Evidence for involvement of microtubules in the action of vasopressin in toad urinary bladder

Summary The antimitotic agents colchicine, podophyllotoxin, and vinblastine inhibit the action of vasopressin and cyclic AMP on osmotic water movement in the toad urinary bladder. The alkaloids have no effect on either basal or vasopressin-stimulated sodium transport or urea flux across the tissue. Inhibition of vasopressin-induced water movement is half-maximal at the following alkaloid concentrations: colchicine, 1.8×10^–6 m; podophyllotoxin, 5×10^–7 m; and vinblastine, 1×10^–7 m. The characteristics of the specificity, time-dependence and temperature-dependence of the inhibitory effect of colchicine are similar to the characteristics of the interaction of this drug with tubulinin vitro, and they differ from those of its effect on nucleoside transport. Inhibition of the vasopressin response by colchicine, podophyllotoxin, and vinblastine is not readily reversed. The findings support the view that the inhibition of vasopressin-induced water movement by the antimitotic agents is due to the interaction of these agents with tubulin and consequent interference with microtubule integrity and function. Taken together with the results of biochemical and morphological studies, the findings provide evidence that cytoplasmic microtubules play a critical role in the action of vasopressin on transcellular water movement in the toad bladder.     

Effect of Colchicine,Vinblastine, and Cytochalasin B on Cell Surface Anionic Sites of Tritrichomonas foetus1

ABSTRACT. Drugs that interact with microtubules (colchicine and vinblastine) and microfilaments (cytochalasin B) partially inhibited cell growth and motility of Tritrichomonas foetus. Parasites incubated with these substances became rounded and cell division was blocked. Neither colchicine nor vinblastine disrupted the microtubules that form the peltar-axostylar system. Any one of these drugs interfered with the net negative surface charge of T. foetus as evaluated by determination of the cellular electrophoretic mobility (EPM). The decrease in the EPM of cytochalasin B-treated cells was caused by dimethylsulfoxide, which was used as solvent. Untreated cells as well as cytochalasin B-treated cells showed a uniform distribution of anionic sites on the plasma membrane as seen with cationized ferritin particles. In cells treated with colchicine or vinblastine the anionic sites were distributed in patches. These results are discussed in terms of participation of labile cytoplasmic microtubules and microfilaments in the control of the distribution of anionic sitecontaining macromolecules located on the cell surface of T. foetus.     

Effects of inhibitors of tubulin polymerization on GTP hydrolysis

The effects of a number of antimitotic drugs on the GTPase activity of tubulin were examined. The previously reported stimulation with colchicine and inhibition with podophyllotoxin and vinblastine wee confirmed. Maytansine, which competes with vinblastine in binding to tubulin, was comparable to the latter in inhibiting GTP hydrolysis. Nocodazole, which competes with colchicine in binding to tubulin, was significantly superior to colchicine in enhancing GTP hydrolysis. This superiority arose from the more rapid bindng of nocodazole to tubulin, as the two drugs had comparable activity when drug and tubulin were preincubated prior to the addition of GTP. Both colchicine and podophyllotoxin contain a trimethoxybenzene ring, while the closest structural analogy of nocodazole to colchicine includes the trimethoxybenzene ring. To explore this apparent paradox, we examined a number of simpler colchicine analogs for their effects on tubulin-dependent GTP hydrolysis. While tropolone was without effect, 3,4,5-trimethoxybenzaldehyde and 2,3,4-trimethoxybenzaldehyde stimulated the reaction. We therefore conclude that the trimethoxybenzene ring of colchicine is primarily responsible for the drug's stimulation of the GTPase activity of tubulin and that the inhibitory effect of podophyllotoxin must derive from the latter's tetrahydronaphthol moiety.     

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.     

Binding of dolastatin 10 to tubulin at a distinct site for peptide antimitotic agents near the exchangeable nucleotide and vinca alkaloid sites

Dolastatin 10, a potent antimitotic peptide from a marine animal, strongly inhibits microtubule assembly, tubulin-dependent GTP hydrolysis, and the binding of vinca alkaloids to tubulin. In studies of the binding of [3H]vincristine to the protein, with vinblastine as a control for competitive inhibition (Ki, 6.6 microM), we found that the macrolide antimitotic agents maytansine and rhizoxin were also competitive inhibitors (Ki values, 3.1 and 12 microM). Dolastatin 10 and an unrelated peptide antimitotic, phomopsin A, were more potent but noncompetitive inhibitors (Ki values, 1.4 and 2.8 microM). Since maytansine and, to a much lesser extent, vinblastine interfere with nucleotide exchange on tubulin, all drugs were examined for effects on nucleotide interactions at the exchangeable GTP site. Rhizoxin had effects intermediate between those of vinblastine and maytansine. Both peptides inhibited binding of radiolabeled GTP to tubulin even more strongly than did maytansine, but no drug displaced nucleotide from tubulin. The drugs were evaluated for stabilizing effects on the colchicine binding activity of tubulin. The peptides prevented loss of this activity, and vinblastine provided partial protection, while rhizoxin and maytansine did not stabilize tubulin. A tripeptide segment of dolastatin 10 also effectively inhibits tubulin polymerization and GTP hydrolysis. The tripeptide did not significantly inhibit either vincristine binding or nucleotide exchange, nor did it stabilize colchicine binding. These findings are rationalized in terms of a model with two distinct drug binding sites in close physical proximity to each other and to the exchangeable GTP site on beta-tubulin.     

Role of microtubules in the intracellular transport of growth hormone

Summary Pulse-chase experiments utilising(³H)leucine have been used to study the effects of colchicine and vinblastine on intracellular transport and secretion of newly synthesised growth hormone from rat anterior pituitary fragments. Growth hormone was isolated from medium and fragments by polyacrylamide gel electrophoresis. When colchicine or vinblastine, which disrupt microtubules, were added immediately after pulse labelling, inhibition of the subsequent secretion of newly synthesised growth hormone was detected throughout the succeeding 5 h. Similar inhibition was seen if the drugs were added after a 1 h delay. However, if colchicine or vinblastine were added only after a 2 h chase incubation, then no significant effect on subsequent release of labelled growth hormone was seen. The results suggest that these agents may inhibit the transport of newly formed growth hormone storage granules from the Golgi complex to the cytoplasmic pool. Microtubules do not appear to be involved in the mechanism of the final secretion of newly synthesised hormone by exocytosis.These studies were supported by grants from the Medical Research Council and British Diabetic Association     

Effect of colchicine, vinblastine, and cytochalasin B on cell surface anionic sites of Tritrichomonas foetus

Drugs that interact with microtubules (colchicine and vinblastine) and microfilaments (cytochalasin B) partially inhibited cell growth and motility of Tritrichomonas foetus. Parasites incubated with these substances became rounded and cell division was blocked. Neither colchicine nor vinblastine disrupted the microtubules that form the peltar-axostylar system. Any one of these drugs interfered with the net negative surface charge of T. foetus as evaluated by determination of the cellular electrophoretic mobility (EPM). The decrease in the EPM of cytochalasin B-treated cells was caused by dimethylsulfoxide, which was used as solvent. Untreated cells as well as cytochalasin B-treated cells showed a uniform distribution of anionic sites on the plasma membrane as seen with cationized ferritin particles. In cells treated with colchicine or vinblastine the anionic sites were distributed in patches. These results are discussed in terms of participation of labile cytoplasmic microtubules and microfilaments in the control of the distribution of anionic site-containing macromolecules located on the cell surface of T. foetus.     

Gametogenesis in Plasmodium; the inhibitory effects of anticytoskeletal agents

Gametocytes of Plasmodium yoelii were incubated with colchicine, vinblastine sulphate and cytochalasin B and then induced to undergo gametogenesis. All the drugs inhibited gamete formation in a dose and time dependent manner. Electron microscopy revealed that colchicine and vinblastine sulphate inhibited the polymerisation of cytoplasmic- axonemal- and intranuclear mitotic- microtubules. Cytochalasin B did not inhibit microtubule assembly but blocked spindle pole separation and axoneme distribution into the microgamete. All drugs prevented escape of the gametocyte from the erythrocyte. The mechanism of mitotie spindle action is discussed.     

The colR4 and colR15 beta-tubulin mutations in Chlamydomonas reinhardtii confer altered sensitivities to microtubule inhibitors and herbicides by enhancing microtubule stability.

The colR4 and colR15 beta 2-tubulin missense mutations for lysine-350 in Chlamydomonas reinhardtii (Lee and Huang, 1990) were originally isolated by selection for resistance to the growth inhibitory effects of colchicine. The colR4 and colR15 mutants have been found to be cross resistant to vinblastine and several classes of antimitotic herbicides, including the dinitroanilines (oryzalin, trifluralin, profluralin, and ethafluralin); the phosphoric amide amiprophos methyl; and the dimethyl propynl benzamide pronamide. Like colchicine and vinblastine, the antimitotic effects of these plant-specific herbicides have been associated with the depolymerization of microtubules. In contrast to their resistance to microtubule-depolymerizing drugs, the mutants have an increased sensitivity to taxol, a drug which enhances the polymerization and stability of microtubules. This pattern of altered sensitivity to different microtubule inhibitors was found to cosegregate and corevert with the beta-tubulin mutations providing the first genetic evidence that the in vivo herbicidal effects of the dinitroanilines, amiprophos methyl, and pronamide are related to microtubule function. Although wild-type like in their growth characteristics, the colR4 and colR15 mutants were found to have an altered pattern of microtubules containing acetylated alpha-tubulin, a posttranslational modification that has been associated with stable subsets of microtubules found in a variety of cells. Microtubules in the interphase cytoplasm and those of the intranuclear spindle of mitotic cells, which in wild-type Chlamydomonas cells do not contain acetylated alpha-tubulin, were found to be acetylated in the mutants. These data taken together suggest that the colR4 and colR15 missense mutations increase the stability of the microtubules into which the mutant beta-tubulins are incorporated and that the altered drug sensitivities of the mutants are a consequence of this enhanced microtubule stability.     

Morphological anomalies induced by antimicrotubule agents inBryopsis plumosa

Summary Antimicrotubule agents, colchicine, vinblastine, and griseofulvin, induced conspicuous morphological anomalies inBryopsis plumosa. First, following cessation of protoplasmic streaming within 15 minutes, elongation stopped in a few hours. Second, innumerable protrusions or new growth points generated over the cell flank in a few days. Similar phenomena were observed in the cells which were subjected to high pressure or low temperature both of which are known to disrupt microtubule.These phenomena were investigated with light and electron microscopy. It is suggested that inhibition of microtubule dependent protoplasmic streaming which may function as an intracellular transport system causes such morphological anomalies.     

Ciliary Proteins and Cytodifferentiation in Stentor coeruleus*

To elucidate further the molecular events required for cytodifferentiation in Stentor coeruleus, the effects of several chemical metabolic inhibitors were tested on the outgrowth in situ of the membranellar cilia of the oral feeding organelle. The chemicals used included several inhibitors of cytoplasmic and mitochondrial protein synthesis (cycloheximide, emetine, and chloramphenicol), and an antimitotic agent (colchicine). Ciliary growth was affected only by colchicine, suggesting that a pool of “ciliary proteins” exists in interphase Stentor of sufficient size to permit complete reformation of the membranellar cilia. The implication of these observations to an understanding of the more complicated process of oral regeneration is discussed.     

Microtubule inhibitors alter the secretion of beta-glucuronidase by human blood platelets: involvement of microtubules in release reaction II

The effects of the antimicrotubular drugs colchicine and vinblastine on the blood platelet release reaction were studied by measuring release of ¹⁴C-5-hydroxytryptamine (¹⁴C-5-HT, release I) and β-glucuronidase (release II) from gel-filtered human platelets. β-glucuronidase release induced by thrombin was significantly inhibited by colchicine (0.01-1 mM) or vinblastine (0.05–0.1 mM). Release of ¹⁴C-5-HT, however, was unaffected at low concentrations of colchicine and only slightly inhibited at higher concentrations. Inhibition of β-glucuronidase release depended on colchicine or vinblastine concentrations and decreased with longer time intervals (1′, 5′, 20′) after thrombin stimulation. Levels of the cytoplasmic enzyme, lactic acid dehydrogenase, in supernatants of colchicine treated platelets were not significantly different from controls. Colchicine also inhibited β-glucuronidse release, but not ¹⁴C-5-HT release, induced by trypsin and sodium arachidonate. Binding of ¹⁴C-colchicine by platelets was measured and it was found that platelet aggregation and release of 5-HT induced by adenosine diphosphate, epinephrine and collagen proceeded without any alteration in colchicine binding. However, significant increases in the rate and degree of colchicine binding were observed when platelets were stimulated by thrombin, trypsin and arachidonic acid which induced aggregation, release of both 5-HT and β-glucuronidase. The results suggest that an alteration in platelet microtubules is correlated with the physiologic response resulting in release II and that the cellular mechanisms effecting release I and II by platelets differ qualitatively in that the microtubules may facilitate release II.     

Role of P-Glycoprotein in the Blood-Brain Transport of Colchicine and Vinblastine

Abstract: Classically, drug penetration through the blood-brain barrier depends on the lipid solubility of the substance, except for some highly lipophilic drugs, like colchicine and vinblastine, both substrates of P-glycoprotein, a drug efflux pump present at the luminal surface of the brain capillary endothelial cells. Colchicine and vinblastine uptake into the brain was studied in the rat using the in situ brain perfusion technique and two inhibitors of P-glycoprotein, verapamil and SDZ PSC-833. When rats were pretreated with PSC-833 (10 mg/kg, intravenous bolus), colchicine and vinblastine uptake was enhanced 8.42- and 9.08-fold, respectively, in all the gray areas of the rat brain studied. The mean colchicine distribution volume was increased from 0.67 ± 0.41 to 5.64 ± 0.70 µl/g and vinblastine distribution volume from 2.74 ± 1.15 to 24.88 ± 4.03 µl/g. When rats were pretreated with verapamil (1 mg/kg, intravenous bolus), colchicine distribution volume was increased 3.70-fold. The increase in colchicine and vinblastine did not differ between the eight brain gray areas. PSC-833 and verapamil pretreatment had no influence on the distribution volume of either drug in the choroid plexus. Nevertheless, distribution volumes remained small, considering the highly lipophilic nature of the substances. We suggest that P-glycoprotein is either only partially inhibited (difficulty of fully saturating P-glycoprotein, especially under in vivo conditions) or not the only barrier to these two drugs.     

Distinct and overlapping binding sites for IKP104 and vinblastine on tubulin

IKP104 is one of a group of tubulin-binding drugs whose interaction with tubulin suggests that it may bind to the protein at or close to the region where vinblastine binds. By itself IKP104 is a potent enhancer of tubulin decay as evidenced by the fact that it induces the exposure of the sulfhydryl groups and hydrophobic areas on tubulin. In this respect, IKP104 differs from vinblastine and other drugs such as phomopsin A, dolastatin 10, rhizoxin, and maytansine which are competitive or noncompetitive inhibitors of vinblastine binding. In contrast, however, in the presence of colchicine, IKP104 behaves differently and strongly stabilizes tubulin, to an extent much greater than does colchicine alone. IKP104 appears to have two classes of binding site on tubulin, differing in affinity; the acceleration of decay appears to be mediated by the low-affinity site (Chaudhuriet al., 1998,J. Protein Chem., in press). We investigated the relationship of the binding of IKP104 and vinblastine. We found that the high-affinity site or sites of IKP104 overlap with or interact with the vinblastine-binding sites, but that the low-affinity site is distinctly different.     

Distinct and overlapping binding sites for IKP104 and vinblastine on tubulin

IKP104 is one of a group of tubulin-binding drugs whose interaction with tubulin suggests that it may bind to the protein at or close to the region where vinblastine binds. By itself IKP104 is a potent enhancer of tubulin decay as evidenced by the fact that it induces the exposure of the sulfhydryl groups and hydrophobic areas on tubulin. In this respect, IKP104 differs from vinblastine and other drugs such as phomopsin A, dolastatin 10, rhizoxin, and maytansine which are competitive or noncompetitive inhibitors of vinblastine binding. In contrast, however, in the presence of colchicine, IKP104 behaves differently and strongly stabilizes tubulin, to an extent much greater than does colchicine alone. IKP104 appears to have two classes of binding site on tubulin, differing in affinity; the acceleration of decay appears to be mediated by the low-affinity site (Chaudhuriet al., 1998,J. Protein Chem., in press). We investigated the relationship of the binding of IKP104 and vinblastine. We found that the high-affinity site or sites of IKP104 overlap with or interact with the vinblastine-binding sites, but that the low-affinity site is distinctly different.     

Evidence for involvement of microtubules in the action of vasopressin in toad urinary bladder

Colchicine, podophyllotoxin and vinblastine have been found to inhibit the action of vasopressin on water movement in the toad urinary bladder. Tubulin is the major colchicine binding component of toad bladder epithelial cells, accounting for approximately 3.3% of the total cell protein. More than 99% of the tubulin is found in the soluble fraction after sonication, the remainder is in the particulate fraction. Similar to the characteristics of the binding of colchicine to tubulins from other sources, the binding of colchicine to toad bladder tubulin is temperature- and time-dependent, is inhibited competitively by podophyllotoxin (Ki= 5.5 x 10(-7)m), and has a binding constant of 1 X 10(6) liters/mole at 37 degrees. Binding activity decays according to first-order kinetics and is stabilized by vinblastine. The characteristics of the interactions of colchicine and podophyllotoxin with epithelial cell tubulin in vitro closely parallel the ability of these drugs to inhibit the response to vasopressin in vivo. These results, coupled with those of functional and morphological studies, support the view that the ability of these drugs to affect vasopressin-induced water movement across toad bladder epithelial cells is related to the depolymerization of cytoplasmic microtubules.     

The role of cytoplasmic streaming in symplastic transport

The distributing of materials throughout a symplastic domain must involve at least two classes of transport steps: plasmodesmatal and cytoplasmic. To underpin the latter, the most obvious candidate mechanisms are cytoplasmic streaming and diffusion. The thesis will be here advanced that, although both candidates clearly do transport cytoplasmic entities, the cytoplasmic streaming per se is not of primary importance in symplastic transport but that its underlying molecular motor activity (of which the streaming is a readily visible consequence) is. Following brief tutorials on low Reynolds number flow, diffusion, and targeted intracytoplasmic transport, the hypothesis is broached that macromolecular and vesicular transport along actin trackways is both the cause of visible streaming and the essential metabolically driven cytoplasmic step in symplastic transport. The concluding discussion highlights four underdeveloped aspects of the active cytoplasmic step: (i) visualization of the real‐time transport of messages and metabolites; (ii) enumeration of the entities trafficked; (iii) elucidation of the routing of the messages and metabolites within the cytoplasm; and (iv) transference of the trafficked entities from cytoplasm into plasmodesmata.     

Reduced permeability in CHO cells as a mechanism of resistance to colchicine

Colchicine resistant (CH^R) lines of stable phenotype have been isolated from cultured Chinese hamster (CHO) cells. Successive single-step selections for increasing resistance were performed by isolating resistant colonies at each step. Two complementary assays involving [³H] colchicine uptake by whole cells and binding of [³H] colchicine by cytoplasmic extracts were developed to test for altered permeability and altered intracellular target protein, respectively. All clones isolated appeared to have decreased permeability to the drug while their colchicine-binding ability was not reduced. The amount of reduction in colchicine uptake correlated strongly with cellular resistance. The CH^R lines were also cross resistant to other drugs such as actinomycin D, vinblastine and Colcemid; furthermore, the degree of cross resistance was positively correlated with the degree of colchicine resistance. The non-ionic detergent Tween 80 potentiated the cytotoxic action of colchicine on mutant cells as well as its rate of uptake into whole cells.     

Colchicine and oryzalin mediated chromosome doubling in different genotypes of Miscanthus sinensis

Different explant materials were treated with antimitotic agents to induce chromosome doubling in several Miscanthus sinensis clones. In vitro propagated plants established in soil, in vitro shoots, embryogenic callus, shoot apices and leaf explants were treated with different concentrations of colchicine or oryzalin. No tetraploids were obtained after antimitotic treatment of plants established in soil. The percentage of chromosome doubled plants after antimitotic treatment of single in vitro shoots was genotype dependent. Rooted in vitro plantlets were not a suitable target for antimitotic treatment, due to a high frequency of ploidy chimeras. Many tetraploid plants were regenerated after antimitotic treatment at the callus and explant level, but the efficiency was genotype dependent, primarily due to differences in the ability to form regenerable callus and to regenerate plants from embryogenic callus. Treatment of shoot apices with colchicine was the most efficient and reproducible system in the four genotypes tested. It was possible to repeatedly use the same colchicine-containing medium without any reduction in the induction of regenerable callus or in the percentage of tetraploids, thereby minimising the handling of this very toxic compound.     

THE MELANOCYTE MODEL : Colchicine-like Effects of Other Antimitotic Agents

The effect of various agents that cause metaphase arrest in dividing cells was studied on the rapid reversible darkening of frog skin under the influence of melanocyte-stimulating hormone (MSH). Darkening is due to dispersion of melanin granules in melanocytes and is thought to be accompanied by a gel-to-sol cytoplasmic transformation. After subsequent washing the skin lightens, with aggregation of melanin granules and cytoplasmic gelation. As previously shown with colchicine, preincubation of frog skin with vinblastine, vincristine, or colcemid produced an increase in darkening induced by MSH, as compared to control skins, and a dosage-dependent inhibition of subsequent lightening. Preincubation with each drug, without subsequent MSH, produced a gradual, irreversible, dosage-dependent darkening over several hours. On a molar basis, the relative strength of the various agents was vinblastine > vincristine > colcemid > colchicine; vinblastine was about 100 times stronger than colchicine. Preincubation of frog skin with griseofulvin, followed by washing, had no subsequent effects on darkening or lightening. However, effects similar to those of the Colchicum and Vinca alkaloids were seen if griseofulvin was kept in the ambient media. These effects were rapidly reversible on removal of the drug from the media. These findings support the melanocyte model originally proposed for the action of colchicine, and emphasize certain facts that models of melanin granule movement will have to accommodate.