Microtubule-depolymerizing agents inhibit asialo-orosomucoid delivery to lysosomes but not its endocytosis or degradation in isolated rat hepatocytes

Microtubule-depolymerizing drugs, such as colchicine, vinblastine sulfate, colcemide and podophyllotoxin, cause an apparent inhibition of the ability of rat hepatocytes to degrade asialo-orosomucoid. However, the binding of asialo-orosomucoid to the cell surface at 0 degrees C, the endocytosis of pre-bound glycoprotein at 37 degrees C, and the dissociation of internal receptor-glycoprotein complexes are unaffected by these microtubule drugs. Receptor recycling is slowed but still occurs, although degradation is blocked. The rate of degradation is decreased by low concentrations of drugs. (For example, 0.25 microM vinblastine sulfate, colchicine and colcemide inhibited 93%, 79% and 26%, respectively.) Neither beta- nor gamma-lumicolchicine affected any of the processes examined. The degree of inhibition with colchicine could be enhanced by a brief treatment of the cells at low temperature to depolymerize microtubules. However, if cells were allowed to endocytose asialo-orosomucoid at 37 degrees C prior to addition of the microtubule drug, then the inhibition of protein degradation was greatly reduced. The decrease in the inhibition of degradation was proportional to the amount of time that cells were exposed to asialoglycoprotein before addition of the drug. The results indicate that the segregation of protein from receptor after they dissociate and/or the subsequent translocation of internalized asialoglycoprotein from the cell perimeter to the lysosomal region requires intact microtubules.     

Effect of colchicine on rat mast cells

In the mast cell, a well-developed array of microtubules is centered around the centrioles. Complete loss of microtubules is observed when mast cells are treated with 10(-5) M colchicine for 4 h at 37 degrees C. The loss of ultrastructurally evident microtubules is associated with a marked change in the shape of mast cells from spheroids to highly irregular, frequently elongated forms with eccentric nuclei. In colchicine-treated cells the association of nucleus, Golgi apparatus, and centrioles is also lost. Mast cells exposed to 10(-5) M colchicine for 4 h at 37 degrees C retain 80% of their capacity to release histamine when stimulated by polymyxin B. Exocytosis is evident in stimulated cells pretreated with colchicine and lacking identifiable microtubules. When the conditions of exposure of mast cells to colchicine are varied with respect to the concentration of colchicine, the length of exposure, and the temperature of exposure, dissociation between deformation of cell shape and inhibition of histamine secretion is observed. These observations indicate that microtubules are not essential for mast cell histamine release and bring into question the assumption that the inhibitory effect of colchicine on mast cell secretion depends on interference with microtubule integrity.     

Evidence that microtubule depolymerization early in the cell cycle is sufficient to initiate DNA synthesis

Microtubule disrupting drugs initiated DNA synthesis in serum-free cultures of nonproliferating fibroblast-like cells. The addition of colchicine to chick, mouse and human fibroblasts in serum-free medium stimulated thymidine incorporation at least twofold, with a half-maximal concentration of 1 X 10(-7) M. This stimulation represented up to 75% of the maximal stimulation by thrombin and was paralleled by an increase in the percentage of labeled nuclei. Other microtubule disrupting drugs showed similar stimulation, whereas lumicolchicine had no effect. Indirect immunofluorescent staining of tubulin showed a correlation between microtubule depolymerization and initiation of DNA synthesis by these drugs. A 2 hr treatment with 10(-6) M colchicine caused complete disruption of the microtubular network and stimulated thymidine incorporation (measured 28 hr later) to an even greater extent than continuous colchicine exposure. A similar 2 hr exposure to 10(-6) M colcemid also stimulated thymidine incorporation and led to a 50% increase in cell number. Taxol, a drug which stabilizes cytoplasmic microtubules, blocks initiation of DNA synthesis by colchicine, indicating that microtubule depolymerization is necessary for this initiation. To determine if microtubule depolymerization is involved in stimulation of DNA synthesis by other growth factors, highly purified human thrombin was added to cells with or without colchicine. In no case did colchicine plus thrombin increase DNA synthesis above that of the maximal stimulation by thrombin alone. Furthermore, pretreatment of cultures with taxol (5 micrograms/ml) inhibited approximately 30% of the stimulation of thymidine incorporation by thrombin. Together, these studies demonstrate that microtubule depolymerization is sufficient to initiate both DNA synthesis and events leading to cell division and suggest that microtubule depolymerization may be a required step in initiation of cell proliferation by growth factors such as highly purified human thrombin.     

Stimulation of Tubulin-Dependent ATPase Activity in Microtubule Proteins from Porcine Brain by Vinblastin

Vinblastine, a plant alkaloid which inhibits tubulin polymerization, stimulated an ATPase activity in microtubules. When microtubule proteins were separated into microtubule-associated proteins (MAPs) and tubulin by phosphocellulose column chromatography, vinblastine did not stimulate an ATPase activity recovered in the MAPs fraction unless tubulin was present. Therefore, vinblastine is considered to act through its binding to the tubulin molecule on MAPs ATPase. Divalent cations that activate tubulin-dependent MAPs ATPase activity were also required for the stimulation by vinblastine. In the presence of Ca2+ and vinblastine the ATPase activity was most active and the extent of stimulation reached about 200% of the original level in the absence of vinblastine. Half-maximal stimulation was attained when the molar ratio of vinblastine to tubulin was 0.5. The concentration of tubulin for half-maximal stimulation was increased in the presence of vinblastine, while divalent cation requirements were decreased. Several factors such as KCl (100 mM), alkaline pH (pH 7.5), and low temperature (10 degrees C) were not responsible for the disappearance of the stimulation. Vincristine stimulated tubulin-dependent MAPs ATPases activity as vinblastine did, whereas the activity was scarcely affected by colchicine, podophyllotoxin, strychnine, and chlorpromazine. Actin had no effect on MAPs ATPase activity in the absence and presence of vinblastine when it was used in place of tubulin.     

Effect of colchicine on PGE1 stimulation of cAMP1 formation in human lymphoblastoid and normal lymphocytes.

PGE1 increased cAMP level in human lymphoblastoid cells (RPMI 1788) after 5-60 min of incubation at 37 degrees C. A gradual decrease of cAMP concentration was found at the later time intervals. Colchicine significantly potentiated the stimulatory effect of PGE1, although it did not have any effect on cAMP level in control lymphoblastoid cells. The maximal effect of colchicine on PGE1 stimulation of cAMP formation was at the 0.1-1.0 microM level. Human lymphocytes also responded with increased cAMP formation to colchicine addition. In contrast, no stimulatory effect of colchicine was found in human granulocytes.     

4',6-Diamidino-2-phenylindole, a fluorescent probe for tubulin and microtubules

A new fluorophor for tubulin which has permitted the monitoring of microtubule assembly in vitro is reported. DAPI (4',6-diamidino-2-phenylindole), a fluorophor already known as a DNA intercalator, was shown to bind specifically to a unique tubulin site as a dimer (KD(app) = 43 +/- 5 microM at 37 degrees C) or to tubulin associated in microtubules (KD(app) = 6 +/- 2 microM at 37 degrees C) with the same maximum enhancement in fluorescence. When tubulin polymerization was induced with GTP, the change in DAPI affinity for tubulin resulted in an enhancement of DAPI binding and, consequently, of fluorescence intensity. DAPI, whose binding site is different from that of colchicine, vinblastine, or taxol, did not interfere greatly with microtubule polymerization. It induced a slight diminution of the critical concentration for tubulin assembly due to a decrease in the depolymerizing rate constant. Moreover, DAPI did not interfere with GTP hydrolysis correlated with tubulin polymerization, but it decreased the GTPase activity at the steady state of tubulin assembly. Even at substoichiometric levels DAPI can be used to follow the kinetics of microtubule assembly.     

Induction of random microtubule polymerization in cold and drug-treated PtK1 cells following hyperosmotic shock treatment

The effects of hypertonic sucrose on spindle and interphase microtubule (MT) arrays of PtK1 cells were investigated by incubating cells in complete culture medium at 4 degrees or 37 degrees C, with or without hypertonic sucrose, nocodazole or vinblastine (VLB). Results from anti-tubulin immunofluorescence showed that sucrose-induced alterations of spindle morphology seen at 37 degrees C did not occur at cold temperatures, but cold-induced MT loss was diminished. Application of warm hypertonic sucrose following depolymerization of MTs by nocodazole or cold resulted in the formation of a "feltwork" of randomly oriented, short MTs throughout the cytoplasm. These results, and those obtained substituting VLB for nocodazole, suggest that the effects of sucrose depend on the cytoplasmic concentration of soluble tubulin and support the hypothesis that osmotic factors are involved in effects of hypertonic sucrose on MT organization.     

Microtubule Depolymerization Inhibits Ethanol-Induced Enhancement of GABAA Responses in Stably Transfected Cells

Abstract: We studied whether microtubule organization is important for actions of ethanol on GABA_A ergic responses by testing the effects of microtubule depolymerization on ethanol enhancement of GABA action in mouse L(tk⁻) cells stably transfected with GABA_A receptor α₁β₁γ_2L subunits. The microtubule-disrupting agents colchicine, taxol, and vinblastine completely blocked ethanol-induced enhancement of muscimol-stimulated chloride uptake. β-Lumicolchicine, a colchicine analogue that does not disrupt microtubules, had no effect on ethanol action. Colchicine did not alter the potentiating actions of flunitrazepam or pentobarbital on muscimol-stimulated chloride uptake. Thus, colchicine specifically inhibited the potentiating action of ethanol. From these findings, we conclude that intact microtubules are required for ethanol-induced enhancement of GABA_A responses and suggest that a mechanism involving microtubules produces posttranslational modifications that are necessary for ethanol sensitivity in this cell system.     

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

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.     

Effects of colchicine, vinblastine and nocodazole on polarity, motility, chemotaxis and cAMP levels of human polymorphonuclear leukocytes

We present evidence for intrinsic polymorphonuclear leukocyte (PMN) polarity manifested in presence of microtubule-disrupting drugs. Polarization in response to colchicine correlated with the known dose-dependent effects of this drug on microtubule disassembly. The response to 10(-5) M colchicine, 10(-5) M vinblastine and 10(-6) M nocodazole was associated with stimulated motility and random locomotion. Responses elicited by microtubule-disrupting drugs differed from f-Met-Leu-Phe (fMLP)-induced polarization by functional and morphological criteria. Polarization, motility and orthokinesis responses were much weaker. Furthermore, ruffling was almost absent in PMNs polarized in response to colchicine, vinblastine or nocodazole. The response was inhibited by cytochalasin B, indicating that it is microfilament-dependent. We suggest that microtubule-disrupting drugs induce motility via structural changes in the cytoskeleton which act as signals for the motor apparatus. The intrinsic polarity manifested in the presence of microtubule-disrupting drugs could be reversed by an extracellular chemotactic gradient. Stimulated locomotion and motility in response to microtubule-disrupting drugs was only observed with initially spherical PMNs but not with initially motile cells. The findings provide an explanation for the numerous conflicting statements on the chemokinetic activities of these drugs. The role of cAMP in stimulated polarization and motility has been studied. Colchicine, vinblastine and nocodazole elicited a transient elevation of cAMP levels within 1 min of stimulation. cAMP elevation and stimulated motility were not quantitatively correlated.     

Microtubule-depolymerizing agents inhibit asialo-orosomucoid delivery to lysosomes but not its endocytosis or degradation in isolated rat hepatocytes

Microtubule-depolymerizing drugs, such as colchicine, vinblastine sulfate, colcemide and podophyllotoxin, cause an apparent inhibition of the ability of rat hepatocytes to degrade asialo-orosomucoid. However, the binding of asialo-orosomucoid to the cell surface at 0°C, the endocytosis of pre-bound glycoprotein at 37°C, and the dissociation of internal receptor-glycoprotein complexes are unaffected by these microtubule drugs. Receptor recycling is slowed but still occurs, although degradation is blocked. The rate of degradation is decreased by low concentrations of drugs. (For example, 0.25 μM vinblastine sulfate, colchicine and colcemide inhibited 93%, 79% and 26%, respectively.) Neither β- nor γ-lumicolchicine affected any of the processes examined. The degree of inhibition with colchicine could be enhanced by a brief treatment of the cells at low temperature to depolymerize microtubules. However, if cells were allowed to endocytose asialo-orosomucoid at 37°C prior to addition of the microtubule drug, then the inhibition of protein degradation was greatly reduced. The decrease in the inhibition of degradation was proportional to the amount of time that cells were exposed to asialoglycoprotein before addition of the drug. The results indicate that the segregation of protein from receptor after they dissociate and/or the subsequent translocation of internalized asialoglycoprotein from the cell perimeter to the lysosomal region requires intact microtubules.     

Interplay of cyclic AMP and microtubules in modulating the initiation of DNA synthesis in 3T3 cells

The results presented here show that disruption of the microtubule network acts synergistically with cAMP-elevating agents to stimulate the entry into DNA synthesis of 3T3 cells. Antimicrotubule agents and increased cAMP levels require an additional growth-promoting factor for inducing initiation of DNA synthesis; such requirement can be furnished by insulin, vasopressin, epidermal growth factor, platelet-derived growth factor, or fibroblast-derived growth factor. The involvement of the microtubules is indicated by the fact that enhancement of the DNA synthetic response was demonstrated with the chemically diverse agents colchicine, nocodazole, vinblastine, or demecolcine, all of which elicited the response in a dose-dependent manner. We verified that colchicine and nocodazole, at the doses used in this study, induced microtubule disassembly in the absence as well as in the presence of cAMP-elevating agents as judged by measurement of [3H]colchicine binding of total and pelletable tubulin. The involvement of cAMP was revealed by increasing its endogenous production by cholera toxin or by treatment with 8BrcAMP. The enhancing effects of antimicrotubule drugs and cAMP-elevating agents could be demonstrated by incorporation of [3H]thymidine into acid-insoluble material, autoradiography of labeled nuclei, or flow cytofluorometric analysis. The addition of antimicrotubule drugs does not increase the intracellular level of cAMP nor does addition of cAMP-elevating agents promote disassembly of microtubules (as judged by measuring [3H]colchicine binding of total and pelletable tubulin) in 3T3 cells. In view of these findings and the striking synergistic effects between these agents in stimulating DNA synthesis in the presence of a peptide growth factor, we conclude that increased cAMP levels and a disrupted microtubule network regulate independent pathways involved in proliferative response.     

Effects of antimicrotubular agents in cAMP production and in steroidogenic response of isolated rat Leydig cells

In dispersed rat Leydig cells, colchicine was found to stimulate basal cAMP production and testosterone secretion in a dose and time-dependent manner, but to a lesser extent than LH. However, these drugs are unable to stimulate adenylate cyclase activity in plasma membranes isolated from these cells. The amount of testosterone secreted at 150 min under the influence of colchicine and LH added simultaneously was not different from the amount produced during stimulation by LH alone. It is only after exposure of the cells for 1 hr to colchicine that the accumulation of cAMP in response to LH was inhibited; furthermore, both intracellular and medium testosterone accumulation in response to the hormone were reduced. Similar effects were observed with two other alkaloids, vinblastine and podophyllotoxin. The three drugs also inhibited the stimulation of testosterone secretion by 8-Br-cAMP or choleratoxin. These studies suggest that the state of microtubule polymerization and/or tubulin can influence the process of steroidogenesis in rat Leydig cells.     

Epinephrine stimulates prostaglandin synthesis by bullfrog lung from warm-acclimated, but not cold-acclimated, animals

Exogenous prostaglandins (PGs) have been shown to have differing effects on frog lung contractility. In this study, prostaglandin synthesis was measured in lung tissues from warm-acclimated (WA, 22 degrees C) and cold-acclimated (CA, 5 degrees C) American bullfrogs, Rana catesbeiana, incubated for 30 min at 5 degrees or 22 degrees C. Media were assayed by radioimmunoassay for PGE2, PGF2 alpha, 6-keto PGF 1 alpha (the metabolite of PGI2), and thromboxane (TX)B2 (the metabolite of TXA2). PGE2 was produced in greatest quantity by tissues from WA and CA animals, at both incubation temperatures. Epinephrine stimulated PGE2, PGF2 alpha, and TXB2 synthesis at 22 degrees C but only stimulated PGE2 production at 5 degrees C. In tissues from CA frogs, epinephrine did not stimulate prostaglandin synthesis at either incubation temperature. Ibuprofen (10(-5) M) inhibited basal and epinephrine-stimulated prostaglandin synthesis in tissues from WA frogs incubated at 22 degrees C. The beta receptor antagonist propranolol (10(-6) M) blocked the epinephrine-stimulated synthesis of PGE2, PGF2 alpha and TXB2, suggesting epinephrine stimulates prostaglandin synthesis through beta receptor activation. The absence of stimulation by epinephrine in lung from CA animals, but not in 5 degrees C incubations of tissues from WA animals, suggests that a modification of beta receptors occurs during prolonged cold exposure.     

Anti-microtubular agents as inhibitors of desensitization to catecholamine stimulation of adenylate cyclase in Ehrlich ascites tumor cells.

Adenylate cyclase activity of the homogenate of Ehrlich ascites tumor cells pretreated with catecholamine at 37 degrees C was not stimulated by the addition of the same catecholamine, whereas that of the cells without the pretreatment was stimulated. Such a desensitization was induced hardly at all when the pretreatment was performed at low temperature. The desensitization of adenylate cyclase activity to catecholamine stimulation was prevented by pre-pretreatment of the cells with colchicine prior to the catecholamine pretreatment. The effect of colchicine was dependent on the period of the treatment and concentration of colchicine. Vinblastine had a similar effect, whereas cytochalasin B was without effect. Thus, involvement of microtubules was suggested in the desensitization of the membrane-associated enzyme to external stimulation.     

The interaction of cystamine with bovine brain tubulin

Microtubule assembly in vitro is sensitive to a variety of non-physiological sulfhydryl-oxidizing agents, but the physiological significance of this phenomenon is unknown, since no physiological sulfhydryl-oxidizing agent has been shown to affect microtubule assembly in vitro. We have accordingly investigated the interaction of tubulin with cystamine. We have found that millimolar concentrations of cystamine inhibit microtubule assembly and induce an abnormal form of tubulin polymerization. Cystamine-induced polymerization does not occur at cold temperature. Formation of the polymer requires reaction of cystamine with two sulfhydryls which become available at 37 degrees C. In addition, cystamine reacts with about three sulfhydryls at 0 degrees C without inducing polymerization. This latter set of sulfhydryls appear to include one or both of the previously defined beta s sulfhydryls whose reaction with N, N'-ethylene-bis(iodoacetamide) is markedly inhibited by GTP, maytansine and vinblastine [Roach, M. C. & Luduena, R. F. (1984) J. Biol. Chem. 259, 12063-12071]. Cystamine's specific manner of interacting with tubulin suggests that it may mimic an endogenous sulfhydryl-directed regulator of microtubule assembly.     

Colchicine binding in the free-living nematode Caenorhabditis elegans

The [3H]colchicine-binding activity of a crude supernatant of the free-living nematode Caenorhabditis elegans was resolved into a non-saturable component and a tubulin-specific component after partial purification of tubulin by polylysine affinity chromatography. The two fractions displayed opposing thermal dependencies of [3H]colchicine binding, with non-saturable binding increasing, and tubulin binding decreasing, at 4 degrees C. Binding of [3H]colchicine to C.elegans tubulin at 37 degrees C is a pseudo-first-order rate process with a long equilibration time. The affinity of C. elegans tubulin for [3H]colchicine is relatively low (Ka = 1.7 x 10(5) M(-1)) and is characteristic of the colchicine binding affinities observed for tubulins derived from parasitic nematodes. [3H]Colchicine binding to C. elegans tubulin was inhibited by unlabelled colchicine, podophyllotoxin and mebendazole, and was enhanced by vinblastine. The inhibition of [3H]colchicine binding by mebendazole was 10-fold greater for C. elegans tubulin than for ovine brain tubulin. The inhibition of [3H]colchicine binding to C. elegans tubulin by mebendazole is consistent with the recognised anthelmintic action of the benzimidazole carbamates. These data indicate that C. elegans is a useful model for examining the interactions between microtubule inhibitors and the colchicine binding site of nematode tubulin.     

Cyclic AMP-independent stimulation of steroidogenesis in Y-1 adrenal tumor cells by antimitotic agents

The stimulation of steroidogenesis by antimitotic drugs has been studied in wild-type (Y-1) and cAMP-dependent protein kinase-deficient (kin-8) mouse adrenal tumor cell lines. Unlike some other cells, Y-1 cells do not increase their cAMP output upon exposure to antimitotic drugs such as colchicine, vinblastine or podophyllotoxin, which readily increase steroidogenesis. Moreover, no increase in cAMP can be detected over an extended time span. Stabilization of tubulin polymers by taxol or high concentrations of vinblastine blocks ACTH-, cholera toxin- or colchicine-stimulated steroidogenesis without major effects on cAMP levels. Colchicine and podophyllotoxin stimulate steroidogenesis in the cAMP-dependent protein kinase-deficient mutant to the same degree as in the wild-type Y-1 cells, although absolute steroid yields are lower in the mutant cells. We suggest that the antimitotic agents stimulate adrenal steroidogenesis by a cAMP-independent pathway that may involve facilitation of cholesterol access to the mitochondrion.     

Modulation of the distribution of plasma membrane intramembranous particles in contact-inhibited and transformed cells.

The intrinsic organization of the plasma membrane differs in normal and transformed cells. With the technique of freeze fracture and electron microscopy contact inhibited 3T3 cells have been shown to contain aggregated plasma membrane intramembranous particles, while transformed cells demonstrate a uniform particle distribution. The distribution of intramembrous particles in transformed cells can be affected by colchicine or vinblastine which induces a dose- and time-dependent particle aggregation. These observations suggest that microtubules and other membrane-associated colchicine-sensitive proteins probably influence the distribution of intrinsic membrane proteins and intramembranous particles in nucleated mammalian cells. An aggregated particle distribution has been observed in 3T3 cells or colchicine-treated transformed cells frozen in media, phosphate-buffered saline or following brief exposure to glycerol, sucrose or dimethyl sulfoxide containing solutions, independent of whether specimens were rapidly frozen from 37 degrees C, room temperature or 4 degrees C incubations. Cells briefly stabilized in 1% formaldehyde yields similar patterns of particle distribution as cells rapidly frozen in media or cryoprotectants. Glutaraldehyde fixation of cells, however, appears to alter the fracturing process in these cells, as visualized by an altered fracture face appearance, decreased numbers of particles, and no particle aggregates. Differences in membrane organization between normal and transformed cells have therefore been demonstrated using a series of preparative methods and colchicine and vinblastine have been shown to modulate intramembranous particle distribution in transformed 3T3 cells.