Cytochalasin inhibits the rate of elongation of actin filament fragments

Submicromolar concentrations of cytochalasin inhibit the rate of assembly of highly purified dictyostelium discoideum actin, using a cytochalasin concentration range in which the final extent of assembly is minimally affected. Cytochalasin D is a more effective inhibitor than cytochalasin B, which is in keeping with the effects that have been reported on cell motility and with binding to a class of high-affinity binding sites from human erythrocyte membranes (Lin and Lin. 1978. J. Biol. CHem. 253:1415; Lin and Lin. 1979. Proc. Natl. Acad. Sci. U.S.A. 76:2345); 5x10(-7) M cytochalasin B lowers it to 70 percent of the control value, whereas 10(-7) M cytochalasin B lowers the rate to 25 percent. Fragments of F-actin were used to increase the rate of assembly fivefold by providing more filament ends on to which monomers could add. Under these conditions, cytochalasin has an even more dramatic effect on the assembly rate; the concentrations of cytochalasin B and cytochalasin D required for half-maximal inhibition are 2x10(-7) M and 10(-8) M, respectively. The assembly rate is most sensitive to cytochalasin when actin assembly is carried out in the absence of ATP (with 3 mM ADP present to stabilize the actin). In this case, the concentrations of cytochalasin B and cytochalasin D required for half-maximal inhibition are 4x10(-8) M and 1x10(-9) M, respectively. A scatchard plot has been obtained using [(3)H]cytochalasin B binding to F-actin in the absence of ATP. The K(d) from this plot (approximately 4x10(-8) M) agrees well with the concentration of cytochalasin B required for half-maximal inhibition of the rate of assembly under these conditions. The number of cytochalasin binding sites is roughly one per F-actin filament, suggesting that cytochalasin has a specific action on actin filament ends.     

Cytochalasin inhibition of hexose transport by platelets

Previously we described a two-transporter model (T1, T2) for galactose uptake by platelets (Horne, M.K. and Hart, J.S. (1986) Biochim. Biophys. Acta 856, 448-456). In the current work we have sought corroborative evidence for this model by studying the effects of cytochalasins on this transport system. Of the various cytochalasins tested, cytochalasin B was the most potent inhibitor (I) of galactose transport, whereas cytochalasin A was less inhibitory and dihydrocytochalasin B and cytochalasin E had no inhibitory effect. The same order of potency was observed for the inhibition of L-glucose diffusion into platelets. The mechanism of cytochalasin B inhibition was investigated in detail. Inhibition of T1 was competitive and required a higher concentration of cytochalasin B (Ki1 approximately 1.7 microM) than inhibition of T2, which was of a mixed type (Ki2 approximately 0.8 microM). The effect of cytochalasin B on T2 could be accounted for by a membrane alteration which enhanced the affinity of the transporter for galactose while simultaneously preventing passage of the TSI complex into the cell. Since a similar effect on membrane permeability would also explain cytochalasin B inhibition of L-glucose diffusion, it is hypothesized that cytochalasin B binds to a membrane structure shared by T2 and the passage for L-glucose. The differences in cytochalasin B sensitivity and mechanism of inhibition manifested by T1 and T2 support our original hypothesis that galactose is indeed transported by kinetically distinct agencies and suggest that these may be physically distinct as well.     

Stimulation of B cells by sequential addition of anti-immunoglobulin antibody and cytochalasin

B cells are stimulated to initiate DNA synthesis by modest doses of anti-immunoglobulin antibody in combination with cytochalasin. The ability of these agents to stimulate B cells in a sequential fashion was evaluated. Anti-immunoglobulin prepared cells to respond to subsequently added cytochalasin, but cytochalasin did not prepare cells to respond to anti-immunoglobulin. Only brief exposure to anti-immunoglobulin was needed to activate B cells for cytochalasin responsiveness; once activated, B cells remained responsive to cytochalasin for hours. The ability of cytochalasin to supply a second signal to activated B cells suggests that the cytoskeleton may be involved in generating or transducing growth-promoting signals for B lymphocytes.     

Cytochalasin induces an increase in cytosolic free calcium in murine B lymphocytes

Cytochalasin promotes the progression of anti-immunoglobulin-treated B lymphocytes to S phase. However, the intracellular events induced by cytochalasin which may mediate signaling for progression have not been elucidated. In this study, the effect of cytochalasin on the level of intracellular free calcium in murine splenic B lymphocytes was assessed by using the fluorescent calcium indicator Indo-1. Cytochalasins A, B, D, and E induced a rapid and sustained elevation of intracellular free calcium. The calcium response to cytochalasin derived largely from the influx of extracellular calcium, although a small, transient elevation in intracellular calcium persisted when the suspension medium was made calcium-free with EGTA, implicating an intracellular source for a portion of the calcium response. Single cell fluorescence studies revealed that cytochalasin elicited a calcium response in most splenic B cells in suspension, indicating that this phenomenon is not restricted to a subpopulation of responding B cells. Phorbol esters inhibited the B cell calcium response to cytochalasin, and an established response to cytochalasin was rapidly and completely reversed by subsequently administered phorbol ester. T cells that lack the cytochalasin pathway showed a markedly diminished calcium response that was only apparent at higher cytochalasin concentration. However, B cells from xid-defective [CBA/N X DBA/2]F1 males, which fail to respond to anti-immunoglobulin plus cytochalasin, showed a calcium response to cytochalasin similar to that of phenotypically normal F1 females. These data, along with the finding that the rise in intracellular calcium occurred in naive B cells as well as B cells previously treated with anti-immunoglobulin, suggest that there is no clear association between the calcium response induced by cytochalasin and the ability of cytochalasin to stimulate progression to S phase. However, this effect of cytochalasin may suggest a connection between actin filaments and calcium influx in B cells.     

Effect of cytochalasin B on the adhesion of mouse peritoneal macrophages

The adhesion of normal mouse macrophages to glass surfaces was reduced by nontoxic levels (1-50 mug/ml) of cytochalasin B in combination with a centrifugal force (1,000-8,000 g). Macrophages nonspecifically activated by Corynebacterium acnes were also detached by this treatment, but less effectively. The effects of cytochalasin B treatment on these cells were shown to be reversible. After detachment, the cells reattached to glass, appeared morphologically normal, and behaved like untreated cells as judged by adhesion, acid phosphatase levels, and phagocytosis. The effect of cytochalasin B on several parameters of phagocytosis by normal macrophages was also examined. The results demonstrate that cytochalasin B can be used to detach macrophages from surfaces and suggest a functional relationship between phagocytosis and macrophage adhesion to surfaces. Furthermore, the effect of cytochalasin B on adhesion of phagocytic cells provides a probe for further investigation of the adhesion of cells to surfaces.     

Cytochalasin B inhibits phosphoinositide hydrolysis in rat hippocampal slices

The effect of cytochalasin B on phosphoinositide (PI) hydrolysis was examined in rat hippocampal slices. Pretreatment of the slices with cytochalasin B caused a significant decrease in PI hydrolysis elicited by carbachol, norepinephrine, or by high K⁺. This effect was cytochalasin B dose- and time-dependent and was not mimicked by cytochalasin D, vinblastine, colchicine, or phloretin. In contrast, in [³H]inositol-prelabeled hippocampal membranes, cytochalasin B did not affect PI hydrolysis elicited by GTPS and GTPS plus carbachol. Similar result was obtained using the membranes prepared from the slices pretreated with cytochalasin B. The inhibitory effect of cytochalasin B on the carbachol-response was observed in SK-N-SH human neuroblastoma cells, but not in cultured rat astrocytes. These results indicate that cytochalasin B inhibits PI hydrolysis in neuron-specific manner and that its action may be an indirect cellular mechanism other than interaction with cytoskeleton elements.     

Effects of cytochalasin B and enucleation on EGF receptor down regulation

The loss of epidermal growth factor (EGF) binding activity on cultured murine 3T3 cells exposed to EGF (EGF receptor down regulation) was determined in colchicine treated cells, cytochalasin B treated cells, and untreated cells. Neither colchicine nor cytochalasin B altered the affinity of the receptor for EGF, but colchicine decreased maximal EGF binding activity by 20%. The maximal extent of EGF receptor down regulation was similar in colchicine treated cells and cytochalasin B treated cells, but the rate of receptor down regulation was higher in cytochalasin B treated cells. Cytoplasts produced by subjecting cytochalasin B treated cells adhering to the substratum to centrifugal force responded to EGF with nearly normal down regulation kinetics. The results suggest that the cytoskeleton is not obligatorily involved in EGF-induced EGF receptor down regulation.     

PKB phosphorylation and survivin expression are cooperatively regulated by disruption of microfilament cytoskeleton

Changes in cell shape can lead to detachment and cell death, and the disruption in the actin cytoskeletal network, as one marker of cell shape changes, can itself induce apoptosis. In this study, the effects of cytochalasin B on the apoptosis-related proteins, protein kinase B and survivin were investigated. Apoptosis induced by disruption of microfilaments with cytochalasin B was found, although it happened at a low level, to simultaneously occur with G2/M arrest in 50% of the cytochalasin B-treated cells. During apoptosis, PKB phosphorylation and survivin expression was decreased by cytochalasin B, and the decline in survivin expression were preceded by PKB dephosphorylation, which implicated that survivin may be a target of PKB protein. The G2/M arrest of cytochalasin B-treated cells may be the direct function of cytochalasin B to microfilaments or the subsequent inhibition of survivin expression, or both. These results suggest that PKB/survivin signaling pathway may be responsible for the apoptosis induced by the disruption of actin cytoskeleton.     

Microfilaments and FSH stimulation of rat granulosa cell steroidogenesis in vitro

The secretion of progesterone and 20 alpha-hydroxypregn-4-en-3-one (20 alpha-dihydroprogesterone) by granulosa cells from 30-day-old rats pretreated with PMSG (4 i.u.; i.p.) was significantly increased in a time- and concentration-dependent manner by FSH or cytochalasin B. Whereas FSH markedly stimulated progestagen secretion during 3 h of incubation, a significant enhancement of the steroidogenic response was not noted until 12 h of exposure to the inhibitor in vitro. Although cytochalasin B also enhanced the submaximal stimulation of progestagen production by FSH (15 ng/ml), it was ineffective in the presence of maximal stimulatory concentration of the gonadotrophin (150 ng/nl). With increasing concentrations of cytochalasin B, the ability of FSH to further stimulate progestagen secretion was progressively reduced. Granulosa cells cultured in medium alone contained a prominent cytoplasmic array of microfilaments which was markedly reduced by FSH or cytochalasin B. FSH and, to a greater extent, cytochalasin B elicited concentration-dependent reductions in the mean area occupied by the cells on the culture surface, the contour index (a size-independent representation of cell profile irregularity) and cell perimeter, indicating that the cells underwent less spreading and were more spherical and regular in outline in the presence of either agent. The FSH-induced reductions in the three shape-related parameters were augmented by cytochalasin B although the influence of the FSH on the mean area and perimeter was progressively reduced in the presence of higher concentrations of cytochalasin B.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibitions of sugar transport produced by ligands binding at opposite sides of the membrane. Evidence for simultaneous occupation of the carrier by maltose and cytochalasin B

This study examines inhibitions of human erythrocyte D-glucose uptake at ice temperature produced by maltose and cytochalasin B. Maltose inhibits sugar uptake by binding at or close to the sugar influx site. Maltose is thus a competitive inhibitor of sugar uptake. Cytochalasin B inhibits sugar transport by binding at or close to the sugar efflux site and thus acts as a noncompetitive inhibitor of sugar uptake. When maltose is present in the uptake medium, Ki(app) for cytochalasin B inhibition of sugar uptake increases in a hyperbolic manner with increasing maltose. When cytochalasin B is present in the uptake medium, Ki(app) for maltose inhibition of sugar uptake increases in a hyperbolic manner with increasing cytochalasin B. High concentrations of cytochalasin B do not reverse the competitive inhibition of D-glucose uptake by maltose. These data demonstrate that maltose and cytochalasin B binding sites coexist within the glucose transporter. These results are inconsistent with the simple, alternating conformer carrier model in which maltose and cytochalasin B binding sites correspond to sugar influx and sugar efflux sites, respectively. The data are also incompatible with a modified alternating conformer carrier model in which the cytochalasin B binding site overlaps with but does not correspond to the sugar efflux site. We show that a glucose transport mechanism in which sugar influx and sugar efflux sites exist simultaneously is consistent with these observations.     

Effects of hyaluronic acid in culture and cytochalasin B treatment before freezing on survival of cryopreserved bovine embryos produced in vitro

Summary One limitation to the widespread use of in vitro-produced embryos in cattle is their poor survival following cryopreservation. Two approaches for enhancing survival of in vitro-produced bovine embryos following cryopreservation were evaluated: culture in the presence of hyaluronic acid and alterations in the cytoskeleton through cytochalasin B treatment. The experiment was a 2×2 factorial design to test main effects of hyaluronic acid added to culture at day 5 after insemination (+or−) and cryopreservation treatment (control or cytochalasin B). Embryos used for cryopreservation were blastocysts and expanded blastocysts harvested on day 7 after insemination. Cytochalasin B increased the percent of embryos that re-expanded (P<0.0001) and that hatched following thawing (P<0.05). The hatching percent was 29.6% for embryos treated with cytochalasin B versus 9.1% for control embryos. There was no significant effect of hyaluronic acid on survival although there was a tendency for embryos cultured with hyaluronic acid to have higher percent hatching if not treated with cytochalasin B (12.7% for hyaluronic acid versus 4.5% for control; hyaluronic acid x cytochalasin B interaction; P=0.09). In conclusion, cytochalasin B treatment before freezing improved cryosurvival of bovine embryos produced in vitro. Such a treatment could be incorporated into methods for cryopreservation of bovine embryos provided post-transfer survival is adequate. In contrast, culture with hyaluronic acid was of minimal benefit—the increased cryosurvival in the absence of cytochalasin B was not sufficient to allow an adequate number of embryos to survive.     

The red blood cell glucose transporter presents multiple, nucleotide-sensitive sugar exit sites.

At any instant, the human erythrocyte sugar transporter presents at least one sugar export site but multiple sugar import sites. The present study asks whether the transporter also presents more than one sugar exit site. We approached this question by analysis of binding of [3H]cytochalasin B (an export conformer ligand) to the human erythrocyte sugar transporter and by analysis of cytochalasin B modulation of human red blood cell sugar uptake. Phloretin-inhibitable cytochalasin B binding to human red blood cells, to human red blood cell integral membrane proteins, and to purified human red blood cell glucose transport protein (GluT1) displays positive cooperativity at very low cytochalasin B levels. Cooperativity between sites and K(d(app)) for cytochalasin B binding are reduced in the presence of intracellular ATP. Red cell sugar uptake at subsaturating sugar levels is inhibited by high concentrations of cytochalasin B but is stimulated by lower (<20 nM) concentrations. Increasing concentrations of the e1 ligand forskolin also first stimulate then inhibit sugar uptake. Cytochalasin D (a cytochalasin B analogue that does not interact with GluT1) is without effect on sugar transport over the same concentration range. Cytochalasin B and ATP binding are synergistic. ATP (but not AMP) enhances [3H]cytochalasin B photoincorporation into GluT1 while cytochalasin B (but not cytochalasin D) enhances [gamma-32P]azidoATP photoincorporation into GluT1. We propose that the red blood cell glucose transporter is a cooperative tetramer of GluT1 proteins in which each protein presents a translocation pathway that alternates between uptake (e2) and export (e1) states but where, at any instant, two subunits must present uptake (e2) and two subunits must present exit (e1) states.     

Effect of cytochalasin B on formation and properties of muscle F-actin.

Cytochalasin B stimulated polymerization and decreased the concentration of G-actin remaining in equilibrium with F-actin filaments. Polymerization in the presence of cytochalasin B gave rise to a smaller increase of viscosity but to the same increase in light scattering, compared to polymerization in the absence of cytochalasin B. Cytochalasin B reduced the viscosity of F-actin and caused the appearance of ATP hydrolysis by F-actin. The cytochalasin B-induced ATPase activity was inhibited by concentrations of KCl higher than 50 mM. The cytochalasin B-induced ATPase activity was enhanced by ethyleneglycol bis(alpha-aminoethyl ether)-N,N'-tetraacetic acid and reduced by MgCl2 at concentrations higher than 0.75 mM. The findings suggest that the stability of actin filaments is reduced by cytochalasin B.     

A variant form of beta-actin in a mutant of KB cells resistant to cytochalasin B

Lines of KB cells resistant to cytochalasin B have been isolated and characterized. The mutant Cyt 1 exhibits increased cross-resistance to cytochalasin E. Cyt 1 cells bind less cytochalasin B than parental KB cells. Two-dimensional gel analysis shows that Cyt 1 cells carry an alteration in beta-actin. This is further confirmed by one-dimensional peptide analysis. The altered beta-actin (beta'-actin) is synthesized when poly(A)+ RNA from Cyt 1 cells is translated in a reticulocyte-cell-free translation system. These results suggest that the primary site of action of cytochalasin B on cellular motility processes is beta-actin.     

Cell cycle and cell protein content of Chinese hamster cells grown in culture with daily renewal of medium

The effects of cytochalasin B on cytokinesis, karyokinesis and DNA synthesis of various cells transformed by DNA viruses (SV40, polyoma and adeno 12) and of non-transformed cells were studied. Cytokinesis of all cell lines tested was completely inhibited by cytochalasin B at the concentration 0.5–2.0 μg/ml. After treatment by cytochalasin B, non-transformed cells became bi- or trinucleated without the division of cytoplasm. Three of the virally transformed cells also became bi- or trinucleated with a small number of multinucleate cells. On the other hand, in two SV40-transformed mouse cells, the number of nuclei per cell increased significantly and cells with 5–10 nuclei were frequently observed. Upon removal of cytochalasin B, cytoplasmic division recovered rapidly and consequently mono- or binucleate cells were formed. In all transformed cells, DNA synthesis was not inhibited by cytochalasin B, while DNA synthesis was inhibited in non-transformed cells.     

Cytochalasin B does not serve as a marker of glucose transport in rabbit erythrocytes

Glucose inhibitable cytochalasin B binding to erythrocyte membranes has been used as a marker of the glucose transporter. Glucose transport and cytochalasin B binding in rabbit erythrocytes differ from those activities found in human erythrocytes. We evaluated the uptake of 3-0-methylglucose and found similar Km (4.81 +/- 1.20 mM (SEM) and 6.59 +/- 0.72 mM) though significantly different Vmax (5.2 +/- 0.7 nM . min-1/10(9) cells and 234 +/- 13 nM X min -1/10(9) cells, p less than 0.001) for rabbit and human erythrocytes, respectively. Equilibrium binding of cytochalasin B to human erythrocyte membranes demonstrates a high affinity cytochalasin B binding site (Kd 38.6 +/- 22.7 nM) which is displaced by glucose. No comparable glucose inhibitable cytochalasin B site exists for rabbit erythrocyte membranes. Photoaffinity labeling of cytochalasin B confirms the presence of a glucose inhibitable cytochalasin B binding site in human, but not rabbit erythrocyte membranes. Cytochalasin B binding is a useful method in the identification of the glucose transporter in human cells, but the technique may be less useful in other species.     

Cytochalasin B enhances T cell mitogenesis by promoting expression of an interleukin 2 receptor

Low concentrations of cytochalasin B enhanced the T cell mitogenesis induced by concanavalin A (Con A) and interleukin 2 (IL-2). Mitogenesis was augmented by cytochalasin B given in the Con A-dependent early phase, or through T cell mitogenesis. Cytochalasin B did not enhance T cell mitogenesis when given only in the IL-2-dependent late phase. Use of the monoclonal antibody that directs the IL-2 receptor showed that cytochalasin B increased the expression of the IL-2 receptor induced by Con A. We concluded that cytochalasin b acts on an early phase of T cell mitogenesis and augments the expression of IL-2 receptor which enables certain nonresponsive T cells to respond to IL-2.     

Cytochalasin B-sensitive, sodium ion-dependent glucose transport in intestinal microvillous membrane

It was found that sodium ion-dependent glucose uptake by microvillous membrane (MVM) vesicles was partially inhibited by cytochalasin B with a half-maximum inhibition at ca. 10 microM. The MVM was photolabeled with [3]cytochalasin B. The Kd value and the maximum number of binding sites for cytochalasin B were ca. 8 microM and 70 pmol/mg protein, respectively. SDS-PAGE of the photolabeled MVM revealed 2 binding components. One was 86 K in Mr and the other 42 K. The binding of cytochalasin B to the 86 K component was affected neither by cytochalasin E nor by the presence of 0.5 M NaCl, but was depressed in the presence of 2-deoxy-D-glucose or phlorizin, which had no effect on the labeling of the 42 K component. These and other data suggested that the 86 K component might be responsible for a cytochalasin B-sensitive glucose transport in intestinal epithelial MVM.     

The effects of cytochalasin B on the testosterone synthesis by interstitial cells of rat testis

The present study examined the effects of cytochalasin B on various steps in the luteinizing hormone (LH)-stimulated increase in testosterone synthesis by collagenase-dispersed interstitial cells of adult rat testis. Cytochalasin B at a concentration range of 0.1–50 μM inhibited the LH-stimulated increase in testosterone synthesis in a dose-dependent manner. Both intracellular and medium (released) testosterone levels were reduced, thus indicating that the decrease was not due to the accumulation of testosterone inside the cell as a result of cytochalasin B treatment. Cytochalasin B also inhibited the 8-bromocyclic AMP and pregnenolone-stimulated testosterone synthesis in a similar dose-dependent manner. Cytochalasin B at the two higher doses (10 and 50 μM) also inhibited the LH-stimulated generation of cyclic AMP by interstitial cells. However, this drug had no effect on basal testosterone synthesis except at the highest concentration added.Previous studies on adrenocorticotropic hormone (ACTH)- and LH-stimulated increase in glucocorticoid and testosterone synthesis in adrenal and Leydig cells, respectively, demonstrated that cytochalasin B or anti-actin inhibited the transport of cholesterol into mitochondria. The present studies suggest that cytochalasin B inhibits at least two additional steps in the LH-stimulated increase in testosterone synthesis: (1) the generation of cyclic AMP at the level of the plasma membrane, and (2) the conversion of pregnenolone to the testosterone at the level of the smooth endoplasmic reticulum. It remains to be established whether these are direct effects of cytochalasin B, or whether they are mediated by disruption of microfilaments by cytochalasin B.