Depolymerization of microtubules alters membrane potential and affects the motional freedom of membrane proteins

Two independent lines of evidence were obtained indicating that microtubule depolymerization affects the functions and the physical state of membranes in intact Chinese hamster ovary cells. The first type of evidence was obtained by using the dye dihexyloxacarbocyanine iodide to measure membrane potential before and after treatment with several microtubule active agents. Microtubule depolymerization resulted in a decrease in cell fluorescence, whereas stabilization of microtubules with taxol resulted in an increase in cell fluorescence. These effects of the drugs were due to their interactions with microtubules and not to direct effects of the drugs on the plasma membranes for the following reasons: effects were time dependent and required entry into the cells as indicated by the lack of fluorescence change in a multi-drug-resistant mutant that does not accumulate antimicrotubule drugs and a colcemid-resistant tubulin mutant did not show these effects on cell fluorescence. Evidence for altered motional freedom of membrane proteins in the plasma membrane was obtained by using electron spin resonance analysis of maleimide spin probe labeled cells. This study showed that depolymerization of microtubules results in increased motional freedom of maleimide-labeled sulfhydryl group containing proteins. Taken together, these data argue that microtubules function in mammalian cells to regulate the physical state of membranes and modulate membrane potential generated across cell membranes.     

Effect of vinblastine sulfate, colchicine and lumicolchicine on membrane organization of normal and transformed cells

Differences in the distribution of plasma membrane intramembranous particles (PMP) have been demonstrated in normal and transformed fibroblasts using freeze fracture and electron microscopy. Transformed 3T3 cells contain randomly distributed PMP and contact-inhibited 3T3 cells have aggregated PMP when frozen in medium, glycerol, sucrose, or following stabilization in 1 % formaldehyde. To define some of the mechanisms controlling the organization of PMP in this system we have examined the effects of microtubule disruptive drugs including vinblastine sulfate and colchicine on SV3T3 cells. These drugs were observed to induce a dose- and time-dependent aggregation of PMP at concentrations between 10⁻⁹ and 10⁻⁵ M. These results suggest that modulation of PMP distribution in these cells may be influenced by an interaction of microtubules with plasma membrane components. However, the observation that lumicolchicine, a derivative of colchicine which does not disrupt microtubules, also promotes PMP aggregation, suggests that these drugs may also have a primary effect on the plasma membrane in addition to the disruption of microtubules. This is supported by the observation that reduced temperature (4 °C) which is known to disrupt microtubules fails to induce PMP aggregation in SV3T3 cells, suggesting the hypothesis that changes in the interaction of plasma membrane or plasma membrane associated constituents may control the distribution of PMP in this cell system.     

The influence of microtubule integrity on plasma membrane fluidity in L929 cells

The aim of this work was to examine the possible influence of the integrity of the microtubule network on the plasma membrane fluidity of L929 mouse fibroblasts. The L929 cell line was selected for the ease of culture and the stability of its characteristics. The cells were treated with colchicine, nocodazole and vinblastine, three microtubule-depolymerizing drugs, at various concentrations and for various times. Membrane fluidity was assessed from fluorescence depolarization measurements with the plasma membrane probe TMA-DPH. Each of the drugs induced a significant, dose-dependent decrease in fluorescence anisotropy. The effect levelled off (5-7% decrease) after ~ 90 min of treatment, and could be unambiguously interpreted as resulting from an increase in membrane fluidity. The cumulative action of the drugs did not significantly increase the effect. The effects of colchicine and nocodazole could be reversed by incubation in drug-free medium, but not that of vinblastine. The results are discussed in correlation with the kinetics of the three drugs interaction with tubulin or microtubules. It is concluded that the microtubule integrity contributed to the high plasma membrane lipidic order, but less than other factors, like the lipid composition and the cholesterol content.     

The influence of microtubule integrity on plasma membrane fluidity in L929 cells

The aim of this work was to examine the possible influence of the integrity of the microtubule network on the plasma membrane fluidity of L929 mouse fibroblasts. The L929 cell line was selected for the ease of culture and the stability of its characteristics. The cells were treated with colchicine, nocodazole and vinblastine, three microtubule-depolymerizing drugs, at various concentrations and for various times. Membrane fluidity was assessed from fluorescence depolarization measurements with the plasma membrane probe TMA-DPH. Each of the drugs induced a significant, dose-dependent decrease in fluorescence anisotropy. The effect levelled off (5-7% decrease) after approximately 90 min of treatment, and could be unambiguously interpreted as resulting from an increase in membrane fluidity. The cumulative action of the drugs did not significantly increase the effect. The effects of colchicine and nocodazole could be reversed by incubation in drug-free medium, but not that of vinblastine. The results are discussed in correlation with the kinetics of the three drugs interaction with tubulin or microtubules. It is concluded that the microtubule integrity contributed to the high plasma membrane lipidic order, but less than other factors, like the lipid composition and the cholesterol content.     

Local anesthetic-induced inhibition of collagen secretion in cultured cells under conditions where microtubules are not depolymerized by these agents

Tertiary amine local anesthetics previously have been shown to influence some microtubule-dependent cellular functions. Since several cell secretion processes, including secretion of collagen, have been shown to be inhibited by microtubule-disrupting drugs such as colchicine, we determined whether local anesthetics affect collagen secretion. Six local anesthetics inhibited collagen and non-collagen protein secretion (up to 98%) into the extracellular medium of 3T3 cells and human fibroblasts, an effect apparently independent of influences on proline transport and total protein synthesis. A combination of colchicine and cytochalasin B did not duplicate the effects of local anesthetics. The effects of subsaturating concentrations of colchicine and procaine on secretion were additive, suggesting that both drugs act on the secretory pathway at the level of microtubules, but other effects of the two types of drugs were strikingly different. In comparing the mechanisms of action of colchicine and local anesthetics, it was seen that, in contrast to colchicine, radioactive procaine and lidocaine were slowly transported into 3T3 cells, did not bind to the tubulin-containing TCA-insoluble fraction, and did not bind to purified tubulin in vitro. The fraction of cellular tubulin present as microtubules (47% in normal cells) was determined by measuring tubulin in stabilized, sedimentable microtubules compared to total tubulin, using a [3H]colchicine binding assay. Pretreatment of cells in the cold or with colchicine led to depolymerization of microtubules, but pretreatment with five local anesthetics tested did not. Therefore, in contrast to colchicine, local anesthetics in concentrations that inhibit secretion do not directly interact with or depolymerize microtubules. These drugs, however, do affect a microtubule-dependent process and may do so by detaching the microtubular system from the cell membrane.     

Differential effect of lipid peroxidation on membrane fluidity as determined by electron spin resonance probes

The effect of lipid peroxidation on membrane fluidity was examined in sonicated soybean phospholipid vesicles. Following iron/ascorbate dependent peroxidation, the vesicles were labeled with a series of doxyl stearate spin probes which differed in the site of attachment of the nitroxide free radical to the fatty acid. Comparison of motional and partitioning parameters derived from electron spin resonance spectra of the probes indicated that the membranes were less fluid following peroxidation. However, the magnitude of the fluidity decrease was markedly dependent on the intramembrane location, as well as on the extent of lipid peroxidation. The effect of lipid peroxidation on fluidity was maximal in the membrane microenvironment sampled by 12-doxyl stearate, whereas other regions of the bilayer were less affected. These findings indicate that lipid peroxidation leads to an alteration of the transbilayer fluidity gradient.     

Influence of colchicine and vinblastine on the intracellular migration of secretory and membrane glycoproteins: III. Inhibition of intracellular migration of membrane glycoproteins in rat intestinal columnar cells and hepatocytes as visualized by light and electron-microscope radioautography after 3H-fucose injection

In the first paper of this series (Bennett et al., 1984), light-microscope radioautographic studies showed that colchicine or vinblastine inhibited intracellular migration of glycoproteins out of the Golgi region in a variety of cell types. In the present work, the effects of these drugs on migration of membrane glycoproteins have been examined at the ultrastructural level in duodenal villous columnar cells and hepatocytes. Young (40 gm) rats were given a single intravenous injection of colchicine (4.0 mg) or vinblastine (2.0 mg). At 10 min after colchicine and 30 min after vinblastine administration, the rats were injected with 3H-fucose. Control rats received 3H-fucose only. All rats were sacrificed 90 min after 3H-fucose injection and their tissues processed for radioautography. In duodenal villous columnar cells, 3H-fucose labeling of the apical plasma membrane was reduced by 51% after colchicine and by 67% after vinblastine treatment; but there was little change in labeling of the lateral plasma membrane. Labeling of the Golgi apparatus increased. This suggests that labeled glycoproteins destined for the apical plasma membrane were inhibited from leaving the Golgi region, while migration to the lateral plasma membrane was not impaired. In hepatocytes, labeling of the sinusoidal plasma membrane was reduced by 83% after colchicine and by 85% after vinblastine treatment. Labeling of the lateral plasma membrane also decreased, although not so dramatically. Labeling of the Golgi apparatus and neighboring secretory vesicles increased. This indicates that the drugs inhibited migration of membrane glycoproteins from the Golgi region to the various portions of the plasma membrane. Accumulation of secretory vesicles at the sinusoidal front suggests that exocytosis may also have been partially inhibited. In both cell types, microtubules almost completely disappeared after drug treatment. Microtubules may, therefore, be necessary for intracellular transport of membrane glycoproteins, although the possibility of a direct action of these drugs on Golgi or plasma membranes must also be considered.     

Microtubules and cyclic amp in human leukocytes: on the order of things

We have shown previously that the β-adrenergic agonist isoproterenol (2μM) and the phosphodiesterase inhibitor isobutylmethylxanthine (1 mM) produce a much greater increase in cyclic AMP in human leukocytes that have been pretreated with colchicine (or with other agents that affect microtubule assembly) than in control leukocytes. The effects of colchicines were both time- and dose-dependant. These and other data suggested that the generation of cyclic AMP is normally restricted by an intact system of cytoplasmic microtubules. If so, then the same time and dose dependencies might apply to other colchicines-induced changes in leukocyte function. We have now assayed the distribution of concanavalin A (Con A)-receptor complexes on the leukocyte membrane, taking into account that leukocytes competent to assemble microtubules show a uniform distribution of surface- bound Con A whereas microtubule-deficient cells accumulate Con A in surface caps. We have found that the effect of colchicine on capping is also both time- and dose dependent, and that the dose-response relationships conform to those required to increase cyclic AMP levels. These findings provide further evidence that both colchicine-induced Con-A capping and colchicine- induced cyclic AMP generation depend upon the relaxation of constraints normally imposed by cytoplasmic microtubules upon the plasma membrane, which limit, respectively, lateral mobility of the lectin-receptor complexes, and expression of hormone-sensitive adenylate cyclase. Moreover, colchicine-induced Con-A cap formation is not affected even by very large changes in leukocyte cyclic AMP levels. Thus, elevated cyclic AMP levels do not appear to promote the dissolution of microtubules; rather, the dissolution of microtubules permits the generation of increased amounts of cyclic AMP.     

Comparison of perturbation effect of propranolol, verapamil, chlorpromazine and carbisocaine on lecithin liposomes and brain total lipid liposomes. An EPR spectroscopy study.

Effect of verapamil, propranolol, chlorpromazine and carbisocaine on dynamics and/or order of liposomes (perturbation effect), prepared from different molar ratios of lecithin (PC) and rat brain total lipids (TL) was studied by EPR spectroscopy using spin probes 16-doxyl stearic acid and 14-doxyl phosphatidylcholine. The PC liposomes had higher dynamics and/or lower order than the TL liposomes. The perturbation effect of the drugs depended largely on the lipid composition of the liposomes. The drugs at the drug/lipid molar ratios from 0.1 to 1 increased membrane dynamics and/or decreased membrane order. The drugs had the most pronounced perturbation effect in the liposomes prepared from brain total lipids. The effect of the drugs decreased with decreasing the TL/PC ratio in the liposomes and was lowest, almost diminished, in the PC liposomes. Increasing concentration of the drugs decreased the difference between the dynamics and/or order of the PC and TL liposomes and so eliminated the influence of lipid composition on these membrane parameters. The results emphasize the role of lipid composition in studies concerning drug-lipid interactions in model and biological membranes.     

The effects of microtubule dissociating agents on the physiology and cytology of the sensory neuron in the femoral tactile spine of the cockroach, periplaneta americana L

Microtubules are prominent cellular components of the mechanosensory and chemosensory sensilla associated with the insect cuticle, and a range of hypotheses have been proposed to account for their role in sensory transduction. Chemical agents such as colchicine and vinblastine, which dissociate microtubules, also interfere with transduction in these sensilla, and this has been attributed to their anti-microtubule activity. We have now examined the dynamic properties of sensory transduction in the mechanosensitive neuron of the cockroach femoral tactile spine, after the application of colchicine, vinblastine and lumicolchicine. Concurrently we have examined the ultrastructure of the same sensory ending by transmission electron microscopy. All of the drugs reduced the mechanical sensitivity o the receptor. Colchicine and vinblastine achieved this reduction without altering the dynamic properties of the receptor but lumicolchicine changed the dynamic response, and increased the relative sensitivity to rapid movements. Conduction velocity, another measure of neuronal function, which relies upon ionic currents flowing through the membrane, was reduced by all three drugs. The effects of the drugs upon the ultrastructure of the sensory ending were also disparate. In the case of colchicine there was complete dissociation of microtubules in the tubular body and distal dendrite before a total loss of mechanical sensitivity. Vinblastine was less effective in dissociating microtubules, although more effective in the reduction of mechanical sensitivity. With lumicolchicine the dominant morphological effect was a severe disruption of the dendritic membrane. We conclude from these experiments that microtubules are not essential in the transduction of mechanical stimuli by cuticular receptors and that the effects of these drugs upon mechanosensitivity are not directly related to their dissociation of the microtubules in the tubular body, but are more likely to arise from actions upon the cell membrane. These actions could include effects upon tubulin in the membrane or upon other membrane components.     

The role of spindle microtubules in the timing of the cell cycle in echinoderm eggs

Spindle microtubules play an important role in the mechanisms that control the timing of cell cycle events in the eggs of the sea urchins L. variegatus and L. pictus. However, recent work which used colchicine to block microtubule assembly in the eggs of two other echinoderms, S. purpuratus and D. excentricus, has raised serious questions about the generality of this role for spindle microtubules. Thus, we have systematically examined the role of spindle microtubules in the timing of the cell cycle in the fertilized eggs of these latter species. We treated eggs of both species with 5-10 microM Colcemid for several minutes starting 30 min after fertilization to completely prevent spindle microtubule assembly for several h. We used Colcemid, instead of colchicine, because it is effective at lower doses and, at these doses, shows no detectable toxic side effects. We compared for control and treated eggs the time course of nuclear envelope breakdown/reformation and DNA synthesis. We found for both species that the eggs continue to cycle without spindle microtubules; mitosis is up to twice the normal duration while interphase remains essentially unaffected. To test for the possible toxic side effects of the 1-2 mM colchicine used earlier on S. purpuratus and D. excentricus, we treated eggs of these two species, and also those of L. variegatus, with 1 mM lumi-colchicine. This photo-inactivated form of colchicine, which does not bind to tubulin, substantially prolongs mitosis and, to a lesser extent, interphase. Thus, the results of the earlier work are most easily explained by the combination of specific and nonspecific effects of the 1-2 mM colchicine used. Our present results indicate that the importance of spindle microtubules in the mechanisms that control the timing of the mitosis portion of the cell cycle is a general phenomenon.     

Fluidizing effects of centrophenoxine in vitro on brain and liver membranes from different age groups of mice

This study examined the effects of different concentrations of centrophenoxine on physical properties of synaptic plasma membranes and liver microsomes using electron spin resonance procedures. Membranes of different age groups of mice were labeled with the 5-doxyl stearic acid spin-label and membrane fluidity determined in the presence and absence of different concentrations of centrophenoxine. Centrophenoxine had a direct effect on membranes as shown by a significant increase in membrane fluidity. This effect was greatest in liver microsomes as compared to synaptic plasma membranes. Age differences were not observed in centrophenoxine-induced fluidization. Effects of centrophenoxine in vivo may be due in part to the drug acting directly on the physical properties of the membrane lipid environment.     

Role of membrane colchicine binding proteins in the transmission of prolactin message to casein genes in the rabbit mammary gland

Previous work demonstrated that tubulin binding drugs specifically inhibit the capacity of prolactin to initiate casein and DNA synthesis in the mammary cell. It was concluded that microtubules or other tubulin containing cellular structures were involved in the transmission of the prolactin message to genes. In the present work, it is shown that griseofulvin, an antimitotic drug which alters microtubule structure and function, does not prevent prolactin actions. Autoradiographic studies showed that [3H]colchicine binds preferentially to plasma and Golgi membranes in the mammary cell. Short term cultures of mammary explants with [3H]colchicine demonstrated that the labelled drug binds to membranous cellular structures which were isolated from explants at the end of the culture. Fractions containing plasma and Golgi membranes contained the highest amount of radioactivity. Solubilisation of the membranes by Triton X-100 dissociated the [3H]colchicine from the prolactin receptors as judged by a chromatography of the soluble fraction on a Sepharose 6 B column. On the column, the labelled colchicine remains associated with a molecular entity which may be free tubulin. In all cases, the binding of [3H]colchicine was greatly attenuated by an excess of unlabelled colchicine but was only slightly affected by the competition with lumicolchicine. These results suggest that mammary membranes contain tubulin and that binding of drugs to this molecule inhibits the generation of the prolactin second messengers eliciting the hormonal actions in the mammary cell. This also suggests that microtubules are probably not involved in the mechanism of prolactin action.     

Microtubules and the regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase.

Cultured C-6 glial cells were utilized to evaluate the effect of antimicrotubular drugs on 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and cholesterol synthesis. Colchicine, Colcemid, and vinblastine (1.0 muM) caused a marked reduction in HMG-CoA reductase activity and, as a consequence, the rate of cholesterol synthesis in these cells. No effect was observed with lumicolchicine, a mixture of colchicine isomers with no effect on microtubules. The effect of colchicine was apparent within 1 h after addition to the culture medium, and, after 6 h, HMG-CoA reductase activity in treated cells was only approximately 15 to 30% of that in untreated cells. Reductase activity was very sensitive to the concentration of drug added, i.e. cells treated with just 0.1 muM colchicine for 6 h exhibited a 50% lower enzymatic activity than did untreated cells. The lack of a generalized, nonspecific toxic effect on the cells was indicated by the finding of no change in the activities of fatty acid synthetase and NADPH-cytochrome c reductase and the rate of total protein synthesis in cells treated with colchicine (1 muM) for 6 h. A close temporal and quantitative correlation was observed between the effects of colchicine on HMG-CoA reductase and on a parameter of microtubular function, i.e. maintenance of glial cell shape. The data suggest that microtubules are involved in the regulation of HMG-CoA reductase and cholesterol synthesis in C-6 glial cells.     

Development of the Na(+)-dependent hexose carrier in LLC-PK1 cells is dependent on microtubules

The Na(+)-dependent hexose carrier, an endogenous apical marker, develops during differentiation of LLC-PK1, an established cell line with characteristics of the proximal tubule. This development was inhibited by the microtubule-disrupting drugs, colchicine and nocodazole, while it was insensitive to lumicolchicine. This strongly suggests that microtubules are involved in the plasma membrane expression of the Na(+)-dependent hexose carrier. We also analyzed the increase in activity of endogenous apical and basolateral membrane proteins during the polarization process. The development of three apical (Na(+)-dependent hexose carrier, gamma-glutamyltransferase and alkaline phosphatase) and one basolateral membrane protein (Na+/K(+)-ATPase) was studied during the reorganization of LLC-PK1 cells into a polarized epithelium. Colchicine inhibited the rapid, transient increase in the expression of the Na(+)-dependent hexose carrier during this polarization process. A similar result was observed for the development of the other apical proteins, while the development of Na+/K(+)-ATPase seemed to be largely insensitive to colchicine. Our results are in agreement with the model that the vesicles containing the apical membrane proteins use microtubules as tracks to reach the plasma membrane. The transport of vesicles containing basolateral membrane proteins clearly occurs by a different pathway which is independent on an intact microtubular network. Since the inhibition by the microtubule-disrupting drugs was complete, it can be concluded that after disruption of microtubules, the apical vesicles do not use the basolateral pathway by default.     

Shedding of Phosphatidylserine from Developing Erythroid Cells Involves Microtubule Depolymerization and Affects Membrane Lipid Composition

Phosphatidylserine (PS), which is normally localized in the cytoplasmic leaflet of the membrane, flip-flops to the external leaflet during aging of, or trauma to, cells. A fraction of this PS undergoes shedding into the extracellular milieu. PS externalization and shedding change during maturation of erythroid cells and affect the functioning, senescence and elimination of mature RBCs. Several lines of evidence suggest dependence of PS shedding on intracellular Ca concentration as well as on interaction between plasma membrane phospholipids and microtubules (MTs), the key components of the cytoskeleton. We investigated the effect of Ca flux and MT assembly on the distribution of PS across, and shedding from, the membranes of erythroid precursors. Cultured human and murine erythroid precursors were treated with the Ca ionophore A23187, the MT assembly enhancer paclitaxel (Taxol) or the inhibitor colchicine. PS externalization and shedding were measured by flow cytometry and the cholesterol/phospholipids in RBC membranes and supernatants, by ¹H-NMR. We found that treatment with Taxol or colchicine resulted in a marked increase in PS externalization, while shedding was increased by colchicine but inhibited by Taxol. These results indicate that PS externalization is mediated by Ca flux, and PS shedding by both Ca flux and MT assembly. The cholesterol/phospholipid ratio in the membrane is modified by PS shedding; we now show that it was increased by colchicine and A23187, while taxol had no effect. In summary, the results indicate that the Ca flux and MT depolymerization of erythroid precursors mediate their PS externalization and shedding, which in turn changes their membrane composition.     

Calcium and chlorpromazine interactions in rat synaptic plasma membranes. A spin-label and fluorescence probe study.

The effects of calcium and of the psychoactive drug chlorpromazine (CPZ) on the rat synaptic plasma membrane have been studied using two stearic nitroxide spin labels having their doxyl groups in positions 5 and 16 and the fluorescent probe 1-anilinonaphtalene-8-sulfonate (ANS). The mobility of the 5-doxyl stearic spin label which probes the membrane phospholipids in the vicinity of their polar heads is decreased in the presence of both compounds. Calcium is more efficient in this respect than CPZ. In spite of this qualitative similarity of action, CPZ inhibits the effect of calcium and vice versa. No modification of the 16-doxyl stearic spectrum has been observed even at high calcium or CPZ concentrations. An increase in fluorescence intensity and a blue shift in the emission wavelength of ANS-probed membranes are observed with very low CPZ concentrations (10^?7 to 10^?5m). With higher concentrations, a further intensity increase and a further blue shift are due to direct interaction between ANS and CPZ. Calcium also increases the fluorescence intensity of ANS-labeled membranes in the concentration range 10^?5–10^?2m. As for the spin-label data, the effects of both compounds are mutually competitive. It is concluded that calcium interacts principally with the phospholipid polar heads of this type of membrane. However, the competition with CPZ suggests indirectly that this ion is also bound to membrane proteins. CPZ has an affinity for membrane lipids only at high concentrations. In its pharmacologically active concentration range, it is located preferentially on the membrane proteins.     

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.     

MECHANISMS OF LYSOSOMAL ENZYME RELEASE FROM HUMAN LEUKOCYTES : I. Effect of Cyclic Nucleotides and Colchicine

In order to study mechanisms underlying selective enzyme release from human leukocytes during phagocytosis, the effects were studied of compounds which affect microtubule integrity or the accumulation of cyclic nucleotides. Human leukocytes selectively extrude lysosomal enzymes (β-glucuronidase) from viable cells during phagocytosis of zymosan or immune complexes, or upon encounter with immune complexes dispersed along a non-phagocytosable surface such as a millipore filter. In each circumstance, lysosomal enzyme release was reduced by previous treatment of cells with pharmacological doses of drugs which disrupt microtubules (e.g. 10^-3–10^-5 M colchicine) or with agents which affect accumulation of adenosine 3'5'-monophosphate (cAMP) (e.g. 10^-3 M cyclic nucleotides and 2.8 x 10^-4–2.8 x 10^-6 M prostaglandin E (PGE) and A (PGA) compounds). Preincubation of cells with 5 µg/ml cytochalasin B resulted in complete inhibition of zymosan ingestion, but not of adherence of zymosan particles to plasma membranes or selective enzyme release. In this system, in which enzyme release was independent of particle uptake, preincubation of cells with colchicine, vinblastine, dibutyryl cAMP, or PGE₁ also reduced extrusion of lysosomal enzymes. When cell suspensions were incubated with membrane-lytic crystals of monosodium urate (MSU), cytoplasmic as well as lysosomal enzymes were released with subsequent death of the cells. However, enzyme release followed phagocytosis of crystals (as measured by enhanced C-1 oxidation of glucose) and was due to "perforation from within" of the lysosomal membrane, rather than lysis by crystals of the plasma membrane. Enzyme release after MSU ingestion was also reduced when cells were treated with pharmacological doses of the test agents. When cells were killed by Triton X-100, acting on the plasma membrane, C-1 oxidation of glucose was abolished and enzyme release could not be inhibited pharmacologically. These observations suggest that lysosomal enzyme release from human phagocytes can be an active process which accompanies plasma membrane stimulation, is independent of cell death, and may be controlled by cyclic nucleotides and agents which affect microtubules.     

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.