/ADP ratios, plasma membrane electrical activity, calcium levels, and insulin secretion have been well documented. Because the mitochondrion is the major site of ATP synthesis and NADH oxidation and the only site of citrate synthesis, mitochondria need to be synchronized for these factors to oscillate. In suspensions of mitochondria from various organs, most of the citrate is exported from the mitochondria. In addition, citrate inhibits its own synthesis. We propose that this enables citrate itself to act as one of the cellular messengers that synchronizes mitochondria. Furthermore, because citrate is a potent inhibitor of the glycolytic enzyme phosphofructokinase, the pacemaker of glycolytic oscillations, citrate may act as a metabolic link between mitochondria and glycolysis. Citrate oscillations may coordinate oscillations in mitochondrial energy production and anaplerosis with glycolytic oscillations, which in the beta cell are known to parallel oscillations in insulin secretion.     

Enzyme Basis for pH Regulation of Citrate and Pyruvate Metabolism by Leuconostoc oenos

Citrate and pyruvate metabolism by nongrowing cells of Leuconostoc oenos was investigated. (sup13)C nuclear magnetic resonance (NMR) spectroscopy was used to elucidate the pathway of citrate breakdown and to probe citrate or pyruvate utilization, noninvasively, in living cell suspensions. The utilization of isotopically enriched substrates allowed us to account for the end products derived from the metabolism of endogenous reserves. The effect of environmental parameters, e.g., pH, gas atmosphere, and presence of malate, on the end products of citrate utilization was studied. Approximately 10% of the citrate supplied was converted to aspartate which remained inside the cells. A metabolic shift with pH was observed, with acetoin production being favored at pH 4, whereas lactate and acetate production increased significantly at higher pH values. The information obtained with NMR was complemented with studies on the relevant enzyme activities in the metabolic pathway of citrate breakdown. The intracellular pH of the cells was strongly dependent on the external pH; this result, together with the determination of the pH profile of the enzymic activities, allowed us to establish the basis for pH regulation; lactate dehydrogenase activity was optimal at pH 7, whereas the acetoin-forming enzymes displayed maximal activities below pH 5. Citrate utilization was also monitored in dilute cell suspensions for comparison with NMR experiments performed with dense suspensions.     

Preparation of cell populations with stabilized erythropoietic potential from the primitive streak chick blastodisc: some implications for control of gastrulation.

Improved methods for preparation from primitive streak chick blastodiscs of cell suspensions capable of forming erythroid cells in culture have been developed. When blastodiscs were preincubayed with hyaluronidase in the absence of collagenase before cell dispersion and a high concentration of methyl-alpha-mannoside was present in all media, the yields of cells were some 10-fold higher than those obtained by former procedures. Cell suspensions obtained consisted almost entirely of viable cells, yielded large numbers of free mature erythrocytes in liquid culture, and formed erythroid colonies and bursts in solidified medium. The capacity to form differentiated cells after resedimenrtation through Ficoll density gradients was partly stabilized. Addition of gee yolk homogenate to the blastodiscs immediately following treatment with hyaluronidase and to all media used thereafter largely stabilized the capacity to form erythroid cells during resedimentation through Ficoll density gradients. Possible relevance of observations made during development of the procedures to the control of onset of cell migration in the process of gastrulation is indicated.     

High pH during trisodium phosphate treatment causes membrane damage and destruction of Salmonella enterica serovar enteritidis

Trisodium phosphate (TSP) is now widely used during the processing of poultry and red meats, but the mechanism whereby it inactivates gram-negative bacteria such Salmonella spp. remains unclear. Thus, Salmonella enterica serovar Enteritidis (ATCC 4931) cells were treated with different concentrations of TSP (1.5, 2.0, and 2.5% [wt/vol]) and compared with (i) cells treated with the same pH as the TSP treatments (pH 10.0, 10.5, and 11.0, respectively) and (ii) cells treated with different concentrations of TSP (1.5, 2.0, and 2.5% [wt/vol]) adjusted to a pH of 7.0 +/- 0.2 (mean +/- standard deviation). Cell viability, loss of membrane integrity, cellular leakage, release of lipopolysaccharides, and cell morphology were accordingly examined and quantified under the above treatment conditions. Exposure of serovar Enteritidis cells to TSP or equivalent alkaline pH resulted in the loss of cell viability and membrane integrity in a TSP concentration- or alkaline pH-dependent manner. In contrast, cells treated with different concentrations of TSP whose pH was adjusted to 7.0 did not show any loss of cell viability or membrane integrity. A 30-min pretreatment with 1.0 mM EDTA significantly enhanced the loss of membrane integrity only when followed by TSP or alkaline pH treatments. Measuring the absorbance at 260 nm, agarose gel electrophoresis, Bradford assay, and Tricine-sodium dodecyl sulfate gel electrophoresis of filtrates of treated cell suspensions revealed considerable release of DNA, proteins, and lipopolysaccharides compared to controls and pH 7.0 TSP treatments. Electron microscopic examination of TSP- or alkaline pH-treated cells showed disfigured cell surface topology and wrinkled appearance and showed evidence of a TSP concentration- and pH-dependent disruption of the cytoplasmic and outer membranes. These results demonstrate that TSP treatment permeabilizes and disrupts the cytoplasmic and outer membranes of serovar Enteritidis cells because of the alkaline pH, which in turn leads to release of intracellular contents and eventual cell death.     

Isolation of pig colonic crypts for cytotoxic assay of luminal compounds: effects of hydrogen sulfide,ammonia, and deoxycholic acid

Some colonic luminal molecules resulting from bacterial metabolism of alimentary or endogenous compounds are believed to exert various effects on the colonic epithelial cell physiology. We isolated surface epithelial cells and intact colonic crypts in order to test bacterial metabolites in the pig model, which is often considered relevant for extrapolation to the physiopathology of the human gastrointestinal tract. Using colonocytes isolated with EDTA, we found that the initial cell viability, estimated by the membrane integrity and oxidative capacity measurement, fell rapidly despite several experimental attempts to preserve it such as the use of a medium designed to increase the adherence of epithelial cells and of a coated extracellular matrix, the presence in the culture medium of the oxidative substrate butyrate, and the use of an inhibitor of the caspases involved in cell apoptosis. In contrast, using dispase and collagenase as proteolytic agents, we were able to obtain pig colonic crypts that maintain an excellent membrane integrity after 4 h. Using this preparation, we were able to test the presumably cytotoxic luminal compounds hydrogen sulfide, ammonia, and deoxycholic acid on colonic crypt viability. Of these, only deoxycholic acid was found to significantly alter the cellular membrane integrity. It is concluded that pig colonic crypts can be useful for thein vitro appraisal of the cytotoxic properties of luminal compounds. This revised version was published online in July 2006 with corrections to the Cover Date.     

Isolated epithelial cells of the toad bladder. Their preparation, oxygen consumption, and electrolyte content

Epithelial cells of the toad bladder were disaggregated with EDTA, trypsin, hyaluronidase, or collagenase and were then scraped free of the underlying connective tissue. In most experiments EDTA was complexed with a divalent cation before the tissue was scraped. Q _OO2, sucrose and inulin spaces, and electrolytes of the isolated cells were measured. Cells disaggregated by collagenase or hyaluronidase consumed O₂ at a rate of 4 µl hr^-1 dry wt^-1. Q _OO2 was increased 50% by ADH (100 U/liter) or by cyclic 3'',5''-AMP (10 mM/liter). Na⁺-free Ringer''s depressed the Q _OO2 by 40%. The Q _OO2 of cells prepared by trypsin treatment or by two EDTA methods was depressed by Na⁺-free Ringer''s but was stimulated relatively little by ADH. Two other EDTA protocols produced cells that did not respond to Na⁺ lack or ADH. The intracellular Na⁺ and K⁺ concentrations of collagenase-disaggregated cells were 32 and 117 mEq/kg cell H₂O, respectively. Cation concentrations of hyaluronidase cells were similar, but cells that did not respond to ADH had higher intracellular Na⁺ concentrations. Cells unresponsive to ADH and Na⁺ lack had high sucrose spaces and low transcellular membrane gradients of Na⁺, K⁺, and Cl^-. The results suggest that trypsin and EDTA disaggregation damage the active Na⁺ transport system of the isolated cell. Certain EDTA techniques may also produce a general increase in permeability. Collagenase and hyaluronidase cells appear to function normally.     

High pH during Trisodium Phosphate Treatment Causes Membrane Damage and Destruction of Salmonella enterica Serovar Enteritidis

Trisodium phosphate (TSP) is now widely used during the processing of poultry and red meats, but the mechanism whereby it inactivates gram-negative bacteria such Salmonella spp. remains unclear. Thus, Salmonella enterica serovar Enteritidis (ATCC 4931) cells were treated with different concentrations of TSP (1.5, 2.0, and 2.5% [wt/vol]) and compared with (i) cells treated with the same pH as the TSP treatments (pH 10.0, 10.5, and 11.0, respectively) and (ii) cells treated with different concentrations of TSP (1.5, 2.0, and 2.5% [wt/vol]) adjusted to a pH of 7.0 ± 0.2 (mean ± standard deviation). Cell viability, loss of membrane integrity, cellular leakage, release of lipopolysaccharides, and cell morphology were accordingly examined and quantified under the above treatment conditions. Exposure of serovar Enteritidis cells to TSP or equivalent alkaline pH resulted in the loss of cell viability and membrane integrity in a TSP concentration- or alkaline pH-dependent manner. In contrast, cells treated with different concentrations of TSP whose pH was adjusted to 7.0 did not show any loss of cell viability or membrane integrity. A 30-min pretreatment with 1.0 mM EDTA significantly enhanced the loss of membrane integrity only when followed by TSP or alkaline pH treatments. Measuring the absorbance at 260 nm, agarose gel electrophoresis, Bradford assay, and Tricine-sodium dodecyl sulfate gel electrophoresis of filtrates of treated cell suspensions revealed considerable release of DNA, proteins, and lipopolysaccharides compared to controls and pH 7.0 TSP treatments. Electron microscopic examination of TSP- or alkaline pH-treated cells showed disfigured cell surface topology and wrinkled appearance and showed evidence of a TSP concentration- and pH-dependent disruption of the cytoplasmic and outer membranes. These results demonstrate that TSP treatment permeabilizes and disrupts the cytoplasmic and outer membranes of serovar Enteritidis cells because of the alkaline pH, which in turn leads to release of intracellular contents and eventual cell death.     

Effects of hyaluronidase, trypsin, and EDTA on surface composition and topography during detachment of cells in culture

Cultured human embryo fibroblasts (HLM18) were labeled with [3H]glucosamine and Na35SO4, and then treated with testicular hyaluronidase, trypsin, or EDTA. Macromolecular material from the surface of these cells was characterized by DEAE-cellulose chromatography and cetylpyridinium chloride precipitation while the associated morphology of cell detachment was studied by phase contrast and scanning electron microscopy. Release of surface glycosaminoglycans by testicular hyaluronidase did not cause cell rounding or detachment. EDTA did not release cell-surface components, but caused cell contraction and detachment morphologically similar to that caused by trypsin. Large amounts of cell-surface glycoproteins and glycosaminoglycans were released by trypsin. From these observations it is concluded that hyaluronic acid is not a principal adhesive agent in the attachment of cells to a substrate. It is suggested that both EDTA and trypsin may have their primary effect upon the cytoskeleton.     

Concurrent isolation of granulocytes and lymphocytes with unaltered permeability,energy state,and metabolic capacity in vitro

A method has been developed based on sedimentation, discontinuous density gradient centrifugation, and differential lysis for the concurrent isolation from the same sample of blood of granulocytes and lymphocytes with yields of 27 and 38%, respectively. The isolated cellular fractions were characterized by a lack of cross-contamination and by the virtual absence of erythrocytes and platelets. Plasma membrane integrity, energy status, and metabolic capacity of the isolated cells were assessed by the cell contents of potassium, sodium, ATP, and by the uptake of oxygen. The respective values for these parameters of cell viability compared favorably with previously reported data for separately isolated granulocytes and lymphocytes. The cellular characteristics determined immediately after the isolation of the cells were maintained throughout a 2-hr period at room temperature.     

STUDIES ON DISPERSED PANCREATIC EXOCRINE CELLS : I. Dissociation Technique and Morphologic Characteristics of Separated Cells

A procedure for dissociation of the guinea pig pancreas into individual cells is described which employs enzymatic digestion with pure collagenase, chymotrypsin, and hyaluronidase, utilizes an interposed chelation of divalent cations by EDTA, and is terminated by gentle shearing. Yields of cells are 50–60%, based on DNA recovered. The population comprises ~95% exocrine cells, the remainder consisting of endocrine, duct, and vascular endothelial cells. The exocrine cells, though spherical, retain the structural attributes of their in situ counterparts, including differentiation of the plasmalemma into zones corresponding to the former apical and basal plasmalemma, polarized distribution of organelles indicated by fields of zymogen granules in the cytoplasm underlying the former apex, central location of the Golgi complex, and placement of the rough endoplasmic reticulum and nucleus in the former basal pole of the cell. Electron microscope study of the effects of individual treatments used during dissociation indicates that digestion of basement membrane and collagen is solely due to collagenase activity and that separation of desmosomes (and possibly of zonulae adherentes) results only from exposure to low [Ca⁺⁺] and EDTA and is not effected by the enzymes used. Gap junctions are resistant to enzymes and EDTA; tight junctions resist enzyme treatment but undergo rearrangement upon exposure to EDTA. Both junctions require mechanical shear for complete cell separation. Neither chymotrypsin nor hyaluronidase produces visible alterations in stromal or junctional elements. Dissociation requires the concerted action of enzymes, chelation of divalent cations, and mechanical shear, since the individual treatments are alone ineffective.     

Inhibition of elicitor-induced phytoalexin formation in cotton and soybean cells by citrate

Addition of an elicitor preparation from Verticillium dahliae to soybean or cotton cell suspension cultures induces the formation of the phytoalexins, glycelollin or sesquiterpene aldehydes, respectively. Recent work (PS Low, PF Heinstein 1986 Arch Biochem Biophys 249: 472-479) has shown that the induction of phytoalexin biosynthesis in these cells is preceded by rapid changes in the plant cell membrane which can be conveniently monitored by membrane associated fluorescent probes. Using this elicitation assay, we have found that citrate, a common metabolite of higher plants, acts as a potent inhibitor of elicitation when added prior to treatment with elicitor. The citrate concentration required to obtain a 50% inhibition of the elicitor-induced fluorescence transition in cultured cotton cells was found to be about 2 millimolar, while the concentration of citrate observed to inhibit elicitor-induced sesquiterpene aldehyde formation in the same cell suspensions was also 2 millimolar. Curiously, in the presence of elicitor, citrate at less than ID₅₀ concentrations increased cell mass accumulation significantly above control incubations without elicitor. A similar inhibition of glyceollin formation with an increase in cell mass accumulation was also observed upon addition of 1 to 5 millimolar citrate to soybean cell suspension cultures. The physiological significance of the inhibition by citrate of phytoalexin formation in plant cell suspensions was supported by the observation that a similar inhibition of sesquiterpene aldehyde formation occurs in cotton plantlets elicited by cold shock or V. dahliae stress. The specificity of citrate as an inhibitor of phytoalexin formation was demonstrated by data showing that other di- and tricarboxylic-hydroxy acids did not inhibit, with the exception of malate which inhibited phytoalexin formation in soybean cells with roughly half the potency of citrate. These experiments not only demonstrate that citrate can act as a specific inhibitor of elicitation, but they further confirm the validity of monitoring elicitation and its modulation with fluorescent probes.     

Differential response of embryonic cells to culture on tissue matrices.

Cell responses to different natural substrates have been followed by scanning microscopy in order to evaluate the role of these substrates in morphogenesis. Matrix has been isolated then repopulated with suspensions of embryonic cells from chick skin, spinal ganglia, duodenal epithelium and heart. In some cases outgrowth from amphibian embryonic tissue was used. Basal lamina of the Xenopus tail may be exposed by freezing and thawing the tissue, or by EDTA treatment. The underlying lamella of orthogonally oriented collagen fibers may be exposed by use of trypsin or hyaluronidase. Trypsin causes more clumping of collagen fibers and a coarser texture of the matrix. On trypsin isolated basement lamella, nerve cell processes grow out on the surface and show no strong tendency to penetrate the lamella while skin mesenchymal cells commonly burrow among the collagen plies. Epithelial cells remain on the surface. On the basal lamina mesenchymal cells ruffle in early stages of culture, then flatten. Epithelial cells flatten rapidly on the lamina. These differences in cell response are in some cases closely related to cell behavior in vivo and suggest that cells show a selective response to the chemical composition of the substrate as well as to its physical conformation.     

The release of radioactive nucleic acids and mucoproteins by trypsin and ethylenediaminetetra-acetate treatment of baby-hamster cells in tissue culture

Monolayers of baby-hamster kidney cells were grown on glass in tissue culture and harvested with trypsin or EDTA in order to investigate the cell surface macromolecules removed by these cell-disaggregating agents. The release of nucleic acids from the cells during the harvesting procedure was monitored by labelling the cellular RNA with [5-(3)H]uridine and the cellular DNA with [2-(14)C]thymidine. Treatment of the cells with EDTA was found to cause an increase in the permeability of the plasma membrane with 7.6% of the cellular RNA, but less than 1% of the cellular DNA, being released. Moreover, 61% of the cells harvested with EDTA were permeable to Trypan Blue. With crude trypsin, lysis of the cell occurred with the release of similar amounts of RNA and DNA amounting to about 11% of the total cellular nucleic acid. In contrast, crystalline trypsin released only 1% of the cellular nucleic acids. Since virtually all the cells (99%) after harvesting in crystalline trypsin were impermeable to Trypan Blue, this method was suitable for obtaining cell surface macromolecules without contamination by intracellular damage. [1-(14)C]Glucosamine was incorporated by the cells only into bound hexosamines and sialic acids. [By monitoring the release of radioactivity in high-molecular-weight material in such experiments a measure of the release of macromolecules containing amino sugars was obtained.] Of the total macromolecules containing amino sugars in the cells 33%, 24% and 13% were released when the cells were harvested with crude trypsin, crystalline trypsin or EDTA respectively. Crystalline trypsin also released 39% of the total sialic acid of the cell, whereas less than 1% of the cellular sialic acid was present in the EDTA-treated fraction. It is concluded that the macromolecules containing amino sugars released with crude trypsin and EDTA are likely to be heavily contaminated with intracellular material. However, the macromolecules released by crystalline trypsin appear to come from the cell surface.     

Preparation and purification of merozoites of Eimeria tenella.

Second-generation merozoites of Eimeria tenella were obtained from both infected cecal tissue and infected chorioallantoic membranes of embryonated eggs. The merozoites were harvested from the tissue by incubation with hyaluronidase, yielding approximately 4 times 10(7) merozoites per cecum and 3 times 10(6) merozoites per chorioallantoic membrane. Subsequent purification of the merozoites by density centrifugation and glass bead filtration resulted in a 54% overall yield and a final preparation of approximately 95% purity. The viability of such preparations was established by inoculation of the merozoites to the ceca of chickens, resulting in oocyst production by 48 hr. This purification procedure allows for a rapid preparation of E. tenella during its second asexual stage in sufficient quantity and purity for biochemical study.     

Structural and functional integrity of chondrocytes immediately following isolation

Chondrocytes were isolated from costal cartilage in young rats after digestion with collagenase and hyaluronidase. The immediate survival of the cells was investigated with the use of different criteria for viability, namely structural integrity and metabolic activity. Structural integrity was studied by transmission and scanning electron microscopy, trypan blue exclusion and NADH oxidation. Metabolic activity was measured both as O2 consumption and as proline and sulphate incorporation, as indicators of collagen and proteoglycan synthesis. The cellular content of glutathione was also measured. The chondrocytes isolated were found to be structurally intact and metabolically active. Early after isolation the chondrocytes varied considerably in size similarly to the native tissue. A selective loss of the larger sized cells was observed during further incubation for 24 h.     

Hyaluronate binding and CD44 expression in human glioblastoma cells and astrocytes.

CD44 is an integral membrane glycoprotein of approximately 90 kDa which has been implicated in the binding of hyaluronate to the cell surface. The expression of CD44 in astrocytes was investigated by means of indirect immunofluorescence on cultured cells. The vast majority of these cells were found to express CD44. Western blot analysis of these cells revealed a highly polydisperse species having an M(r) corresponding to 74-86 kDa. In order to visualize hyaluronate-binding cells, living cultures were probed with fluorescein-conjugated hyaluronate (FI-HA). Some astrocytes were able to bind FI-HA, provided that they were first treated with hyaluronidase. Streptomyces hyaluronidase, which is hyaluronate-specific, was effective in exposing the hyaluronate-binding capacity of these cells. This leads one to conclude that hyaluronate is bound to the surface of these cells and that it masks their capacity to bind hyaluronate. Provided that they were first treated with hyaluronidase, the U-87 MG (glioblastoma-astrocytoma), U-373 MG (glioblastoma), and Hs 683 (glioma) cell lines were also able to bind FI-HA. The U-138 MG (glioblastoma) cell line was unable to bind FI-HA, with or without prior hyaluronidase treatment. A quantitative assay was developed with the use of [3H]hyaluronate ([3H]HA). This revealed the binding to be highly specific, inasmuch as the addition of unlabeled hyaluronate, but not other glycosaminoglycans, was effective in inhibiting the binding of the [3H]HA. An anti-CD44 monoclonal antibody, 50B4, was able to inhibit the binding of the [3H]HA to the U-373 MG cell line. In this cell line, then, CD44 functions as a hyaluronate receptor and one may infer that this is also the case in some astrocytes.