Effects of Multidrug Resistance-Related ATP-Binding-Cassette Transporter Proteins on the Cytoskeletal Activity of Cytochalasins

Cytochalasins are microfilament-active mould metabolites, widely utilized to study the involvement of the actin cytoskeleton in cellular processes as well as in genotoxicity and cell kinetic research. In this study we have investigated whether multidrug-resistance phenotypes, caused by overexpression of the ATP-binding-cassette transporter proteins P-glycoprotein (P-gp) or multidrug-resistance-associated protein (MRP), influence the microfilament-depolymerizing effect of cytochalasins. Using four well-characterized multidrug-resistance cell models, we have shown that both the microfilament-disrupting (phalloidine staining) and the cytotoxic (MTT-assay) activity of cytochalasins are reduced in parallel with increased P-gp expression and restorable by P-gp-modulating agents. This also applied to the cytochalasin D-mediated induction of polykaryons (microscopic evaluation) which arise as a consequence of impaired cytokinesis but unaffected karyokinesis. The reduced cellular activity of cytochalasins in P-gp-positive cell lines was correlated with decreased intracellular accumulation ([³H]cytochalasin B accumulation) which was also restorable by P-gp modulators. Moreover, the dose-dependent inhibition of P-gp photoaffinity labeling ([³H]azidopine) suggested cytochalasins as P-gp-binding agents. In contrast, MRP overexpression had no effect on either cytochalasin microfilament activity or cytotoxicity. In conclusion, data indicate that the microfilament-destructive effects of cytochalasins are impaired due to a reduction of the intracellular cytochalasin accumulation by P-gp but not by MRP. Results are discussed with regard to P-gp as a resistance factor when cytochalasins are utilized to study microfilament dynamics, cell cycle kinetics or chromosomal damage. Moreover, the polykaryon-inducing activity of cytochalasin D is suggested as a specific indicator for a P-gp-mediated multidrug-resistance phenotype and the reversing potency of chemosensitizers.     

Wide-ranging effects of eight cytochalasins and latrunculin A and B on intracellular motility and actin filament reorganization in characean internodal cells

Numerous forms of cytochalasins have been identified and, although they share common biological activity, they may differ considerably in potency. We investigated the effects of cytochalasins A, B, C, D, E, H and J and dihydrocytochalasin B in an ideal experimental system for cell motility, the giant internodal cells of the characean alga Nitella pseudoflabellata. Cytochalasins D (60 microM) and H (30 microM) were found to be most suited for fast and reversible inhibition of actin-based motility, while cytochalasins A and E arrested streaming at lower concentrations but irreversibly. We observed no clear correlation between the ability of cytochalasins to inhibit motility and the actual disruption of the subcortical actin bundle tracks on which myosin-dependent motility occurs. Indeed, the actin bundles remained intact at the time of streaming cessation and disassembled only after one to several days' treatment. Even when applied at concentrations lower than that required to inhibit cytoplasmic streaming, all of the cytochalasins induced reorganization of the more labile cortical actin filaments into actin patches, swirling clusters or short rods. Latrunculins A and B arrested streaming only after disrupting the subcortical actin bundles, a process requiring relatively high concentrations (200 microM) and very long treatment periods of >1 d. Latrunculins, however, worked synergistically with cytochalasins. A 1 h treatment with 15 nM latrunculin A and 4 microM cytochalasin D induced reversible fragmentation of subcortical actin bundles and arrested cytoplasmic streaming. Our findings provide insights into the mechanisms by which cytochalasins and latrunculins interfere with characean actin to inhibit motility.     

Differentiation of rat lens epithelial cells in tissue culture. II. Effects of cytochalasins B and D on actin organization and differentiation

Cytochalasins B and D were used to investigate the involvement of microfilaments in the differentiation of rat lens epithelial cells in tissue culture. Two questions were asked: (1) Does the organization of microfilaments change upon morphological differentiation of the lens epithelial cell? (2) Is the change in the organization of microfilaments required for the production of the differentiation-specific protein, γ-crystallin? Cytochalasin B arborized differentiating lens epithelial cells and had no effect on the undifferentiated cells. Immunofluorescent staining of these two types of cells revealed significant differences in the organization of actin. Actin appeared as longitudinal filaments in the differentiating cells, while it appeared in a diffuse nonfibrillar form in the undifferentiated cells. This indicated changes in the organization of actin during differentiation. Cytochalasin B caused a decline in cell number at 10^?6–10^?5M. However, only that concentration which caused arborization of cells and disruption of microfilaments (10^?5M) inhibited morphological differentiation and production of γ-crystallin. Cytochalasin D (10^?7–10^?5M) did not cause a dramatic decrease in cell number; nevertheless, it induced the arborization of cells and disruption of microfilaments at lower concentrations (10^?7–10^?6M) and inhibited morphological differentiation and production of γ-crystallin at lower concentrations (10^?7–10^?6M) than did cytochalasin B. Thus, only those concentrations of cytochalasins which disrupt microfilaments and prevent their organization into filamentous form seem to inhibit differentiation. This suggests that the organization of actin is required for the program of differentiation of the lens epithelial cells.     

Binding of [3H]cytochalasin B and [3H]colchicine to isolated liver plasma membranes

The binding to isolated hepatocyte plasma membranes of radioactively labelled inhibitors of microfilamentous and microtubular protein function ([³H]cytochalasin B and [³H]colchicine, respectively) was studied as one means of assessing the degree of association of these proteins with cell surface membranes. [³H]Cytochalasin B which behaved identically to the unlabelled compound with respect to binding to these membranes was prepared by reduction of cytochalasin A with NaB³H₄. The binding was rapid, readily reversible, proportional to the amount of membrane and relatively insentive to changes of pH or ionic strength. At 10^?6 M [³H]cytochalasin B, glucose or p-chloromercuribenzoate, an inhibitor of glucose transport inhibited binding by about 20%; treatment of membranes with 0.6 M KI which depolymerizes F actin to G actin caused about 60% inhibition of binding. These two types of inhibition were additive indicating two separate classes of binding sites, one associated with sugar transport and one with microfilaments. Filamentous structures with the diameter of microfilaments (50 Å) were seen in electron micrographs of thin sections of the membranes. At concentrations greater than 10^?5 M [³H]cytochalasin B, binding was proportional to drug concentration, characteristic of non-specific adsorption or partitioning. Intracellular membranes of the hepatocyte also bound [³H]cytochalasin B, those of the smooth endoplasmic reticulum to a greater extent than plasma membranes.[³H]Colchicine bound to plasma membranes in proportion to the amount of membrane and at a rate compatible with binding to tubulin. However, other properties of the binding including effects of temperature, drug concentration and antisera against tubulin were different from those of binding to tubulin. Hence, no evidence was obtained for association of microtubular elements with these membranes. Despite this there appeared to be an interdependence between microtubule and microfilament inhibitors: vinblastine sulfate stimulated [³H]cytochalasin B binding and cytochalasin B stimulated ³H colchicine binding. [³H]Colchicine also bound to intracellular membranes, especially smooth microsomes.     

Anti-immunoglobulin in combination with cytochalasin stimulates proliferation of murine B lymphocytes

The ability of cytochalasin to influence the stimulation of murine B lymphocytes through surface immunoglobulin was assessed during short term cultures. Modest doses of anti-immunoglobulin alone did not stimulate proliferation of mouse spleen cells at 2 days. Cytochalasin B alone also had no effect. However, anti-immunoglobulin in combination with cytochalasin B stimulated substantial proliferation as judged by [3H]thymidine incorporation. Cytochalasins A, E, and D, and dihydrocytochalasin B were all effective in promoting B cell proliferation. Spleen cells from xid-defective (CBA/N X DBA/2)F1 male mice failed to proliferate in response to anti-immunoglobulin plus cytochalasin, suggesting that this treatment affects the same subset of B cells as anti-immunoglobulin plus B cell growth factor. Moreover, proliferation that was stimulated by anti-immunoglobulin plus cytochalasin B was not affected by T cell depletion. Cytochalasin may circumvent the need for, or replace, a second signal for proliferation.     

Cytochalasin sensitive structures and lymphocyte activation.

In purified human peripheral blood lymphocytes, low (0.01–10 μM) concentrations of cytochalasins A B, E and D (CA, CB, CD, CE) produced marked augmentation of transport and metabolic responses to phytohemagglutinin (PHA) and concanavalin A (ConA) including effects on DNA synthesis, cAMP accumulation, phosphatidylinositol turnover and sodium-dependent amino acid transport. At high concentrations (10–100 μM) these same responses were inhibited. Cytochalasin effects were minimal or absent if lectin was not present indicating that these agents are acting by modulating the action of the lectin rather than through a direct effect on cell metabolism. Using [¹²⁵I]ConA, the number of lectin molecules bound per cell was shown not to be altered by the cytochalasins. Taken together with the previously reported effects of the cytochalasins on calcium uptake in lectin stimulated lymphocytes, these observations suggest that microfilaments (or related cytochalasin sensitive structures) play an important role in the modulation of lymphocyte activation.     

The cytoskeleton and rat granulosa cell steroidogenesis: possible involvement of microtubules and microfilaments

The participation of both microtubules and microfilaments in granulosa cell steroidogenesis was assessed by monitoring the effects of colchicine (0-250 microM) and/or cytochalasin B (0-10 micrograms/ml) or dihydrocytochalasin B (0-2.0 micrograms/ml) on cellular morphology and production of progestins during 24 h of culture. Both colchicine and the cytochalasins increased granulosa cell production of progesterone and of 20 alpha-hydroxy-pregn-4-en-3-one (20 alpha-OH-progesterone) in a dose-dependent manner. The largest increase in steroidogenesis (about 2- to 3-fold) was observed at 4-250 microM colchicine and at 2-10 micrograms/ml cytochalasin. Those concentrations of the inhibitors of microtubule or microfilament polymerization that stimulated basal progestin production also markedly influenced cell spreading. Whereas cells cultured for 24 h in medium alone became very flattened with numerous cytoplasmic extensions, those cultured with colchicine (0.2-250 microM) or cytochalasin (0.4-2 micrograms/ml) were much less spread and progressively became more rounded and regular in outline. These changes in cell morphology were reflected by decreases in the mean area occupied by the cells on the culture surface of up to 60-65% and reductions in mean contour index values from 5.7 +/- 0.1 (control) to 3.9 +/- 0.1 (250 microM colchicine), 4.2 +/- 0.1 (2 micrograms/ml cytochalasin B), or 4.1 +/- 0.1 (2 micrograms/ml dihydrocytochalasin B). Cultures containing both colchicine and cytochalasin B exhibited a greater steroidogenic response than that elicited by either inhibitor alone. For example, granulosa cell progesterone production was stimulated almost 2-fold by 4 microM colchicine or 2 microM/ml cytochalasin B, but 5.5-fold by 4 microM colchicine plus 2 micrograms/ml cytochalasin B.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence that forskolin binds to the glucose transporter of human erythrocytes

Binding of [4-3H]cytochalasin B and [12-3H]forskolin to human erythrocyte membranes was measured by a centrifugation method. Glucose-displaceable binding of cytochalasin B was saturable, with KD = 0.11 microM, and maximum binding approximately 550 pmol/mg of protein. Forskolin inhibited the glucose-displaceable binding of cytochalasin B in an apparently competitive manner, with K1 = 3 microM. Glucose-displaceable binding of [12-3H]forskolin was also saturable, with KD = 2.6 microM and maximum binding approximately equal to 400 pmol/mg of protein. The following compounds inhibited binding of [12-3H]forskolin and [4-3H]cytochalasin B equivalently, with relative potencies parallel to their reported affinities for the glucose transport system: cytochalasins A and D, dihydrocytochalasin B, L-rhamnose, L-glucose, D-galactose, D-mannose, D-glucose, 2-deoxy-D-glucose, 3-O-methyl-D-glucose, phloretin, and phlorizin. A water-soluble derivative of forskolin, 7-hemisuccinyl-7-desacetylforskolin, displaced equivalent amounts of [4-3H]cytochalasin B or [12-3H]forskolin. Rabbit erythrocyte membranes, which are deficient in glucose transporter, did not bind either [4-3H]cytochalasin B or [12-3H]forskolin in a glucose-displaceable manner. These results indicate that forskolin, in concentrations routinely employed for stimulation of adenylate cyclase, binds to the glucose transporter. Endogenous ligands with similar specificities could be important modulators of cellular metabolism.     

Relative Potencies of Different Cytochalasins for the Inhibition of Phagocytosis in Ciliates

Ciliate actins have been reported to exhibit an unusual degree of sequence divergence, within the phylum Ciliophora, and when compared to actins from other organisms. To determine whether these primary structural differences are correlated with pharmacological differences, we investigated the effects of seven cytochalasins on phagocytosis, which has been shown to be actin-dependent in eukaryotic cells. The relative potencies of cytochalasin inhibition of phagocytosis in Spirostomun ambiguum and Paramecium multimicronucleatum were similar. Dihydrocytochalasin B and cytochalasin A were the most potent of the seven cytochalasins in both ciliates, and strongly inhibited phagocytosis at 20-40 μM. Dihydrocytochalasin B was considerably more potent than either cytochalasins B or D, a result unexpected on the basis of reports utilizing other organisms. However, even at concentrations up to 100 μM, dihydrocytochalasin B did not inhibit the rate of Spirostomum defecation. After long-term treatment of Spirostomum with 50 μM dihydrocytochalasin B, the sensitivity of phagocytosis to the drug was reduced. These results indicate that the relative cytochalasin sensitivities of actin-dependent functions in ciliates differ from those of other organisms, and are consistent with the hypothesis that sequence differences might give rise to substantial differences in the pharmacological properties of ciliate actins.     

Cytochalasins Z1, Z2 and Z3, three 24-oxa[14]cytochalasans produced by Pyrenophora semeniperda

Three new cytochalasans, named cytochalasins Z1, Z2 and Z3, were isolated from the wheat culture of Pyrenophora semeniperda, a fungus proposed to biologically control grass weeds. Other cytochalasins isolated from the same organic extract were identified as the already known cytochalasins F, T, deoxaphomin and cytochalasins B, the latter being produced in very large amounts. All three new cytochalasins were characterized as 24-oxa[14]cytochalasans by extensive use of NMR and MS techniques. Cytochalasins Z1 and Z2 proved to be structurally related to cytochalasin T, whereas cytochalasin Z3 was related to cytochalasin B. When assayed on wheat and tomato seedlings, cytochalasin Z3, in comparison to the new cytochalasins, cytochalasin B, its 21,22-dihydroderivative, cytochalasin F and deoxaphomin showed a remarkable ability to inhibit root elongation. The possibility of using these metabolites in biological control strategies is discussed.     

Cytochalasin binding in lymphocytes and polymorphonuclear leukocytes

The binding of [³H]cytochalasin B (CB) to intact cells was compared in lymphocytes, polymorphonuclear leukocytes (PMNLs) and erythrocytes over a broad range of cytochalasin concentrations. Binding curves consistent with the presence of high and low affinity binding sites were demonstrated in all three cell types. However, in contrast to observations in erythrocytes, in lymphocytes and PMNLs CB binding was unaffected by d-glucose. p-Hydroxymercuribenzoate and p-hydroxymercurisulfonate were only partially inhibitory and unlabeled cytochalasins E, D and A (CE, CD, CA) inhibited [³H]CB binding more effectively than unlabeled CB. While attempts to demonstrate that plasma membrane-rich subcellular fractions from lymphocytes selectively bind [³H]CB were inconclusive, radioautographic studies on unbroken cells indicated that most or all of the high affinity CB-binding sites in lymphocytes and PMNLs were in close proximity to the cell surface.     

Cytochalasins inhibit nuclei-induced actin polymerization by blocking filament elongation

Polylysine was found to induce polymerization of muscle actin in a low ionic strength buffer containing 0.4 mM MgCl2. The rate of induced polymerization was dependent on the amount and on the molecular size of the polylysine added. A similar effect was obtained by adding actin nuclei (containing about 2-4 actin subunits) cross-linked by p-N,N'- phenylenebismaleimide to G-actin under the same conditions, suggesting that the effect of polylysine is due to promotion of the formation of actin nuclei. Polymerization induced by polylysine and by cross-linked actin nuclei was inhibited by low concentrations (10(-8)-10(-6)M) of cytochalasins. Binding experiments showed that actin filaments, but not actin monomers, contained high-affinity binding sites for [3H]cytochalasin B (one site per 600 actin monomers). The relative affinity of several cytochalasins for these sites (determined by competitive displacement of [3H]dihydrocytochalasin B) was: cytochalasin D greater than cytochalasin E approximately equal to dihydrocytochalasin B. The results of this study suggest that cytochalasins inhibit nuclei-induced actin polymerization by binding to highly specific sites at the point of monomer addition, i.e., the elongation site, in actin nuclei and filaments.     

Activation of superoxide production and differential exocytosis in polymorphonuclear leukocytes by cytochalasins A,B, C,D and E: Effects of various ions

All of the common cytochalasins activate superoxide anion release and exocytosis of β-N-acetylglucosaminidase and lysozyme from guinea-pig polymorphonuclear leukocytes (neutrophils) incubated in a buffered sucrose medium. Half-maximal activation of both processes is produced by approx. 2 μM cytochalasin A, C >μM cytochalasin B ? 4–5 μM cytochalasin D, E. While maximal rates of O₂^? release and extents of exocytosis require extracellular calcium (1–2 mM), replacing sucrose with monovalent cation chlorides is inhibitory to neutrophil activation by cytochalasins. Na⁺, K⁺ or choline inhibited either cytochalasin B- or E-stimulated O₂^? production with IC₅₀ values of 5–10 mM and inhibition occurs whether Cl^?, NO₃^? or SCN^? is the anion added with Na⁺ or K⁺. Release of β-N-acetylglucosaminidase in control or cytochalasin B-stimulated cells is inhibited by NaCl (IC₅₀ ≈ 10 mM), while cytochalasin E-stimulated exocytosis is reduced less and K⁺ or choline chloride are ineffective in inhibiting either cytochalasin B- or E-stimulated exocytosis. Release of β-glucuronidase, myeloperoxidase or acid phosphatase from neutrophils incubated in buffered sucrose is not stimulated by cytochalasin B. Stimulation of either O₂^? or β-N-acetylglucosaminidase release by low concentrations of cytochalasin A is followed by inhibition of each at higher concentrations. It appears that all cytochalasins can activate both NAD(P)H oxidase and selective degranulation of neutrophils incubated in salt-restricted media and that differential inhibition of these two processes by monovalent cations and/or anions is produced at some step(s) subsequent to cytochalasin interaction with the cell.     

The effects of microtubule and microfilament disrupting agents on cytoskeletal arrays and wall deposition in developing cotton fibers

Summary The effects of various cytoskeletal disrupting agents (cholchicine, oryzalin, trifluralin, taxol, cytochalasins B and D) on microtubules, microfilaments and wall microfibril deposition were monitored in developing cotton fibers, using immunocytochemical and fluorescence techniques. Treatment with 10^–4 M colchicine, 10^–6 M trifluralin or 10^–6 M oryzalin resulted in a reduction in the number of microtubules, however, the drug-stable microtubules still appear to influence wall deposition. Treatment with 10^–5 M taxol increased the numbers of microtubules present within 15 minutes of application. New microtubules were aligned parallel to the existing ones, however, some evidence of random arrays was observed. Microtubules stabilized with taxol appeared to function in wall organization but do not undergo normal re-orientations during development. Microtubule disrupting agent had no detectable affect on the microfilament population. Exposure to either 4×10^–5 M cytochalasin B or 2×10^–6M cytochalasin D resulted in a disruption of microfilaments and a re-organization of microtubule arrays. Treatment with either cytochalasin caused a premature shift in the orientation of microtubules in young fibers, whereas in older fibers the microtubule arrays became randomly organized. These observations indicate that microtubule populations during interphase are heterogeneous, differing at least in their susceptibility to disruption by depolymerizing agents. Changes in microtubule orientation (induced by cytochalasin) indicate that microfilaments may be involved in regulating microtubule orientation during development.     

Effects of (dihydro)cytochalasin B, colchicine, monensin and trifluoperazine on uptake and processing of liposomes by Kupffer cells in culture

We investigated the effects of (dihydro)cytochalasin B, colchicine, monensin and trifluoperazine on uptake and processing of large unilamellar liposomes by rat Kupffer cells in maintenance culture. The phospholipid vesicles were labeled in the lipid moiety with phosphatidyl[14C]choline and contained [3H]inulin or [125I]iodoalbumin as nondegradable and degradable markers of the aqueous vesicle content, respectively. Cytochalasin B and dihydrocytochalasin B, inhibitors of microfilament function, reduced inert inulin label uptake by 75% maximally, but residual uptake was not followed by release of lipid degradation products from the cells. By contrast, colchicine, an inhibitor of microtubule assembly, reduced uptake of liposomal inulin by maximally 55% but could not inhibit release of lipid degradation products from the cells. It is concluded that the cytochalasins partly inhibit uptake but fully prevent the arrival of internalized liposomes in the lysosomal compartment, while the action of colchicine is to slow down the overall process of uptake and subsequent transportation to the lysosomes. Monensin reduced inulin uptake to an extent similar to that found with colchicine, but reversibly blocked degradation of liposomal lipid and encapsulated protein. The kinetics of degradation of liposomal constituents suggests that residual uptake in the presence of monensin represents accumulation in an intracellular compartment. Trifluoperazine did not affect binding, internalization or degradation of encapsulated protein at low concentration (6 microM), but completely inhibited release of liposomal lipid degradation products under these conditions. At intermediate concentration (14 microM), the drug also reduced the internalization, while a high concentration (22 microM) was required to inhibit protein degradation as well. We conclude that trifluoperazine has multiple sites of action in the uptake and processing of liposomal constituents by Kupffer cells.     

Inhibition of surfactant release from isolated type II cells by compound 4880

Release of [³H]phosphatidylcholine from pulmonary Type II epithelial cells was stimulated by terbutaline, forskolin and cytochalasin D. Compound $\text{48}\text{80}$ inhibited both basal and agonist-stimulated release of [³H]PC. The IC₅₀ for inhibition by compound $\text{48}\text{80}$ was 1–2 μg/ml, and was similar for inhibition of both basal and stimulated release of [³H]phosphatidylcholine. Inhibitory effects of $\text{48}\text{80}$ were noted following a 1 h exposure to compound $\text{48}\text{80}$ and persisted up to 3 h. The inhibitory effect of compound $\text{48}\text{80}$ was entirely reversed by removing compound $\text{48}\text{80}$ from the external milieu. Compound $\text{48}\text{80}$ had no effect on cytosolic cyclic AMP levels or lactate dehydrogenase release. Inhibition of surfactant release produced by compound $\text{48}\text{80}$ was unaffected by changes in extracellular calcium concentrations. Compound $\text{48}\text{80}$ is a non-toxic inhibitor of phosphatidylcholine release from Type II epithelial cells.     

N-formylpeptide-receptor dynamics, cytoskeletal activation, and intracellular calcium response in human neutrophil cytoplasts

Cytoplasts (enucleated neutrophils which are depleted of dense granules) were prepared from human neutrophils with a modified procedure which employed dihydrocytochalasin B instead of cytochalasin B. These cytoplasts retained an activatable cytoskeletal network similar to cells in that filamentous actin polymerization in response to an N-formylpeptide (fluoresceinated N-formyl-nle-leu-phe-nle-tyr-lys, FLPEP) occurred with similar dose-response characteristics and was inhibitable by cytochalasin B and dihydrocytochalasin B. Cytoplasts had the same number of receptors per surface area as cells and binding constants and dissociation kinetics were the same for cells and cytoplasts. The conversion of receptors from a rapidly dissociating form to a slowly dissociating form was comparable in cells and cytoplasts. This conversion was not inhibited by cytochalasins and thus did not require actin polymerization. Cytoplasts were capable of internalizing 30% of bound FLPEP after 3 min of binding. Cytochalasins did not block this internalization which thus did not appear to require actin polymerization. After 5 min of binding, [3H]-N-formyl-met-leu-phe cosedimented with the Golgi marker enzymes when cytoplasts were fractionated on sucrose density gradients after N2 cavitation. These results indicate that the internalization mechanism is functional in cytoplasts. The Indo-1-detectable calcium response in cytoplasts had a ED50 similar to cells, though the maximum increase in Ca2+ concentration was about one-half that of cells. The response recovered with time after stimulation and the calcium detected was primarily from intracellular stores. The decay of responses after addition of formylpeptide antagonists was parallel for cells and cytoplasts, and leukotriene B4-induced responses in both cells and cytoplasts. Thus the regulation of the responses in cells and cytoplasts was analogous.     

Disruption of filamentous actin inhibits human macrophage fusion.

The foreign body reaction to implanted biomaterials, characterized by the presence of macrophages and foreign body giant cells (FBGC), can result in structural and functional failure of the implant. Recently, we have shown that interleukin-4 and interleukin-13 can independently induce human macrophage fusion to form FBGC via a macrophage mannose receptor (MR) -mediated pathway. The MR is believed to mediate both endocytosis of glycoproteins and phagocytosis of microorganisms, which bear terminal mannose, fucose, N-acetylglucosamine, or glucose residues. Polarization of microfilaments to closely apposed macrophage membranes as observed with fluorescence confocal microscopy led us to ask whether MR-mediated fusion occurred via a filamentous actin-dependent pathway. Cytochalasins B and D and latrunculin-A, agents that disrupt microfilaments, inhibited macrophage fusion in a concentration-dependent manner. The concentrations of cytochalasins D and B that inhibited fusion did not significantly decrease macrophage adhesion, spreading, or motility but did inhibit internalization of Candida albicans during interleukin-13-enhanced, MR-mediated phagocytosis. Very low concentrations of cytochalasin B (< 2 microM) induced a slight enhancement of macrophage fusion. Taken together, the results of this study suggest that cytokine-induced, MR-mediated macrophage fusion requires an intact F-actin cytoskeleton and that the mechanism of fusion is similar to phagocytosis.--DeFife, K. M., Jenney, C. R., Colton, E., Anderson, J. M. Disruption of filamentous actin inhibits human macrophage fusion.     

Effect of cytochalasins on cytosolic-free calcium concentration and phosphoinositide metabolism in leukocytes

Cytochalasins are routinely used to stimulate a variety of functions in eukaryotic cells even though their precise mode of action remains to be elucidated. In the present work we used the fluorescent Ca2+ indicator quin2 to study the effect of various cytochalasins, cytochalasins A, B, C, D, E (CA, CB, CC, CD, CE) and dihydrocytochalasin B (dhCB) on the intracellular Ca2+ concentration ([Ca2+]i) in various types of leukocytes, viz, neutrophils and lymphocytes. In human neutrophils, cytochalasins increase [Ca2+]i mainly by releasing Ca2+ from membrane-bound, intracellular stores. Thus, in order to readily appreciate the effect of cytochalasins on [Ca2+ )i, these cells must be loaded with low intracellular quin2 concentrations. On the other hand, in peripheral blood lymphocytes, splenocytes and thymocytes, the increase in [Ca2+]i is predominantly due to an increased Ca2+ influx from the extracellular medium. In addition, we found that in neutrophils these drugs prolong the increase in [Ca2+]i induced by chemotactic peptides, probably by increasing the cell permeability to Ca2+. Finally, in thymocytes, cytochalasins potentiate the production of inositol phosphates induced by the polyclonal mitogen concanavalin A (conA).