scholarly journals Concanavalin A induces microtubule assembly and specific granule discharge in human polymorphonuclear leukocytes.

1976 ◽  
Vol 68 (3) ◽  
pp. 781-787 ◽  
Author(s):  
S Hoffstein ◽  
R Soberman ◽  
I Goldstein ◽  
G Weissmann
Keyword(s):  
Concanavalin A ◽  
Binding Sites ◽  
Polymorphonuclear Leukocytes ◽  
Cytochalasin B ◽  
Microtubule Assembly ◽  
Human Neutrophils ◽  
Con A ◽  
Specific Granules ◽  
Specific Granule ◽  
Human Polymorphonuclear Leukocytes

Human neutrophils stimulated by concanavalin A (Con A, 100 microng/ml) contained markedly enhanced numbers of microtubules and discharged peroxidase-negative (specific) but not peroxidase-position (azurophile) granules. Release of lysozyme from specific granules was dose and time dependent, could be inhibitied by alpha-methyl-D-mannoside, and enhanced by cytochalasin B. Many microtubules were associated with internalized plasma membrane bearing Con A binding sites.

scholarly journals Effects of glutathione-oxidizing agents on microtubule assembly and microtubule-dependent surface properties of human neutrophils.

1976 ◽  
Vol 71 (3) ◽  
pp. 921-932 ◽  
Author(s):  
J M Oliver ◽  
D F Albertini ◽  
R D Berlin
Keyword(s):  
Concanavalin A ◽  
Peripheral Blood ◽  
Polymorphonuclear Leukocytes ◽  
Human Peripheral Blood ◽  
Microtubule Assembly ◽  
Human Neutrophils ◽  
Hexose Monophosphate ◽  
Con A ◽  
Oxidizing Agents ◽  
The Face

In human peripheral blood polymorphonuclear leukocytes and lymphocytes, GSH-oxidizing agents promote the movement of surface-bound concanavalin A (Con A) into caps and inhibit the assembly of microtubules (MT) that is normally induced by Con A binding. Con A capping and inhibition of MT assembly occur when GSH levels in cell suspensions are decreased by 30-70%, and return to GSH to control levels is accompanied by the appearance of cytoplasmic MT and by inhibition of the capping response with Con A. Oxidation of GSH markedly stimulates the hexose monophosphate shunt, and regeneration of GSH occurs rapidly. The data indicate that MT cannot be assembled or maintained in the face of decreased GSH levels. Thus, GSH homeostasis becomes critical during physiological events such as phagocytosis which simultaneously induce the assembly of MT and the production of agents like H2O2 that can oxidize GSH.

scholarly journals Antagonism by dibutyryl adenosine cyclic 3',5'-monophosphate and testololactone of concanavalin A capping.

1975 ◽  
Vol 66 (2) ◽  
pp. 392-403 ◽  
Author(s):  
B Storrie
Keyword(s):  
Concanavalin A ◽  
Binding Sites ◽  
Cytochalasin B ◽  
Cold Treatment ◽  
Intracellular Camp ◽  
Camp Concentration ◽  
Con A ◽  
Cell Rounding ◽  
Intracellular Camp Concentration

Exposure of CHO-K1 cells in vitro to dibutyryl adenosine cyclic 3',5'-monophosphate (DBcAMP) plus testololactone produces a rapid, reversible antagonism of ligand-induced collection of initially dispersed concanavalin A (Con A) binding sites into a caplike mass. Morphologically, as Con A capping occurs, the cells become less spread and then round completely. With prolonged Con A exposure, cells cultured in either the absence or the presence of DBcAMP plus testololactone cap and round. Capping is blocked by cold treatment and respiratory inhibitors. Colcemid at concentrations greater than 1 muM promotes both Con A capping and cell rounding. Cytochalasin B at similar concentrations inhibits both capping and cell rounding. Treatment of cells with Con A has little effect on intracellular cAMP concentration. Possible mechanisms by which cAMP may modulate the movement of Con A binding sites are discussed.

scholarly journals Role of microtubule assembly in lysosomal enzyme secretion from human polymorphonuclear leukocytes. A reevaluation.

1977 ◽  
Vol 73 (1) ◽  
pp. 242-256 ◽  
Author(s):  
S Hoffstein ◽  
I M Goldstein ◽  
G Weissmann
Keyword(s):  
Plasma Membrane ◽  
Dose Response ◽  
Lysosomal Enzyme ◽  
Polymorphonuclear Leukocytes ◽  
Cytochalasin B ◽  
Microtubule Assembly ◽  
Enzyme Release ◽  
Thin Sections ◽  
Human Polymorphonuclear Leukocytes ◽  
In Cells

The dose-related inhibition by colchicine of both lysosomal enzyme release and microtubule assembly was studied in human polymorphonuclear leukocytes (PMN) exposed to the nonphagocytic stimulus, zymosan-treated serum (ZTS). Cells were pretreated with colchicine (60 min, 37 degrees C) with or without cytochalasin B (5 microng/ml, 10 min) and then stimulated with ZTS (10%). Microtubule numbers in both cytochalasin B-treated and untreated PMN were increased by stimulation and depressed below resting levels in a dose-response fashion by colchicine concentrations above 10(-7) M. These concentrations also inhibited enzyme release in a dose-response fashion although the inhibition of microtubule assembly was proportionately greater than the inhibition of enzyme release. Other aspects of PMN morphology were affected by colchicine. Cytochalasin B-treated PMN were rounded, and in thin sections the retracted plasma membrane appeared as invaginations oriented toward centrally located centrioles. Membrane invaginations were restricted to the cell periphery in cells treated with inhibitory concentrations of colchicine, and the centrioles and Golgi apparatus were displaced from their usual position. After stimulation and subsequent degranulation, the size and number of membrane invaginations greatly increased. They remained peripheral in cells pretreated with greater than 10(-7) M colchicine but were numerous in the pericentriolar region in cells treated with less than 10(-7) M. Similarly, untreated PMN that were permitted to phagocytose immune precipitates had many phagosomes adjacent to the centriole. After colchicine treatment, phagosomes were distributed randomly, without any preferential association with the centrioles. These data suggest that microtubules are involved in maintaining the internal organization of cells and the topologic relationships between organelles and the plasma membrane.

scholarly journals FACTORS AFFECTING THE REDISTRIBUTION OF SURFACE-BOUND CONCANAVALIN A ON HUMAN POLYMORPHONUCLEAR LEUKOCYTES

1974 ◽  
Vol 62 (2) ◽  
pp. 351-365 ◽  
Author(s):  
Graeme B. Ryan ◽  
Joan Z. Borysenko ◽  
Morris J. Karnovsky
Keyword(s):  
Concanavalin A ◽  
Polymorphonuclear Leukocytes ◽  
Cytochalasin B ◽  
Immobilized Cells ◽  
Fluorescein Isothiocyanate ◽  
Cross Linking ◽  
Con A ◽  
Factors Affecting ◽  
Motile Cells ◽  
Cellular Locomotion

Human neutrophil polymorphonuclear leukocytes (PMN) were studied to determine the influence of cellular locomotion upon the redistribution and capping of concanavalin A (Con A). Con A was detected by fluorescence (using Con A conjugated to fluorescein isothiocyanate [Con A-FITC]), or on shadow-cast replicas (using Busycon canaliculatum hemocyanin as a marker for Con A). After labeling with Con A 100 µg/ml at 4°C and warming to 37°C, locomotion occurred, and the Con A quickly aggregated into a cap at the trailing end of the cell. When locomotion was inhibited (with cytochalasin B, or by incubation in serum-free medium at 18°C) Con A rapidly formed a cap over the central region of the cell. Iodoacetamide inhibited capping. PMN labeled with FITC, a monovalent ligand, developed caps at the tail only on motile cells; FITC remained dispersed on immobilized cells. PMN exposed to Con A 100 µg/ml at 37°C bound more lectin than at 4°C, became immobilized, and showed slow central capping. The Con A soon became internalized to form a perinuclear ring. Such treatment in the presence of cytochalasin B resulted in the quick formation of persistent central caps. Colchicine (or prior cooling) protected PMN from the immobilizing effect of Con A, and tail caps were found on 30–40% of cells. Immobilization of colchicine-treated cells caused Con A to remain in dispersed clusters. Thus, capping on PMN is a temperature- and energy-dependent process that proceeds independently of cellular locomotion, provided a colchicine-sensitive system is intact and the ligand is capable of cross linking receptors. On the other hand, if the cell does move, it appears that ligands may be swept into a cap at the tail whether cross-linking occurs or not.

Regulation of enzyme release from human polymorphonuclear leukocytes: further evidence for the independent regulation of granule subpopulations

1987 ◽  
Vol 65 (12) ◽  
pp. 1007-1015 ◽  
Author(s):  
Patricia Murphy ◽  
David A. Hart
Keyword(s):  
Polymorphonuclear Leukocytes ◽  
Second Messenger ◽  
Cytochalasin D ◽  
Enzyme Release ◽  
Specific Granules ◽  
Second Messenger Systems ◽  
Specific Granule ◽  
Human Polymorphonuclear Leukocytes

Addition of 5–20 mM LiCl to purified human polymorphonuclear leukocytes led to the release of lysozyme, the specific granule constituent, but not the release of elastase which is in azurophilic granules. In contrast, 2.5–10 μg cytochalasin D/mL induced the release of both lysozyme and elastase. Addition of lipopolysaccharide to leukocytes did not induce enzyme release but primed cells for enhanced release induced by cytochalasin D. Lipopolysaccharide also primed cells for enhanced release of lysozyme by either N-formylmethionylleucylphenylalanine (fMLP) or Li+ but did not prime cells for elastase release by these stimuli. In contrast, fMLP + cytochalasin D interacted synergistically, leading to enhanced elastase release but not lysozyme release from the cells. Additional experiments with combinations of secretogogues and lipopolysaccharide yielded results consistent with the hypothesis that specific granules and subpopulations of azurophilic granules are under separate regulation and, thus, may be influenced by separate elements of intracellular second messenger systems.

scholarly journals Mechanisms of lysosomal enzyme release from human polymorphonuclear leukocytes. Effects of phorbol myristate acetate.

1975 ◽  
Vol 66 (3) ◽  
pp. 647-652 ◽  
Author(s):  
I M Goldstein ◽  
S T Hoffstein ◽  
G Weissmann
Keyword(s):  
Phorbol Myristate Acetate ◽  
Lysosomal Enzyme ◽  
Polymorphonuclear Leukocytes ◽  
Cytochalasin B ◽  
Enzyme Release ◽  
Myristate Acetate ◽  
Azurophil Granule ◽  
Cytoplasmic Microtubules ◽  
Specific Granule ◽  
Human Polymorphonuclear Leukocytes

PMA enhanced release of the azurophil granule enzyme, beta-glucuronidase, as well as lysozyme, from cytochalasin B-treated PMN's exposed to either zymosan particles or C5a. PMA was active at nanomolar concentrations, was not toxic to the cells, and was most effective when present for brief durations (0-1 min) before exposure of the cells to the stimuli. Beta-glucuronidase was not released in significant amounts from PMN's exposed to PMA alone, in the absence of stimuli such as zymosan or C5a. In contrast, only the specific granule enzyme, lysozyme, was released from unstimulated cells. Electron micrographs of cells exposed to PMA revealed an increase in the number of visible cytoplasmic microtubules as compared to control cells. Enhancement of lysosomal enzyme (beta-glucuronidase) release by PMA appears to be independent of effects on release of specific granule enzymes (lysozyme), but rather is likely due to PMA-induced elevations of cellular cGMP.

scholarly journals EFFECTS OF COLCHICINE, CYTOCHALASIN B, AND 2-DEOXYGLUCOSE ON THE TOPOGRAPHICAL ORGANIZATION OF SURFACE-BOUND CONCANAVALIN A IN NORMAL AND TRANSFORMED FIBROBLASTS

1974 ◽  
Vol 61 (1) ◽  
pp. 70-82 ◽  
Author(s):  
Thomas E. Ukena ◽  
Joan Z. Borysenko ◽  
Morris J. Karnovsky ◽  
Richard D. Berlin
Keyword(s):  
Binding Site ◽  
Concanavalin A ◽  
Binding Sites ◽  
Cytochalasin B ◽  
Simian Virus 40 ◽  
Colchicine Treatment ◽  
Simian Virus ◽  
Transformed Cells ◽  
Con A ◽  
Primary Organization

The distribution of surface-bound concanavalin A on the membranes of 3T3, and simian virus 40-transformed 3T3 cultured mouse fibroblasts was examined using a shadow-cast replica technique with a hemocyanin marker. When cells were prefixed in paraformaldehyde, the binding site distribution was always random on both cell types. On the other hand, labeling of transformed cells with concanavalin A (Con A) and hemocyanin at 37°C resulted in the organization of Con A binding sites (CABS) into clusters (primary organization) which were not present on the pseudopodia and other peripheral areas of the membrane (secondary organization). Treatment of transformed cells with colchicine, cytochalasin B, or 2-deoxyglucose did not alter the inherent random distribution of binding sites as determined by fixation before labeling. However, these drugs produced marked changes in the secondary (but not the primary) organization of CABS on transformed cells labeled at 37°C. Colchicine treatment resulted in the formation of a caplike aggregation of binding site clusters near the center of the cell, whereas cytochalasin B and 2-deoxyglucose led to the formation of patches of CABS over the entire membrane, eliminating the inward displacement of patches observed on untreated cells. The distribution of bound Con A on normal cells (3T3) at 37°C was always random, in both control and drug-treated preparations. Pretreatment of cells with Con A enhanced the effect of colchicine on cell morphology, but inhibited the morphological effects of cytochalasin B. The mechanisms that determine receptor movement and disposition are discussed.

scholarly journals Visualization of intracellular concanavalin A binding sites in retinal photoreceptors.

1978 ◽  
Vol 26 (10) ◽  
pp. 822-828 ◽  
Author(s):  
I Nir
Keyword(s):  
Concanavalin A ◽  
Binding Sites ◽  
Photoreceptor Cells ◽  
Con A ◽  
Thin Sections ◽  
Glycol Methacrylate ◽  
Outer Segments ◽  
Retinal Photoreceptors ◽  
Carbohydrate Components ◽  
The Relationship

Localization of carbohydrate components in retinal photoreceptor cells and membranes was studied. Frog and rat retinas were fixed with glutaraldehyde and embedded in glycol methacrylate or in a mixture of glycol methacrylate, glutaraldehyde and urea. Thin sections were incubated with ferritin-labeled concanavalin A (F-Con A) and stained with osmium vapors. Intensive binding was observed in both rod and cone outer segments. In the rod inner segment, differential binding of F-Con A was demonstrated. While numerous ferritin granules were observed in the myoid zone, only a few were seen in the ellipsoid zone, except for a local accumulation along the plasma membrane. In the rod outer segment, Con A binding sites were closely associated with the disk membranes. Ferritin granules were observed on both sides of the membranes. The relationship between the localization of Con A binding sites and the orientation of visual pigment molecules within the rod outer segments disk membranes was discussed.

scholarly journals Thrombospondin receptor expression in human neutrophils coincides with the release of a subpopulation of specific granules

1992 ◽  
Vol 284 (2) ◽  
pp. 513-520 ◽  
Author(s):  
S J Suchard ◽  
M J Burton ◽  
S J Stoehr
Keyword(s):  
Cytochalasin B ◽  
Anion Channel ◽  
Polymorphonuclear Leucocyte ◽  
Receptor Expression ◽  
Human Neutrophils ◽  
Ionophore A23187 ◽  
Surface Receptors ◽  
Specific Granules ◽  
Azurophil Granule ◽  
Disulphonic Acid

The extracellular matrix (ECM) protein thrombospondin (TSP) binds specifically to polymorphonuclear leucocyte (PMN) surface receptors and promotes cell adhesion and motility. TSP receptor expression increases 30-fold after activation with the synthetic chemotactic peptide, N-formylmethionyl-leucylphenylalanine (FMLP) or the Ca2+ ionophore A23187, in combination with cytochalasin B. The expression of TSP receptors was correlated with the exocytosis of both specific and azurophil granules. Newly expressed TSP receptors are not derived from easily mobilized specific granules since agents that trigger some specific granule release [phorbol myristate acetate (PMA), FMLP or ionophore A23187 alone] do not increase TSP receptor expression. In this study we used the anion-channel blocker, 4,4′-di-isothiocyanatostilbene-2,2′-disulphonic acid (DIDS) to investigate the source of these newly expressed receptors. When PMNs were exposed to cytochalasin B and FMLP or to cytochalasin B and ionophore A23187 in the presence of 30-100 microM-DIDS, TSP receptor expression increased coincidently with vitamin B12-binding protein release from specific granules. Under these same conditions, the release of the azurophil granule component, myeloperoxidase, was significantly inhibited. Using agonists that cause release of specific granules, or both specific granules and azurophil granules, we determined that DIDS blocked the release of PMA-mobilized specific granules and cytochalasin B plus FMLP- or cytochalasin B plus ionophore A23187-mobilized myeloperoxidase-containing azurophil granules but not specific granules mobilized by cytochalasin B plus FMLP or cytochalasin B plus ionophore A23187. These results suggested that PMNs contain at least two subpopulations of specific granules: one that is easily mobilized, lacks TSP receptors and is inhibitable by DIDS, and one that is difficult to mobilize, contains a large pool of TSP receptors and the release of which is enhanced in the presence of DIDS.

Differences in density of Concanavalin A-binding sites due to differences in surface morphology of suspended normal and transformed 3T3 fibroblasts

1975 ◽  
Vol 19 (1) ◽  
pp. 21-32
Author(s):  
J.G. Collard ◽  
J.H. Temmink
Keyword(s):  
Surface Morphology ◽  
Concanavalin A ◽  
Binding Sites ◽  
3T3 Cells ◽  
Con A ◽  
3T3 Fibroblasts ◽  
Transformed Fibroblasts ◽  
Transmission Electron ◽  
The Difference ◽  
Electron Microscopes

Calculations of the density of Concanavalin A (Con A)-binding sites on normal and transformed fibroblasts have, as yet, been based on the unproven assumption that suspended cells are smooth spheres. We studied the surface morphology of suspended normal and transformed fibroblasts with scanning and transmission electron microscopes, and found a large difference in surface morphology between suspended normal and transformed 3T3 cells. When this difference in surface morphology was taken into account, the estimated cell surface area of normal 3T3 cells was approximately seven times larger than that of transformed 3T3 cells. Since equal numbers of 3H-Con A molecules are bound on normal and transformed cells, the density of Con A-binding sites is approximately seven times greater on transformed than on normal 3T3 cells. The difference in density of Con A-binding sites between normal and transformed fibroblasts might be sufficient to explain the difference in agglutination response, as originally suggested by Burger, and may also be the cause of the different degrees of clustering of Con A-binding sites on the plasma membrane of these cells.

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