Effect of colchicine on the uptake of prolactin and insulin into Golgi fractions of rat liver.

In previous studies we have shown that 125I-labeled prolactin is taken up by a receptor-dependent process and concentrated in an intact form in Golgi elements from female rat liver (J. Biol. Chem., 1979, 254:209-214). In this study we have examined the effect of colchicine on this uptake process into Golgi elements. Colchicine [25 mumol (10 mg)/100 gm body wt] was injected intraperitoneally in adult female rats, and hepatic Golgi fractions were prepared at 1, 2, and 3 h postinjection. The enzyme recoveries and morphological appearance of fractions from colchicine-treated and control (alcohol alone) animals were similar. At times greater than 1 h after colchicine there was a marked (greater than 60%) inhibition of uptake of 125I-ovine prolactin (125I-oPRL) into Golgi light and intermediate fractions but no inhibition of uptake into Golgi heavy and plasmalemma elements. At times from 2 to 45 min postinjection, 125I-oPRL was extracted from Golgi elements and found to be largely intact as judged by rebinding to receptors. The inhibitory effect of colchicine was seen at doses ranging from 0.25 mumol to 25 mumol/100 g body wt. Vincristine also inhibited 125I-oPRL uptake into the Golgi light and intermediate fractions but lumicolchicine had no inhibitory effect. There was a smaller effect of colchicine both at early (1 h) and later (3 h) times on the extent and pattern of 125I-insulin uptake. Colchicine treatment did not produce a significant change in lactogen receptor levels in the Golgi fractions. These results demonstrate that colchicine treatment inhibited the transfer of prolactin into Golgi vesicular elements. The much smaller effect on insulin uptake suggests that there may be differences in the manner in which the two hormones are handled in the course of internalization.

MT1 melatonin receptor internalization underlies melatonin-induced morphologic changes in Chinese hamster ovary cells and these processes are dependent on Gi proteins, MEK 1/2 and microtubule modulation.

Melatonin induces cellular differentiation in numerous cell types. Data show that multiple mechanisms are involved in these processes that are cell-type specific and may be receptor dependent or independent. The focus of this study was to specifically assess the role of human MT1 melatonin receptors in cellular differentiation using an MT1-Chinese hamster ovary (CHO) model; one that reproducibly produces measurable morphologic changes in response to melatonin. Using multiple approaches, we show that melatonin induces MT1-CHO cells to hyperelongate through a MEK 1/2, and ERK 1/2-dependent mechanism that is dependent upon MT1 receptor internalization, Gi protein activation, and clathrin-mediated endocytosis. Using immunoprecipitation analysis, we show that MT1 receptors form complexes with Gi(alpha) 2,3, Gq(alpha), beta-arrestin-2, MEK 1/2, and ERK 1/2 in the presence of melatonin. We also show that MEK and ERK activity that is induced by melatonin is dependent on Gi protein activation, clathrin-mediated endocytosis and is modulated by microtubules. We conclude from these studies that melatonin-induced internalization of human MT1 melatonin receptors in CHO cells is responsible for activating both MEK 1/2 and ERK 1/2 to drive these morphologic changes. These events, as mediated by melatonin, require Gi protein activation and endocytosis mediated through clathrin, to form MT1 receptor complexes with beta-arrestin-2/MEK 1/2 and ERK 1/2. The MT1-CHO model is invaluable to mapping out signaling cascades as mediated through MT1 receptors especially because it separates out MEK/ERK 1/2 activation by MT1 receptors from that of receptor tyrosine kinases.

Colchicine binding in the rat tapeworm, Hymenolepis diminuta.

A fraction with enriched colchicine-binding properties prepared from Hymenolepis diminuta was found to possess many of the properties of tubulin isolated from other sources. Colchicine was bound by a simple saturable process with a dissociation constant of 13.2 microM. The binding capability decayed with a half-life of about 5 h. Binding was unaffected by lumicolchicine, was competitively inhibited by podophyllotoxin (inhibition constant of 4.8 microM) and showed an apparent stimulation by vinblastine sulphate. Sodium chloride also appeared to stimulate the binding process. The ligand/receptor complex had a molecular weight of approx. 112 000 as determined by gel filtration. On the basis of this biochemical and pharmacological evidence it was concluded that the colchicine receptor in the supernatant fraction of the H. diminuta homogenate was almost certainly tubulin. Refinement of the preparation should facilitate further studies on the mode of action of certain types of anthelmintic compound.

Effect of colchicine on rat liver plasma membrane.

Colchicine effect has been tested on rat liver plasma membrane-bound enzymes after in vitro or in vivo treatment. It appears that the in vitro treatment does not affect 5'-nucleotidase, Mg2+-ATPase and (Na+ + K+)-ATPase, whereas adenylate cyclase is sensitive to both in vitro and in vivo treatment, the latter condition being also effective for 5'-nucleotidase.

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.

Colchicine stimulates the rate of contraction of heart cells in culture.

Microtubules have been demonstrated in intact heart muscle as well as in cultured myocytes. To better understand what role these filaments may be playing in the regulation of cardiac function we have used the microtubule disrupting agent colchicine and examined its effect upon the rate of beating of rat heart cells. Colchicine, but not the inactive stereoisomer lumicolchicine, increased the myocyte rate of spontaneous contraction in a dose dependent manner. This effect was clearly distinguishable from the positive chronotropic effect of isoprenaline and unlike isoprenaline was not blocked by propranolol. Colchicine was without effect on the in vitro activity of adenylate cyclase assayed in a myocyte homogenate. The binding of 3H-colchicine to cultured heart cells increased with a time course consistent with the increase in heart rate. Subcellular distribution and sephadex gel chromatography demonstrated that approximately 30% of the cell associated colchicine comigrated with tubulin. Measurements of total myocyte tubulin by 3H-colchicine binding indicated that tubulin represents about 0.04% of the total heart cell protein.

Intact microtubules are necessary for complete processing, storage and regulated secretion of von Willebrand factor by endothelial cells.

The importance of intact microtubules in the processing, storage and regulated secretion of von Willebrand factor (vWf) from Weibel-Palade bodies in endothelial cells was investigated. Human umbilical vein endothelial cells treated for 1 h with colchicine (10(-6) M) or nocodozole (10(-6) M) lost their organized microtubular network. Stimulation of these cells with secretagogues (A23187, thrombin) produced only 30% release of vWf in comparison to control cells containing intact microtubules. The nocodazole treatment was reversible. One-hour incubation in the absence of the drug was sufficient for microtubules to reform and restore the full capacity of the cells to release vWf. Long-term incubation (24 h) of endothelial cells with microtubule-destabilizing agents had a profound effect on vWf distribution. In control cells, vWf was localized to organelles in the perinuclear region (i.e., endoplasmic reticulum and Golgi apparatus) and to Weibel-Palade bodies. In drug-treated cells vWf staining was dispersed throughout the cytoplasm, and Weibel-Palade bodies were absent. The vWf synthesized in the absence of microtubules contained significantly less large multimers than that produced by control cells. Since Weibel-Palade bodies specifically contain the large multimers, we hypothesize that the structural defect in vWf secreted by cells in the absence of microtubules is due to the lack of Weibel-Palade bodies in these cultures.

Factors involved in the sensitivity of Stentor to colchicine, lumicolchicine, and vinblastine sulfate.

Oral regeneration by the ciliate Stentor coeruleus is inhibited by colchicine (Cc), but only at a relatively high concentration (0.9 mM); moreover, regeneration is inhibited by an even lower concentration of lumicolchicine (LCc) (0.2 mM). Together these results suggest that Cc may not be acting via tubulin binding. To evaluate this possibility we: (1) tested the effect of both drugs, and vinblastine sulfate (Vb) for comparison, on a population of labile cytoplasmic microtubules; and (2) measured the kinetics of association of all three drugs with regenerating cells. We found that Cc and Vb reduced the number of microtubules only at concentrations that blocked regeneration, whereas LCc blocked regeneration without reducing microtubule number. In addition, LCc associated with the cells much more readily than Cc, such that the cell-associated concentration of Cc that blocked regeneration was actually several fold lower than the effective concentration of LCc. We propose that common effects of Cc and LCc unrelated to tubulin binding play no more than a minor role in Cc effects on regeneration and conclude that Cc acts primarily if not exclusively via its antimicrotubule activity.

Motility in the siphonous green alga Bryopsis. II. Chloroplast movement requires organized arrays of both microtubules and actin filaments.

The cortical cytoplasm of the giant cells of Bryopsis contains hundreds of interconnected microtubule (MT) bundles aligned along the cell's long axis. Actin fibers show an extensive but not exclusive superposition with these MT bundles. Chloroplasts move parallel to the bundles. Colchicine (0.5 mM), vinblastine (0.1 mM), and the herbicide ami-prophosmethyl (APM, 1-5 microM) strongly inhibit chloroplast movement and severely disrupt both the MT and the actin network. Additionally, APM leads to the appearance of large actin bundles up to 5 microns in diameter and several tens of micron in length. Erythro-9-[3-(2-hydroxynonyl)]adenine (EHNA, 1 mM) does not block chloroplast movement, but affects chloroplast behavior by causing transient aggregations. The MT network is not significantly changed by EHNA, but actin fibers converge in large, radially symmetric complexes in regions of chloroplast aggregates. Cytochalasin D (CD, 1-10 micrograms/ml) leads to a significant but transient reduction of chloroplast speed within the first 60 min, as the actin network breaks down into small foci. Within the next 1 to 3 h of treatment, these foci segregate into massive clusters where chloroplasts remain immobilized. At the same time, chloroplast movement recovers in other areas of the cell. This recovery coincides with the reappearance of actin filament bundles in these cell regions. The MT cytoskeleton is not significantly affected by CD. These data are inconsistent with a mechanism of chloroplast movement in Bryopsis based solely on either MTs or actin, but instead they suggest an intimate interaction of both cytoskeletal networks in maintaining the spatial organization of the cytoplasm and in supporting chloroplast movement.

Involvement of microtubules in lipoprotein degradation and utilization for steroidogenesis in cultured rat luteal cells.

Cells isolated from superovulated rat ovaries metabolize low density lipoprotein (LDL) and high density lipoprotein (HDL) of human or rat origin and use the lipoprotein-derived cholesterol as a precursor for progesterone production. Under in vitro conditions, both lipoproteins are internalized and degraded in the lysosomes, although degradation of HDL is of lower magnitude than that of LDL. In this report we have examined the role of cellular microtubules in the internalization and degradation of human LDL and HDL in cultured rat luteal cells. The microtubule depolymerizing agents colchicine, podophyllotoxin, vinblastine, and nocodazole as well as taxol, deuterium oxide, and dimethyl sulfoxide, which are known to rapidly polymerize cellular tubulin into microtubules, were used to block the function of microtubules. When these antimicrotubule agents were included in the incubations, degradation of the apolipoproteins of [125I]iodo-LDL and [125I]iodo-HDL by the luteal cells was inhibited by 50-85% compared to untreated control values. Maximum inhibitory effects were observed when the cells were preincubated with the inhibitor for at least 4 h at 37 C before treatment with the labeled lipoprotein. Lipoprotein-stimulated progesterone production by luteal cells was also inhibited by 50% or more in the presence of antimicrotubule agents. However, basal and hCG-stimulated progesterone production were unaffected by these inhibitors. The binding of [125I]iodo-LDL and [125I]iodo-HDL to luteal cell plasma membrane receptors was not affected by the microtubule inhibitors. Although binding was unaffected and degradation was impaired in the presence of the inhibitors, there was no detectable accumulation of undegraded lipoprotein within the cells during the 24 h of study. From this study we conclude that the uptake and utilization of LDL and HDL by cultured rat luteal cells are mediated by cellular microtubules.

Effect of colchicine on drug-induced changes in plasma renin concentration in rats.

Cytoplasmic microtubules appear to play a role in the secretion of a variety of protein and protein hormones. Involvement of microtubules in renin secretion has been hypothesized but not established. The present studies were designed to determine: 1) if the antimicrotubule drug, colchicine, would alter plasma renin concentration (PRC); and 2) if changes in PRC could be related to an effect on cytoplasmic microtubules. Dose response experiments in Sprague-Dawley rats showed that 0.4 or 0.8 mg/kg/day i.p. of colchicine for 3 days significantly increased PRC while a dose of 0.2 mg/kg/day was without effect. The increase in PRC at the higher doses was associated with toxicity of the drug. In other experiments, rats pretreated with colchicine (0.2 mg/kg/day) or saline received either furosemide (5 mg/kg) or isoproterenol (25 micrograms/rat) i.p. to stimulate renin secretion. Colchicine at a dose that did not alter basal PRC significantly inhibited an increase in PRC after stimulation with either isoproterenol or furosemide. Lumicolchicine, a structural isomer of colchicine without antimicrotubule activity, did not alter the response to isoproterenol stimulation. These data suggest that microtubules play a role in the increase in renin secretion following stimulation.

Colchicine acts as a progression factor to initiate DNA synthesis in quiescent Balb/c 3T3 cells.

In quiescent Balb/c 3T3 cells, competence factors such as 12-O-tetradecanoylphorbol-13-acetate (TPA) and platelet-derived growth factor (PDGF) synergize with progression factors such as insulin to initiate DNA synthesis. In this study, we found that colchicine, a microtubule-disrupting agent, acted synergistically with TPA, but not with insulin, to induce the maximal stimulation of DNA synthesis. Colchicine also synergized with PDGF in the presence of epidermal growth factor to elicit nearly the optimal induction of DNA synthesis. Moreover, it acted synergistically with fibroblast growth factor, another competence factor. These results suggest that colchicine acts as a progression factor like insulin in quiescent Balb/c 3T3 cells.

Pharmacological characterization of the slow component of deactivation of guinea-pig isolated ileum to the spasmogenic action of C5adesArg.

The slow component of deactivation of guinea-pig isolated ileum to C5adesArg was studied to analyse the mechanism of loss and subsequent recovery of sensitivity. Neither cycloheximide (10(-3) M) nor colchicine (5 X 10(-5) M), vinblastine, lumicolchicine, or cytochalasin B (each 2 X 10(-5) M) affected significantly the spasmogenic effect of C5adesArg or the course of deactivation produced by repeated applications; chloroquine (2 X 10(-4) M) inhibited the spasmogenic effect unspecifically without interfering with deactivation. Recovery from slow deactivation was totally blocked by chloroquine and considerably diminished by colchicine and vinblastine, but was not affected by the other agents. It is proposed that recovery involves lysosomal processing of C5a receptors (occupied by the peptide) but does not require biosynthesis of new receptors.

Modulation of the hepatic alpha 1-adrenoceptor responsiveness by colchicine: dissociation of free cytosolic Ca(2+)-dependent and independent responses.

1. The cytoskeletal depolymerizing agent, colchicine, prevents the hepatic alpha 1-adrenoceptor-mediated stimulation of respiration, H+ and Ca2+ release to the effluent perfusate, intracellular alkalosis, and glycogenolysis. Unlike the other parameters, colchicine does not perturb the alpha 1-agonist-induced stimulation of gluconeogenesis or phosphorylase 'a' activation, and enhances the increase in portal pressure response. The lack of effect of colchicine on the hepatic alpha 2-adrenoceptor-mediated effects indicates that its actions are alpha 1-specific. 2. Colchicine enhances the acute alpha 1-adrenoceptor-mediated intracellular Ca2+ mobilization and prevents the activation of protein kinase C. This differential effect on the two branches of the alpha 1-adrenoceptor signalling pathway is a distinctive feature of the colchicine action. 3. The lack of effect of colchicine in altering the alpha 1-adrenoceptor ligand binding affinity suggests that it might interact with some receptor-coupled regulatory element(s). 4. The acuteness of the colchicine effect and the ability of its isomer beta-lumicolchicine to prevent all the alpha 1-adrenoceptor-mediated responses but the increase in vascular resistance, indicate that its action cannot be merely ascribed to its effects in depolymerizing tubulin. 5. Colchicine perturbs the hepatic responses to vasoactive peptides. It enhances the vasopressin-induced rise of cytosolic free Ca2+ in isolated hepatocytes and prevents the sustained decrease of Ca2+ in the effluent perfusate. It also inhibits the stimulation of glycogenolysis, without altering the stimulation of gluconeogenesis. 6. It is concluded that there are at least two major alpha 1-adrenoceptor signalling pathways. One is colchicine-sensitive, independent of variations in free cytosolic Ca2+, and protein kinase C-dependent; the other one is colchicine-insensitive, dependent on variations in free cytosolic Ca2+, and protein kinase C-independent.

Involvement of the microtubular system in the endothelin-1 secretion from porcine aortic endothelial cells.

The effects of certain microtubule-disrupting agents on endothelin-1 (ET-1) secretion from porcine aortic endothelial cells were studied. When endothelial cells were treated with thrombin (1 unit/mL), a significant increase in ET-1 secretion was detected in the incubation medium, while ET-1 secretion in the medium was diminished when the cells were treated simultaneously with either colchicine or vinblastine (10(-8)-10(-6) M). In such cases, however, the ET-1 content detected in the cells increased dose-dependently in accordance with the concentrations of the microtubule-disrupting agents. The intracellular accumulation of ET-1 was observed both in mitochondrial and microsomal fractions. On the other hand, thrombin produced a significant increase in polymerized tubulin content without affecting the total tubulin content. A thrombin-induced increase in the intracellular Ca2+ concentration of endothelial cells was inhibited by treatment with either colchicine or vinblastine. These results seem to indicate that the microtubular system may play an important role in ET-1 secretion from endothelial cells.

The effects of colchicine or vinblastine on the blood calcium level in rats.

In order to clarify how microtubule inhibitors induce hypocalcemia, rats were injected with intravenous colchicine (1 mg/kg) or vinblastine (2 mg/kg). The blood calcium levels decreased rapidly, and the minimum values, reached 4 h after the injection, were 7.55 +/- 0.70 mg/100 ml (mean +/- S.D., P < 0.001) for colchicine and 7.61 +/- 0.17 mg/100 ml (P < 0.001) for vinblastine. At 24 h, these values returned to the normal range (10.13 +/- 0.42 mg/100 ml). The blood calcium values in rats fed a low calcium diet and in thyroparathyroidectomized rats were also reduced by colchicine. The incorporation of blood 45Ca into bone was reduced by the injection of colchicine. Histologically, the bone cells of rats injected with either drug were severely damaged 8 h after the injection. These results indicate that hypocalcemia may be mediated by interference with the regulatory mechanisms of bone cell calcium homeostasis, and that the destruction of microtubules may be closely related to the development of the hypocalcemia.

Colchicine injection in the inferior olivary nucleus increases the number of Purkinje cell dendritic spines.

The number of Purkinje cell dendritic spines was evaluated in the cerebellum of rats after injection of colchicine into the inferior olivary nucleus. In these conditions, the spines situated in the proximal (inner) part of the molecular layer were increased as compared to lumicolchicine-injected or non-injected control animals. Most postsynaptic targets for climbing fibers are located in the inner molecular layer and the fact that spines in this region increased when axonal transport was blocked in the climbing fibers suggests that the latter play a role in the control of spine formation.

Microtubule disruption and cognitive defects: effect of colchicine on learning behavior in rats.

Neuropathological findings in Alzheimer's disease (AD) suggest a possible involvement of microtubule dysfunction in neurodegenerative process pathogenesis. Because microtubules have a major role in neuronal plasticity, microtubule disruption could be also directly responsible for cognitive defects in AD. We report that in rats, continuous microtubule disruption induced by chronic colchicine administration results in a dose-dependent learning deficit. In addition, retention is also impaired. These cognitive defects are specific, as chronic colchicine induces no other behavioral toxicity within the study dose range. Colchicine-induced cognitive defects resemble those of AD, which are characterised by amnesia of recent learning and loss of formerly established memories. This new procedure of pharmacologically induced cognitive impairment may prove useful, both towards understanding AD pathogenesis and towards drug screening.

The effects of colchicine on prostaglandin I2 and thromboxane A2 biosynthesis in the rat dental pulp.

Pulp tissues isolated from rats treated with either colchicine (0.5, 0.75 or 1.0 mg/kg) or beta-lumicolchicine (1.0 mg/kg) for 24 h were incubated in Tyrode buffer. Prostaglandin (PG) I2 and thromboxane (TX) A2 released into the medium were determined by radioimmunoassay as their stable metabolites, 6-keto-PGF1 alpha and TXB2, respectively. Colchicine generally enhanced PGI2 and TXA2 production concomitantly with hypocalcaemia, except that 0.5 mg/kg dosage had no significant effects. This enhancement was greater in PGI2 than in TXA2 synthesis, but was not observed in PGI2 release from aortic tissue. beta-Lumicolchicine (1.0 mg/kg) had no effect on PG and TX synthesis, nor on serum calcium. Kinetic studies revealed that after a lag period of more than 12 h, 1.0 mg/kg colchicine caused a progressive increase in the production of PGI2 and TXA2, which reached a maximum at 24 h and decreased thereafter gradually to the basal level within 168 h (7 days). In contrast, the hypocalcaemic effect of colchicine appeared rapidly, reached a maximum within 3 h and gradually recovered to the control level within 7 days. Colchicine at concentrations of 1-1000 microM had no stimulatory effect on PGI2 and TXA2 biosynthesis when incubated in vitro with isolated pulp tissues; it was inhibitory at higher concentrations. The enhancing effect of colchicine treatment on PGI2 and TXA2 production in dental pulp tissue may be caused indirectly by interaction of this agent with microtubules, but the precise mechanisms are unclear. Thus, a drug known to affect dentinogenesis modifies arachidonic-acid metabolism in dental pulp.

Comparison of the effect of colchicine and vinblastine on the inhibiton of dentinogenesis in rat incisors.

The quantitative effect of vinblastine, colchicine and lumicolchicine on the rate of dentine formation was determined by chronologically-labelling dentine with the lead salt of EDTA, before and after the intravenous administration of the test drug. Colchicine and vinblastine produced a dose-dependent inhibition of dentine formation. Lumicolchicine had no adverse effect. Although the log dose-response lines of colchicine and vinblastine were parallel, the effect of vinblastine was more potent. The relative potency ratio of vinblastine to colchicine was 1.25-1.70. These results suggest that both colchicine and vinblastine may act at the same site i.e. microtubules, thereby inhibiting the secretion of new dentine matrix.