Effect of anticalmodulin and antitubulin on LDL-receptor synthesis in aortic smooth muscle cells cultured in vitro

Effect of trifluoperazine and colchicine on LDL-receptor synthesis in smooth muscle cells exposed to hypercholesterolemic medium in vitro have been studied. While trifluoperazine at 25 microM concentration caused stimulation of LDL-receptor synthesis, colchicine acted as a dose-dependent inhibitor of LDL-receptor synthesis. Thus calmodulin down regulates LDL-receptor synthesis independent of microtubular involvement.     

Effect of trifluoperazine and colchicine on smooth muscle cellular proliferative and secretory activity induced by hypercholesterolemic medium in vitro

The present study, addressed to understand the mechanism behind the cholesterol-induced proliferative and collagen secretory activity of smooth muscle cells, revealed that cholesterol-induced smooth muscle cellular DNA synthesis and collagen secretion was mediated through its ability to amplify the intracellular cGMP signal because of the fact that Trifluoperazine (an anticalmodulin and blocker of phospholipase A2) and colchicine (an antitubulin and inhibitor of guanyl cyclase) inhibited DNA synthesis and collagen-secretory activity of smooth muscle cells by their ability to decrease the cGMP levels within smooth muscle cells. From these results we suggest that membrane cholesterol modulated phospholipase 'A2' activity may be the basic mechanism involved in cholesterol-induced proliferative and collagen-secretory activity of smooth muscle cells in vitro.     

Study of possible interactions of tubulin, microtubular network, and STOP protein with mitochondria in muscle cells

We studied possible connections of tubulin, microtubular system, and microtubular network stabilizing STOP protein with mitochondria in rat and mouse cardiac and skeletal muscles by confocal microscopy and oxygraphy. Intracellular localization and content of tubulin was found to be muscle type-specific, with high amounts in oxidative muscles, and much lower in glycolytic skeletal muscle. STOP protein localization and content in muscle cells was also muscle type-specific. In isolated heart mitochondria, addition of 1 μM tubulin heterodimer increased apparent K _m for ADP significantly. Dissociation of microtubular system into free tubulin by colchicine treatment only slightly decreased initially high apparent K _m for ADP in permeabilized cells, and diffusely distributed free tubulin stayed inside the cells, obviously connected to the intracellular structures. To identify the genes that are specific for oxidative muscle, we developed and applied a method of kindred DNA. The results of sequencing and bioinformatic analysis of isolated cDNA pool common for heart and m. soleus showed that in adult mice the β-tubulin gene is expressed predominantly in oxidative muscle cells. It is concluded that whereas dimeric tubulin may play a significant role in regulation of mitochondrial outer membrane permeability in the cells in vivo, its organization into microtubular network has a minor significance on that process.     

Role of microtubules in milk secretion — Action of colchicine on microtubules and exocytosis of secretory vesicles in rat mammary epithelial cells

Summary Effect of colchicine on microtubules was studied in mammary epithelial cells treated both in vivo and in vitro with the alkaloid. Three hours after the intramammary infusion of colchicine, secretory activity of mammary epithelia ceased, milk constituents accumulated and were randomly distributed within the cytoplasm, sometimes leaking into the perialveolar connective tissue, and autophagic vacuoles were prevalent. It appeared that an accelerated involutionary process was occurring. No microtubules were observed after this treatment. In vitro treated cells appeared to be less affected by the alkaloid. Although numerous casein-containing secretory vesicles accumulated in the cytoplasm, lipid droplet accumulation was less, and fewer autophagic vacuoles were observed, although lysosomes were commonly observed. Occasionally, obliquely sectioned microtubules were found in cells treated with low concentrations of colchicine but were absent at higher colchicine concentrations; however, paracrystalline inclusions (tubulin aggregates) were observed in some cells at all concentrations of the drug. These observations provide evidence that drugs which interfere with microtubule integrity reduce the secretory activity in mammary epithelia. This evidence is consistent with the concept of an association of the microtubular system and the secretory process.     

Effect of colchicine on histamine secretion and calcium uptake in mast cells

The function of contractile system of microtubules on the mechanism of mast cell exocytosis by using colchicine, a depolymerizing alkaloid of the microtubular system, has been studied. The response of histamine release and 45Ca-uptake in isolated rat mast cells treated with colchicine has been determined. The incubation of mast cells in the presence of 10(-8)-10(-3) M colchicine slightly inhibits histamine secretion induced by the stimulant concentration 50 micrograms/ml of compound 48/80 (35 +/- 5%). Similarly colchicine does not significantly affect histamine values spontaneously elicited in unstimulated mast cells; the percentages of secretion are never greater than 10%. However, high doses of this alkaloid are found to markedly inhibit entry of calcium ions into the cell. These results suggest that microtubules do not participate in the secretory process of mast cells, although they significantly decrease calcium uptake. The microtubules might be connected to the membrane, so that the depolymerization of this contractile system could damage the membrane structures through which Ca2+ is transported.     

Aldehyde-induced modifications of the microtubular system in 3T3 fibroblasts.

The molecular structure of aldehydes is closely related to their antimicrotubular effect. Morphological modifications of the microtubular system in living cells after incubation with certain aldehydes are consistent with biochemical alterations detected in previous research. The microtubular arrangement was visualized by an immunofluorescence technique with antitubulin antibodies, while the content of tubulin in the cells was evaluated by a colchicine binding assay. 2-Nonenal behaved similarly to 4-hydroxynonenal, a lipid peroxidation product, disorganizing microtubular network in 3T3 fibroblasts and decreasing the amounts of tubulin able to bind labelled colchicine. Nonanal did not significantly impair the tubulin characteristics in the cells, despite the fact that it has been shown to be active on the purified microtubular system; benzaldehyde was ineffective. This would appear to explain the mechanisms of interaction of aliphatic aldehydes which might be suitable for use as antimicrotubular drugs.     

Passive mechanical behavior of human neutrophils: effects of colchicine and paclitaxel.

The role of microtubules in determining the mechanical rigidity of neutrophils was assessed. Neutrophils were treated with colchicine to disrupt microtubules, or with paclitaxel to promote formation of microtubules. Paclitaxel caused an increase in the number of microtubules in the cells as assessed by immunofluorescence, but it had no effect on the presence or organization of actin filaments or on cellular mechanical properties. Colchicine at concentrations <1.0 microM caused disruption of microtubular structures, but had little effect on either F-actin or on cellular mechanical properties. Higher concentrations of colchicine disrupted microtubular structure, but also caused increased actin polymerization and increases in cell rigidity. Treatment with 10 microM colchicine increased F-actin content by 17%, the characteristic cellular viscosity by 30%, the dependence of viscosity on shear rate by 10%, and the cortical tension by 18%. At 100 microM colchicine the corresponding increases were F-actin, 25%; characteristic viscosity, 50%; dependence of viscosity on shear rate, 20%; and cortical tension, 21%. These results indicate that microtubules have little influence on the mechanical properties of neutrophils, and that increases in cellular rigidity caused by high concentrations of colchicine are due to a secondary effect that triggers actin polymerization. This study supports the conclusion that actin filaments are the primary structural determinants of neutrophil mechanical properties.     

The effects of colchicine and cytochalasin B on uptake and degradation of asialo-glycoproteins in isolated rat hepatocytes.

We have studied the effects of colchicine, an inhibitor of microtubular function, and cytochalasin B (CB), an inhibitor of microfilaments, on the uptake and degradation of asialo-glycoproteins in isolated rat hepatocytes. CB inhibited degradation only, while colchicine inhibited uptake as well as degradation. When the two were combined, no additive effect on degradation was found. The inhibition of uptake by colchicine could be accounted for by a reduction in the binding capacity of the plasma membrane for asialo-glycoprotein. The intracellular distribution of endocytosed asialo-glycoprotein was examined by isopycnic centrifugation in sucrose gradients. The results suggest that in cells treated with colchicine or CB, access of the endocytosed protein to the lysosomes is impeded.     

Desmosome assembly in MDCK epithelial cells does not require the presence of functional microtubules.

Desmosomes, complex multisubunit structures that assemble at sites of cell-cell contact, are important components of the epithelial junctional complex. Desmosome assembly requires the coordinated interaction at the plasma membrane of at least 8 cytoplasmic and integral membrane proteins organized into two structurally and functionally distinct domains, the cytoplasmic plaque and membrane core. Previous studies (Pasdar et al., J. Cell Biol., 113:645-655) provided evidence that cytokeratin filaments and microtubules may regulate transfer and assembly of cytoplasmic plaque and membrane core proteins, respectively. To determine directly the role of microtubules in these processes, Madin-Darby canine kidney (MDCK) cells were treated with nocodazole or colchicine to disrupt the microtubular network. Biochemical analysis of the different components of the cytoplasmic plaque and membrane core domains revealed little or no effect of nocodazole or colchicine on the kinetics of synthesis, post-translational modifications, transfer of proteins to the plasma membrane or their metabolic stability in the presence or absence of cell-cell contact. Likewise, immunofluorescence analysis of desmosome formation demonstrated an apparently normal desmosome assembly in the presence of nocodazole or colchicine upon induction of cell-cell contact. These results indicate that an intact microtubular network is not necessary for the processing or transport of the desmosomal membrane core glycoproteins to the plasma membrane in the absence or presence of cell-cell contact. Furthermore, the integration of the cytoplasmic plaque and membrane core domains induced by cell-cell contact at the plasma membranes of adjacent cells does not require the presence of functional microtubules.     

Characterization of acetylcholine-induced insulin secretion in the isolated perfused dog pancreas.

In the presence of a nonstimulatory concentration of glucose, a 60-min perfusion with 50 muM acetylcholine was shown to elicit a monophasic release of insulin in the isolated dog pancreas preparation. A decline in secretory response, which may be due to desensitization of the beta-cell to acetylcholine, was noted during the latter part of the perfusion interval. The potent insulin secretory response elicited by acetylcholine during the 60-min period was abolished 0y 25 muM atropine. Inhibition of the insulinotropic action of acetylcholine was also noted with administration of the mitotic spindle inhibitor, colchicine. When compared to 20-min control perfusions, addition of 1 mM colchicine resulted in a 50% reduction in acetylcholine-induced insulin release. These results suggest that insulin secretion stimulated by acetylcholine can be considered to be due to a muscarinic action of this agent which is dependent, at least in part, upon the microtubular system of the beta-cell.     

Effects of colchicine- and cytochalasin B-treatment on the intracellular distribution of yolk droplets, lipid bodies, and Golgi apparatus of the chick neuroepithelial cells

Treatment with colchicine (antimicrotubular agent) and cytochalasin B (antimicrofilamentous agent) has been used to investigate the possible role played by the cytoskeleton in the maintenance of intracellular distribution of yolk droplets, lipid bodies, and Golgi apparatus of the chick neuroepithelial cells. On the one hand, embryos treated with colchicine showed modifications in their distribution patterns of yolk droplets and lipid bodies, which suggests the involvement of the microtubular integrity of neuroepithelial cells in the maintenance of normal distribution patterns. On the other hand, the close relationships between vitelline and lipid inclusions and Golgi apparatus observed in untreated embryos seems to be kept in the embryos treated with colchicine and cytochalasin B. Moreover, from the effects of colchicine on Golgi apparatus position a possible functional role for the microtubular system in the maintenance of Golgi apparatus polarity in the chick neuroepithelial cells can be proposed. The results provided here constitute new information about the cellular mechanisms involved in chick neurulation.     

Microtubules and protein secretion in rat lacrimal glands. Inhibitory effect of the tubulin . colchicine complex isolated from lacrimal glands upon brain tubulin polymerization. Identification of the complex by gel electrophoresis.

The specific inhibitory effect of colchicine upon protein secretion by lacrimal glands could be related to the formation of a complex between colchicine and tubulin from the soluble fraction of the gland. By gel electrophoresis under nondissociating conditions, it is shown that this complex is similar to the colchicine . tubulin complex from brain. The complex isolated from lacrimal glands is highly inhibitory upon brain tubulin assembly since as low as 0.07 microM complex impedes the polymerization of 8 microM tubulin by 50%, compared to 3 microM for free colchicine. Therefore, a small percentage of complexed tubulin (0.9%) is enough for polymerization to be blocked. In lacrimal glands the complex might prevent the polymerization of tubulin, and colchicine shift the tubulin in equilibrium microtubules equilibrium to microtubules disassembly. The disorganization of the labile microtubular system could lead to a modification of the transport of the secretory granules and to a perturbation of secretion.     

Microtubules regulate local Ca2+ spiking in secretory epithelial cells

The role of the cytoskeleton in regulating Ca(2+) release has been explored in epithelial cells. Trains of local Ca(2+) spikes were elicited in pancreatic acinar cells by infusion of inositol trisphosphate through a whole cell patch pipette, and the Ca(2+)-dependent Cl(-) current spikes were recorded. The spikes were only transiently inhibited by cytochalasin B, an agent that acts on microfilaments. In contrast, nocodazole (5-100 micrometer), an agent that disrupts the microtubular network, dose-dependently reduced spike frequency and decreased spike amplitude leading to total blockade of the response. Consistent with an effect of microtubular disruption, colchicine also inhibited spiking but neither Me(2)SO nor beta-lumicolchicine, an inactive analogue of colchicine, had any effect. The microtubule-stabilizing agent, taxol, also inhibited spiking. The nocodazole effects were not due to complete loss of function of the Ca(2+) signaling apparatus, because supramaximal carbachol concentrations were still able to mobilize a Ca(2+) response. Finally, as visualized by 2-photon excitation microscopy of ER-Tracker, nocodazole promoted a loss of the endoplasmic reticulum in the secretory pole region. We conclude that microtubules specifically maintain localized Ca(2+) spikes at least in part because of the local positioning of the endoplasmic reticulum.     

Potentiation of lymphocyte activation by colchicine.

Proliferation of human lymphocytes induced by IO4- is potentiated by 30 min exposure to colchicine (10(-6)M), whereas the response to Con A is inhibited. Treatment with colchicine before or after IO4- modification has similar enhancing effects. Lumicolchicine does not alter proliferative responses. In addition to the proliferation of IO4--oxidized cells, irradiated IO4- modified lymphocytes induce proliferation when mixed with untreated lymphocytes. Enhancement occurs in both these conditions only when IO4--modified cells are treated with colchicine. Preliminary data indicate that proliferation in mixed lymphocyte cultures is also potentiated when either stimulating or responding cells are pretreated with colchicine. These findings suggest a selective stimulatory effect of colchicine on lymphocyte responses induced by cell-cell contact. Agents that modify microtubular assemblies might regulate the induction of immune responses that involve cellular interactions.     

Effects of long term denervation on smooth muscle of the chicken expansor secundariorum

Summary Denervation of the expansor secundariorum muscle of the adult and 2 week chicken, by sectioning the brachial plexus, resulted in an approximate twofold increase in dry weight over 8 weeks. Unlike skeletal muscle, no ultrastructural changes were exhibited by the smooth muscle cells for a period of up to 5 months post denervation. No evidence of hypertrophy of the individual muscle cells was observed, but following colchicine treatment a definite increase in the number of mitotic figures was noted within muscle bundles indicating that the increase in dry weight of the expansor muscle is due to hyperplasia of the smooth muscle cells. The results are discussed in relation to in vitro studies of the interaction of sympathetic nerves with smooth muscle.     

Polymerization of tubulin in the presence of colchicine or podophyllotoxin. Formation of a ribbon structure induced by guanylyl-5'-methylene diphosphonate

GTP-dependent in vitro polymerization of rat brain microtubular protein is inhibited to 50% by substoichiometric concentrations of the antimitotic drugs colchicine (0.12 mol/mol of tubulin) and podophyllotoxin (0.14 mol/mol of tubulin). Substitution of pp(CH₂)pG² for GTP, however, results in an extensive microtubular protein polymerization at such concentrations. In the presence of pp(CH₂)pG, suprastoichiometric concentrations of podophyllotoxin (19 mol/mol of tubulin) are required to inhibit the polymerization process by 50%. Colchicine is very ineffective since 3 × 10⁵ moles/mole of tubulin are required to give a 50% inhibition. Electron microscopical analysis shows that the polymers formed by microtubular protein in the presence of suprastoichiometric concentrations of drugs are not the normal short microtubules typical of pp(CH₂)pG-driven polymerization, but are ribbons with three or four protofilaments. The colchicine content of the harvested ribbons has been measured directly and found to be approximately 0.8 moles colchicine/mole of tubulin. Treatment of microtubular protein with substoichiometric concentrations of drugs results in an increase in the number of protofilaments forming the ribbons. Many of the ribbons can close into morphologically normal microtubules when microtubular protein is treated with only 0.05 moles of either colchicine or podophyllotoxin per mole of tubulin.     

The effects of microtubular binding agents on secretion of IgM antibody

Inhibition of antibody secretion was effected by agents which bind microtubular protein, such as colchicine and vincristine. However, cytochalasin B, an inhibitor of microfilaments, was not effective. Recovery of secretory activity could be accomplished by dimethylsulfoxide (DMSO) but not by other agents which altered membrane permeability to a varying extent. Those agents tested were: trypsin, pronase, and a lysolecithin analogue. DMSO did not act by reaggregation of disaggregated microtubules, as studied by electron microscopy. The mechanism of its action remains obscure. Micromanipulated single cells from the center of a plaque included plasma cells, lymphocytes and macrophages. In some of these cells no endoplasmic system comparable to a fully developed plasma cell could be demonstrated. Therefore, some mechanism for secretion must operate in addition to, or other than, through distended endoplasmic reticulum, confirming the evidence for microtubular involvement in this function.     

Inhibition of adenosine 3',5'-cyclic monophosphate phosphodiesterase by colchicine: implications for glucagon and corticosteroid secretion

In the study reported, colchicine, often regarded as a specific inhibitor of microtubular function, was shown to exert a concentration-dependent inhibition of the low Km cyclic AMP phosphodiesterases of the pancreatic islet, adrenal cortex and various other tissues of the rat. The results indicated that colchicine is only slightly less active as an inhibitor of the enzyme than theophylline on a molar basis and kinetic analysis revealed that both inhibitors acted competitively in the case of the liver enzyme. Our results show that the inhibitory effect of colchicine upon cyclic AMP phosphodiesterase is a general property of the alkaloid at concentrations of 5 x 10(-5)M and above in both endocrine and non-endocrine tissues. Thus, results obtained employing colchicine at concentrations significantly greater than those which are known to lead to microtubular disaggregation must be viewed with great caution if incorrect implication of microtubular participation in biological processes is to be avoided. For example, we propose that the previously reported paradoxical stimulatory effects of colchicine on the secretion of glucagon from the rat pancreatic islet and on steroidogenesis in the rat adrenal may be due to cyclic AMP accumulation consequent upon phosphodiesterase inhibition in these endocrine tissues and not to microtubular disaggregation as has hitherto been assumed.     

Phenotype modulation in primary cultures of rat aortic smooth muscle cells

The transition of adult rat aortic smooth muscle cells from a contractile to a synthetic phenotype during the first week of primary culture on a substrate of fibronectin in serum-free medium was studied by light and electron microscopy. The weak base chloroquine and the carboxylic ionophore monensin were both found to inhibit the spreading of the cells and the accompanying changes in cellular fine structure. The exchange of myofilament bundles for a prominent rough endoplasmic reticulum and Golgi complex was delayed and vacuoles filled with incompetely degraded material accumulated in the cytoplasm. The microtubule-disruptive drugs colchicine and nocodazole likewise opposed the spreading and fine structural reorganization of the cells. Most typically, the Golgi stacks were small and widely dispersed. In addition, vacuoles of the type mentioned above increased in number. On the other hand, there was surprisingly little effect of cytochalasin B, a drug that is supposed to interfere with the assembly of actin filaments. The observations suggest that the phenotypic modulation of arterial smooth muscle cells is dependent on: (a) lysosomal degradation of discarded cellular constituents, (b) active vesicular transport along the exocytic pathway to provide the expanding cell surface with new membrane, and (c) a normal microtubular cytoskeleton to ensure the establishment of a new and functionally efficient intracellular organization.     

Evidence for nucleating microtubules in microtubular associations and for an opening polarity under colchicine action.

Bundles of microtubular structures appear in the cytoplasm of germinal cells of the African frog Dicroglossus occipitalis. They are made of several associated microtubules. Every bundle contains one normal singlet and numerous arch-shaped microtubular structures growing in all directions from the singlet wall. The walls of these microtubules are shown to contain 10 to 13 protofilaments. Attempts made with colchicine point out their susceptibility to this antimitotic drug. The formation and opening of these microtubular structures give evidence of complex organization.