Plasma Membrane Of Human Fibroblasts Research Articles

Synthesis and shedding of hyaluronan from plasma membranes of human fibroblasts and metastatic and non-metastatic melanoma cells

The regulation of hyaluronan synthesis and shedding was analysed in human fibroblasts and in two melanoma cells that differed in the metastatic potential and proteolysis of the hyaluronan receptor CD44. Dissociation of nascent hyaluronan from plasma membranes isolated from fibroblasts by high salt concentrations led to activation of hyaluronan synthase. Hyaluronan synthesis was also enhanced in plasma membranes from fibroblasts that had been treated with hyaluronidase or trypsin. Hyaluronan oligosaccharides stimulated hyaluronan production in fibroblast cultures. These results indicated that nascent high-molecular-mass hyaluronan inhibited its own chain elongation, if it was retained in the vicinity of the synthase by cell-surface receptors. The results also indicated that increased hyaluronan synthesis and shedding correlated with proteolysis of CD44 on the melanoma cell lines, which has been observed by others.

Synthesis and shedding of hyaluronan from plasma membranes of human fibroblasts and metastatic and non-metastatic melanoma cells

The regulation of hyaluronan synthesis and shedding was analysed in human fibroblasts and in two melanoma cells that differed in the metastatic potential and proteolysis of the hyaluronan receptor CD44. Dissociation of nascent hyaluronan from plasma membranes isolated from fibroblasts by high salt concentrations led to activation of hyaluronan synthase. Hyaluronan synthesis was also enhanced in plasma membranes from fibroblasts that had been treated with hyaluronidase or trypsin. Hyaluronan oligosaccharides stimulated hyaluronan production in fibroblast cultures. These results indicated that nascent high-molecular-mass hyaluronan inhibited its own chain elongation, if it was retained in the vicinity of the synthase by cell-surface receptors. The results also indicated that increased hyaluronan synthesis and shedding correlated with proteolysis of CD44 on the melanoma cell lines, which has been observed by others.

Transbilayer movement of fluorescent and spin-labeled phospholipids in the plasma membrane of human fibroblasts: a quantitative approach.

All phospholipids in the plasma membrane of eukaryotic cells are subject to a slow passive transbilayer movement. In addition, aminophospholipids are recognized by the so-called aminophospholipid translocase, and are rapidly moved from the exoplasmic to the cytoplasmic leaflet of the plasma membrane at the expense of ATP hydrolysis. Though these principal pathways of transbilayer movement of phospholipids probably apply to all eukaryotic plasma membranes, studies of the actual kinetics of phospholipid redistribution have been largely confined to non-nucleated cells (erythrocytes). Experiments on nucleated cells are complicated by endocytosis and metabolism of the lipid probes inserted into the plasma membrane. Taking these complicating factors into account, we performed a detailed kinetic study of the transbilayer movement of short-chain fluorescent (N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl); NBD) and, for the first time, spin-labeled analogues of phosphatidylcholine (PC), -ethanolamine (PE), -serine (PS), and sphingomyelin (SM) in the plasma membrane of cultured human gingival fibroblasts. At 20 degrees C, the passive transbilayer diffusion of NBD analogues was very slow, and the choline-containing NBD analogues were internalized predominantly by endocytosis. Spin-labeled analogues of PC and SM showed higher passive transbilayer diffusion rates, and probably entered the cell by both passive transbilayer movement and endocytosis. In contrast, the rapid uptake of NBD- and spin-labeled aminophospholipid analogues could be mainly ascribed to the action of the aminophospholipid translocase, since it was inhibited by ATP depletion and N-ethylmaleimide pretreatment. The initial velocity of NBD-aminophospholipid translocation was eight to ten times slower than that of the corresponding spin-labeled lipid, and the half-times of redistribution of NBD-PS and spin-labeled PS were 7.2 and 3.6 minutes, respectively. Our data indicate that in human fibroblasts the initial velocity of aminophospholipid translocation is at least one order of magnitude higher than that in human erythrocytes, which should be sufficient to maintain the phospholipid asymmetry in the plasma membrane.

Influence of obstacles on lipid lateral diffusion: computer simulation of FRAP experiments and application to proteoliposomes and biomembranes.

Fluorescence Recovery After Photobleaching experiments were simulated using a computer approach in which a membrane lipid leaflet was mimicked using a triangular lattice obstructed with randomly distributed immobile and non-overlapping circular obstacles. Influence of the radius r and area fraction c of these obstacles and of the radius R of the observation area on the relative diffusion coefficient D* (Eq. (1)) and mobile fraction M was analyzed. A phenomenological equation relating D* to r and c was established. Fitting this equation to the FRAP data we obtained with the probe NBD-PC embedded in bacteriorhodopsin/egg-PC multilayers suggests that this transmembrane protein rigidifies the surrounding lipid phase over a distance of about 18 A (approximately equal to two lipid layers) from the protein surface. In contrast, analysis of published diffusion constants obtained for lipids in the presence of gramicidin suggests that in terms of lateral diffusion, this relatively small polypeptide does not significantly affect the surrounding lipid phase. With respect to the mobile fraction M, and for point obstacles above the percolation threshold, an increase in R led to a decrease in M which can be associated with the existence of closed domains whose average size and diffusion properties can be determined. Adaptation of this model to the re-interpretation of the FRAP data obtained by Yechiel and Edidin (J Cell Biol (1987) 115:755-760) for the plasma membrane of human fibroblasts consistently leads to the suggestion that the lateral organization of this membrane would be of the confined type, with closed lipid domains of approximately equal to 0.5 microns 2 in area.

Cytosolic calcium responses induced by photolytic release of 1,4,5-inositol trisphosphate in single human fibroblasts

We have used the whole cell technique to microinject human fibroblasts with either 1,4,5-inositol trisphosphate (InsP 3) or ‘caged’ InsP 3 in order to study the mechanisms of transmembrane signalling related to mitogenic stimulations. Cytosolic Ca 2+ elevations in response to 1,4,5 InsP 3 diffusing from the patch pipette were difficult to detect, while 1,4,5 InsP 3, photoreleased after loading the cell with its inactive precursor, was capable of generating not only a single cytosolic Ca 2+ rise but sometimes triggered an oscillatory calcium response, similar to that often observed under mitogenic stimulation. We estimated that less than 100 nM InsP 3 was sufficient to generate Ca 2+ responses. The Ca 2+ rise produced by the photoreleased InsP 3 could fully activate the K + channels present in the plasma membrane of human fibroblasts.

Exchange of cystine and glutamate across plasma membrane of human fibroblasts.

It is found that both the inward and outward transport of cystine and glutamate through the plasma membrane of cultured human fibroblasts is mediated mostly by a single transport system. Cystine and glutamate at one side of the membrane stimulate the passage of these amino acids present at the other side of the membrane. When the concentration of intracellular glutamate is reduced to near zero, cystine hardly enters the cell, and likewise the release of glutamate from the cell ceases when cystine is absent in the medium. Homocysteate and alpha-aminoadipate share this transport system and, when added, similarly participate in the transport process. Since the intracellular pool of cystine is negligibly small whereas that of glutamate is very large, the physiologic flows via this system are the entry of cystine and the exodus of glutamate coupled together. Measurements of the rate of uptake of cystine into the cells and the rate of release of glutamate from the cells indicate that the entry of cystine and the exodus of glutamate occur at a ratio close to 1:1. Since cystine is known to behave as an anionic form in this transport, it is concluded that the transport system for cystine and glutamate in plasma membrane of human fibroblasts is a kind of an anion-exchanging agency.

Detection and characterization of carrier-mediated cationic amino acid transport in lysosomes of normal and cystinotic human fibroblasts. Role in therapeutic cystine removal?

The discovery of a trans-stimulation property associated with lysine exodus from lysosomes of human fibroblasts has enabled us to characterize a system mediating the transport of cationic amino acids across the lysosomal membrane of human fibroblasts. The cationic amino acids arginine, lysine, ornithine, diaminobutyrate, histidine, 2-aminoethylcysteine, and the mixed disulfide of cysteine and cysteamine all caused trans-stimulation of the exodus of radiolabeled lysine from the lysosomal fraction of human fibroblasts at pH 6.5. In contrast, neutral and acidic amino acids did not affect the rate of lysine exodus. trans-Stimulation of lysine exodus was observed over the pH range from 5.5 to 7.6, was specific for the L-isomer of the cationic amino acid, and was intolerant to methylation of the alpha-amino group of the amino acid. The lysosomotropic amine, chloroquine, greatly retarded lysine exodus, whereas the presence of sodium ion was without effect. The specificity and lack of Na+ dependence of this lysosomal transport system is similar to that of System y+ present on the plasma membrane of human fibroblasts. In addition, we find cystine exodus from the lysosomal fraction of cystinotic human fibroblasts to be greatly retarded as compared to that of normal human fibroblasts with half-times of exodus similar to those reported for the lysosomes of cystinotic and normal human leukocytes (Gahl, W. A., Tietze, F., Bashan, N., Steinherz, R., and Schulman, J. D. (1982) J. Biol. Chem. 257, 9570-9575). In contrast, normal and cystinotic human fibroblasts did not show any differences with regard to lysine efflux or its trans-stimulation by cationic amino acids. An important mechanism by which cysteamine treatment of cystinosis allows cystine escape from lysosomes may be the ability of the mixed disulfide of cysteine and cysteamine formed by sulfhydryl-disulfide exchange to migrate by this newly discovered system mediating cationic amino acid transport.

Relationship between lectin monosaccharide specificity and binding to the plasma membrane of human fibroblasts

The binding of concanavalin A to cultured normal human fibroblasts has been investigated. Scatchard analysis of the binding data shows the presence of two classes of concanavalin A binding site on the cell surface. One class of binding site, having a high affinity for concanavalin A, is saturated at 0.25 μg/ml. The other, lower affinity sites are saturated at 100 μg/ml concanavalin A. At 4°C the K a for the high affinity sites is approximately 2.2 · 10 6 M −1. The average number of high affinity sites/cell is 3.1 · 10 6, representing less than 1% of the total receptor sites for the lectin. The rate constants of association of concanavalin A are 5.4 · 10 6 M −1 · s −1 and 1.9 · 10 5 M −1 · s −1 for the high and low affinity sites, respectively. From these data, rate constants of dissociation have been calculated as 2.4 · 10 −3 s −1 for the high affinity sites and 0.8 · 10 −1 s −1 for the low affinity sites. Addition of methyl-α- D-mannopyranoside caused a rapid dissociation of concanavalin A from both binding sites. To further characterize the high and low affinity binding sites, we studied their interaction with other lectins. Nine lectins with a variety of carbohydrate binding speficificities were used. Pea and lentil lentins, which have very similar monosaccharide specificity to concanavalin A, inhibited concanavalin A binding by only 25% while concanavalin A completely inhibited the binding of both pea and lentin lectins. These results suggest that all pea and lentil lectin binding sites are also concanavalin A sites but that only one-quarter of all concanavalin A binding sites are also pea and lentil lectin sites. When lectins with monosaccharide specificities different from concanavalin A were studied, none had any effect on concanavalin A binding except wheat germ agglutinin which inhibited by 30%. We conclude that monosaccharide specificity alone is not sufficient to determine the sites to which a lectin will bind on the cell surface.