Inhibitor Colchicine Research Articles

Modification of cell development in vitro: The effect of colchicine on anther and isolated microspore culture in Brassica napus

In an attempt to discover the biological basis of microspore derived embryogenesis, the effect of the antimicrotubule agent colchicine on anther and free microspore embryogenesis was investigated. The microtubule inhibitor colchicine promoted embryogenesis from cultured anthers, both with regard to the number of anthers responding and the number of embryos being produced per anther. A similar promotional response was also observed with cultured microspores. Although the parameters for cultured anthers and free microspores differed, administration of the drug for a short period immediately prior to pollen mitosis I seems to exert the maximum promotional effect. Of the five cultivars of Brassica napus studied, all responded to colchicine treatment. However, the drug did release more embryogenic potential in poor-responding varieties (i.e. Lirawell and Optima) than in the highest responding variety (Topas). Colchicine also resulted in increased embryogenic response in microspores cultured at lower temperatures.

Characterization of moesin in the sea urchin Lytechinus variegatus: redistribution to the plasma membrane following fertilization is inhibited by cytochalasin B

We have investigated the distribution and function of an ezrin-radixin-moesin-like (ERM) molecule in the sea urchin. A sea urchin homologue of moesin was cloned that shares 75% amino acid similarity in the conserved N-terminal region to other moesin molecules. A 6.3 kb message is transcribed late in embryogenesis and is present in adult tissues. Polyclonal antibodies were generated to proteins expressed by a bacterial expression vector, and affinity purified. These antibodies recognize a single 75 kDa protein that is present throughout development in approximately equal abundance, and specifically they immuno-precipitate a single protein. We show by immunolocalization that SUmoesin has two predominant patterns during development. First, SUmoesin is rapidly redistributed after fertilization from a location throughout the egg cytoplasm to a location in the egg cortex. Later in embryogenesis, SUmoesin is localized to the apical ends of cells in the regions of cell-cell junctions. We show that SUmoesin is present in actin-rich regions of the embryo. Finally, we show that the location of SUmoesin requires an intact actin-based cytoskeleton. SUmoesin fails to localize to the plasma membrane after fertilization in the presence of cytochalasin B. Furthermore, SUmoesin loses its apical position in the region of cell-cell junctions in the presence of cytochalasin B in later stages of embryogenesis. This effect is reversible, and the microtubule inhibitor colchicine has no effect. These results show that SUmoesin becomes associated with apical plasma membrane structures early in development, and that SUmoesin is both coincident with actin and requires the assembly of actin filaments to maintain its localization.

Vesicles and mixed micelles in hypothyroid rat bile before and after thyroid hormone treatment: evidence for a vesicle transport system for biliary cholesterol secretion.

Hypothyroid rats show reduced secretion of biliary lipids, especially cholesterol. Secretion of biliary cholesterol is markedly augmented to levels above euthryroid beginning 12-24 h after administration of thyroid hormone. In the current studies, bile from hypothyroid and triiodothyronine-treated chronic bile-fistula rats was analyzed for vesicles and mixed micelles by metrizamide gradient ultracentrifugation. For euthryoid and hypothyroid animals, less than 12% of biliary cholesterol was in a vesicle gradient fraction. After treatment with triiodothyronine, biliary cholesterol increased markedly, and 50% of total cholesterol, 60% of excess cholesterol secreted, appeared in the vesicle fraction. Triiodothyronine stimulation of vesicle secretion resulted in cholesterol-rich vesicles (cholesterol:phospholipid ratio rose from less than 0.1 to 0.56), but no change in the distinct fatty acid composition of vesicle phospholipids. The microtubule inhibitor colchicine, given 12 h after triiodothyronine, prevented subsequent increase in cholesterol secretion in the form of vesicles. These studies, in a model that allows rapid changes in biliary lipid secretion, support the hypothesis that an important component of cholesterol and phospholipid secretion into bile involves microtubules and may involve a vesicle pathway.

Microtubules are involved in maintaining the cellular polarity in pollen tubes ofNicotiana sylvestris

The polarity of a growing pollen tube is clearly reflected by a distinct zonation of the cytoplasmic content. The vegetative nucleus and the generative cell (GC) are located in the tip region of the tube, and the basal cytoplasmic portion is highly vacuolated. Using pollen tubes ofNicotiana sylvestris Spegazz. & Comes grown in vitro, we examined the effects of varying concentrations of the microtubule inhibitors colchicine and propham. The depolymerization of the cortical microtubules by 25 μM colchicine led to a disorganization of the cytoplasm, i.e., vacuolization of the tip region, and to a deranged position of both the vegetative nucleus and the generative cell. The same concentration of colchicine inhibited tube growth by 10–20% of the control. Mitosis of the GC was not affected. Only from concentrations of 200 μM the configuration of the GC's microtubules was altered and an inhibition of mitosis was observed. At this concentration the disorganization of the cytoplasm was always reversible, but neither inhibition of mitosis nor derangement of the nuclear positioning was. At 1,800 μM colchicine, pollen tube growth was inhibited by 50% of the control. Using propham, the same three steps of action were observed, although propham proved to be about a hundred times more effective than colchicine. We conclude that the cortical microtubules of the pollen tube are involved in maintaining cellular polarity, probably as a part of a heterogeneous cytoskeletal network including also microfilaments and membranous elements. Nuclear positioning seems to be dependent on both, the tube's cortical and the GC's microtubules. A possible involvement of the extracellular matrix in maintaining intracytoplasmic polarity is suggested.

Pathways of intracellular trafficking and release of ferritin by the liver in vivo: The effect of chloroquine and cytochalasin D

We have previously shown that the clearance of exogenous ferritin and the release of endogenous ferritin into both serum and bile are altered by the microtubular inhibitor colchicine. In this study we further examined the role of the lysosome-endosome pathway in ferritin metabolism. We examined the effect of the lysosomotropic agent chloroquine and the microtubular inhibitor cytochalasin D on the uptake and release of ferritin by normal and iron-loaded rats under basal conditions and in the presence of an exogenous tissue ferritin load. Either chloroquine (50 mg/kg body wt) or cytochalasin D (0.9 microgram/100 gm body wt/min) was administered to normal and iron-loaded rats at zero time. Rats were also infused with either saline solution or rat liver ferritin containing a trace amount of 125I-ferritin. The clearance of 125I-ferritin from the circulation was not affected by chloroquine or cytochalasin D either in normal or in iron-loaded rats; however, both chloroquine and cytochalasin D decreased the serum ferritin concentration in normal rats to 39% +/- 9% and 22% +/- 7% of the baseline serum ferritin levels, respectively, implying that both drugs inhibited the release of endogenous ferritin in normal rats. In iron-loaded rats both chloroquine and cytochalasin D decreased the biliary ferritin concentration to 11% +/- 1% and 37% +/- 4% of the baseline ferritin levels, respectively, and the 125I protein-bound counts per minute in the bile to 50% of the control result. This finding is consistent with an inhibitory effect of both drugs on the biliary excretion of endogenous ferritin and the intracellular transport of exogenous ferritin, respectively. In the presence of an exogenous tissue ferritin load, there was no detectable inhibitory effect of either drug on the biliary excretion of either endogenous or exogenous ferritin. These results provide the following evidence: (a) the receptor-mediated endocytosis of ferritin is not dependent on functioning lysosomes or microfilaments; (b) the release of endogenous ferritin into the serum of normal rats and the bile of iron-loaded rats is a chloroquine-sensitive, microfilament-dependent process; (c) the biliary excretion of trace amounts of exogenous ferritin is dependent on both chloroquine-sensitive vesicles and microfilaments; and (d) increased levels of exogenous ferritin are excreted directly into the bile by way of a second microfilament-independent, chloroquine-insensitive pathway. This study provides support for a physiological mechanism for the release of ferritin from the liver.

Cell adhesion mediated by CD4 and MHC class II proteins requires active cellular processes.

Human CD4 is an accessory molecule found on the cell surface of a subset of T lymphocytes. We have previously shown by infection of simian fibroblasts with an SV40-CD4 recombinant virus that CD4 acts as an adhesion molecule by binding to human MHC class II Ag expressed on the surface of human B lymphocytes. This report confirms that human B lymphoblastoid cell lines expressing class II molecules at the cell surface can bind to Chinese hamster ovary cells that have been stably transfected with human CD4. This cellular adhesion is a late event, which is first detected after 2 h, but remains stable for up to 16 h. The association between CD4 and class II is energy dependent, as it is detected at 37 degrees C, but not at 4 degrees C or if either cell type is fixed with paraformaldehyde. ATP is required for the establishment and maintenance of stable CD4/class II-mediated cell conjugates. Cytoskeletal interactions also regulate CD4/class II adhesion as treatment with the microtubule and microfilament inhibitors colchicine, cytochalasin-D, and nocodazole rapidly dissociates even preformed cell conjugates. Our observations also indicate that the Ag-independent engagement of class II by CD4 induces homotypic clustering of human B cells. This effect is blocked by reagents directed against lymphocyte function-associated Ag-1 and may result from the induction of a high affinity phenotype of lymphocyte function-associated Ag-1 induced by class II signaling. Finally, we discuss the implications of CD4/class II-mediated adhesion and the role of CD4 in the regulation of T cell adhesion.

Spinule formulation in the fish retina: is there an involvement of actin and tubulin? An electronmicroscopic immunogold study.

During light-adaptation dendrites of teleost horizontal cells form finger-like processes, called 'spinules', which are characterized by synaptic membrane densities. To investigate the involvement of cytoskeletal elements in the formation and retraction of spinules, effects of the microtubule and actin inhibitors colchicine and cytochalasin D were examined by injection into the vitreous. Both substances inhibited the light-induced spinule formation. The ultrastructural immunolocalization of tubulin revealed labelling of dendrites only in their proximal parts. The distal parts of dendrites which invaginate into cone pedicles were free of label. Treatment with anti-actin revealed immunoreactivity along the entire length of dendrites up to the dendritic terminals. The spinules, however, showed no labelling. This finding does not support the hypothesis that spinules are protruded by actin polymerization. After cytochalasin D treatment the density of label in the dendritic terminals was enhanced by a factor of three, which suggests an accumulation of actin. Thus, spinule inhibition by cytochalasin D is probably caused by distortion of a functional actin network in the dendritic terminals.

Distribution of the surface antigen HAM-4 and cytoskeleton during reformation of bile-canalicular structures in rat primary cultured hepatocytes

The distribution of rat bile-canalicular surface antigen (HAM-4 antigen) and cytoskeletal elements (microtubules, actin filaments, and cytokeratin filaments) was examined during the reformation of bile-canalicular structures (BC-structures) in primary cultures of dissociated hepatocytes obtained following collagenase perfusion. HAM-4 antigen, which initially dispersed after cell dissociation, became focused into regions of cell-to-cell contact even before formation of BC-structures. Typical bile-canalicular microvilli also appeared in these regions before the intercellular spaces were completely closed. Finally, after in vitro reformation of BC, HAM-4 antigen was localized specifically at the BC-surface. The process of BC-reformation and the intracellular organization of actin and cytokeratin filaments were not significantly affected by microtubule inhibitors (nocodazole, colcemid, and colchicine). However, the localization of HAM-4 antigen molecules at the surface of BC was disrupted by these inhibitors, suggesting that the distribution of HAM-4 antigen, which represents a marker for the reconstruction of surface polarity, is dependent on microtubule function.

Acute ozone-induced change in airway permeability: role of infiltrating leukocytes

The role of infiltrating polymorphonuclear leukocytes (PMNs) in acute lung injury and inflammation is still controversial. In inbred mice, acute ozone (O3) exposure induces airway inflammation that is characterized by a maximal influx of lavageable PMNs 6 h after exposure and a maximal increase in lung permeability 24 h after O3. We tested the hypothesis that O3-induced change in airway epithelial permeability of O3-susceptible C57BL/6J mice is due to infiltrating PMNs. Male mice (6-8 wk) were treated with a nonsteroidal anti-inflammatory drug (indomethacin), a chemotactic inhibitor (colchicine), or an immunosuppressant (cyclophosphamide) to deplete or inhibit PMNs from infiltrating the airways. After drug or vehicle treatment, mice were exposed for 3 h to 2 ppm O3 or filtered air, and pulmonary inflammation was assessed by inflammatory cell counts and total protein content (a marker of airway permeability) in bronchoalveolar lavage (BAL) fluid. Filtered air exposure did not affect the parameters of pulmonary inflammation at any time after exposure. Compared with vehicle controls, each of the drug treatments resulted in significant reduction of PMN influx 6 and 24 h after O3. However, total BAL protein content was not attenuated significantly by the three treatments at either 6 or 24 h postexposure. Results of these experiments suggest that the influx of PMNs and the change in total BAL protein are not mutually dependent events in this model and suggest that infiltrating PMNs do not play a major role in acute O3-induced changes in permeability of the murine lung.

Induction of apoptosis in chronic lymphocytic leukemia cells and its prevention by phorbol ester

Chronic lymphocytic leukemia lymphocytes were used to study mechanisms involved in apoptosis (programmed cell death). Apoptosis, which was determined by morphological changes including cell death and by internucleosomal DNA fragmentation, occurred during culture for 1 to 2 days in a portion of the cells from three of the four patients tested. Most of the cells under-went apoptosis and DNA fragmentation was greatly enhanced when cells were cultured in the presence of the microtubule inhibitor colchicine, the topoisomerase II inhibitor etoposide, or the glucocorticoid methylpredni-solone. Tumor-promoting phorbol esters inhibited spontaneous DNA fragmentation and cell death including that induced by colchicine, etoposide, and methylprednisolone, indicating that they act on an event common to apoptosis caused by diverse stimuli. Phorbol esters probably act through protein phosphorylation, since they were effective at concentrations which modulated protein kinase C (PKC) and their action was prevented by H-7, which binds to and inactivates the catalytic site of PKC. In the absence of phorbol ester, H-7 itself induced some apoptosis. These findings implicate PKC in the suppression of apoptosis, but its precise role requires systematic investigation.

Expanded distribution of mRNA for nerve growth factor, brain-derived neurotrophic factor, and neurotrophin 3 in the rat brain after colchicine treatment.

The effect of intracerebroventricular injection of the mitosis inhibitor colchicine on expression of mRNA for nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin 3 was studied in the rat brain with in situ hybridization. Colchicine up-regulates mRNA for NGF and BDNF in many of the neuronal systems normally expressing these factors. In addition, after colchicine treatment NGF and BDNF mRNAs were localized in several brain areas where they normally cannot be detected. Thus, NGF mRNA was present, for example, in many motor nuclei and in the basal forebrain, and BDNF mRNA was seen in many nuclei in the brain stem and in catecholamine neurons, including dopamine neurons in the substantia nigra. The latter neurons have recently been shown to be sensitive to BDNF, and the present results show that these neurons can produce this factor themselves. A decrease in mRNA for BDNF and neurotrophin 3 was seen only in the granular-cell layer of the hippocampal formation. A strong hybridization signal for BDNF and neurotrophin 3 mRNA was also observed over several myelinated tracts in treated rats, supporting the hypothesis that glial cells as well as neurons can produce these trophic factors.

Biliary excretion of polyethylene glycol molecular weight 900: Evidence for a bile salt-stimulated vesicular transport mechanism

Polyethylene glycol molecular weight 900 (PEG-900) has been used as a marker of vectorial water transport into bile canaliculus. However, the mechanisms by which this compound is excreted have not been clarified. To gain more information on this process, we studied the biliary excretion of [ 3H]PEG-900 in rats during choleresis induced by canalicular choleretics. In addition, the effects of the mutubule inhibitors colchicine and vinblastine, and of the acidotroplc agent chloroquine, on PEG-900 excretion were studied to determine whether a vesicular pathway is involved. Continuous i.v. infusion of either dehydrocholate (DHC, a non-micelle forming bile salt choleretic) or 4-methylumbelliferone (4-MU, a non-bile salt canalicular choleretic) at stepwise-increasing rates [0.7, 1.0 and 1.2 μmol·min −1d(100g body wt) −1] induced a gradual increment in bile flow, whereas a transient increment of [ 3H]PEG-900 excretion was observed only during DHC-induced choleresis. Furthermore, studies in which two consecutive i.v. injections of DHC (10 smmol/100 g body wt) were administered showed that [ 3H]PEG-900 excretion induced by a second administration of DHC was 54% lower than that induced by the first one, despite a similar excretion in bile flow. Finally, colchicine (0.5 mol/100 g body wt), vinblastine (0.5 mol/100 g body wt) and chloroquine (50 mg/kg body wt) pretreatments inhibited the DHC-induced increment in biliary [ 3H]PEG-900 output, while DHC-induced choleresis was almost unaffected. Conversely, excretion of [ 14C]sucrose, when coadministered with [ 3H]PEG-900, was not impaired by the treatments. These results suggest that, unlike sucrose, PEG-900 excretion is not associated with canalicular water movements. Instead, it may be related to a vesicular transport process followed by a bile acid-stimulated discharge of secretory vesicles into bile through the lysosomal compartment.

Protein Kinase C (αand β) Immunoreactivity in Rabbit and Rat Retina: Effect of Phorbol Esters and Transmitter Agonists on Immunoreactivity and the Translocation of the Enzyme from Cytosolic to Membrane Compartments

Using a monoclonal antibody against protein kinase C (PKC) that recognises the isoenzymes alpha, beta I, and beta II, positive immunoreactivity was observed throughout the cytoplasm of bipolar cells in both rat and rabbit retinas. PKC immunoreactivity was also associated with the outer segment of photoreceptors in the rabbit retina and presumed amacrine cells in the rat retina. The PKC immunoreactivity in the retina was unaffected in content or localisation in rats kept in continuous dark or light conditions over a period of 6 days. The localisation of PKC immunoreactivity in retinas was similar in 6-day-old, 16 day-old, or adult rabbits. However, the content of PKC was lowest at the youngest stage and highest in the adult rabbit retinas. Of the two active phorbol esters studied, only phorbol 12,13-dibutyrate (PDbut) at a concentration of 1 microM caused the PKC immunoreactivity in rabbit retina bipolar cells to be "transported" from the perikarya towards the axonal terminal processes. Biochemical analyses showed that most of the cytosolic PKC was translocated to the membrane compartment following such treatment. The other phorbol ester, phorbol 12-myristate 13-acetate, even at a concentration of 10 microM did not cause a similar transport of PKC immunoreactivity in the bipolar cells, although a partial translocation of the enzyme could be followed biochemically. Both the translocation and transport of PKC by PDbut could be reversed by simply incubating the retinas in physiological solution for 60 min. The "transport" and translocation processes were not obviously affected by the transport inhibitor colchicine or by known PKC inhibitor such as staurosporine, H-7, sphingosine, or polymyxin B. In addition, agonists known to stimulate inositol phosphates in the retina, viz., carbachol, noradrenaline, and quisqualate, or 4-aminopyridine did not cause a translocation or "transport" of PKC as observed for the phorbol esters.

Effect of antireceptor antibodies in dilated cardiomyopathy on the cycling of cardiac beta receptors

A substantial proportion of patients with dilated cardiomyopathy have circulating autoantibodies directed againts the cardiac β-adrenoceptor. These antireceptor antibodies inhibit both ligand binding to membrane β-receptors and isoproterenol-sensitive adenylate cyclase. The functional consequences of antibody-receptor interactions were further studied by examining their influence on β-adrenoceptor cycling. Sera from eight patients with cardiomyopathy induced a loss of β-receptors from the surface of cardiac myocytes consistent with internalization. This loss was inhibited by concanavalin A, suggesting that receptor sequestration preceded internalization but was unaffected by the cytoskeleton inhibitors colchicine and cytochalasin. In cell-free preparations, serum-induced desensitization of β-receptors was prevented by heparin but not the inhibitor of protein kinase A; this is consistent with a requirement for receptor phosphorylation by the β-receptor kinase. In contrast to isoproterenol-mediated endocytosis, β-receptors internalized under the influence of cardiomyopathic sera do not recycle to the plasma membrane. These results indicate that antireceptor antibodies in human dilated cardiomyopathy induce downregulation by interfering at several steps in the cycling of β-receptors. These effects would dontribute to the reported decline in β-receptor responsiveness in cardiomyopathic myocardium.

Effect of reserpine and colchicine on neuropeptide mRNA levels in the rat hypothalamic paraventricular nucleus

Using in situ hybridization and immunohistochemistry, we have studied mRNA and peptide levels in the hypothalamic paraventricular nucleus (PVN) 24 h after a single large dose of reserpine (10 mg/kg, i.p.) and 24 h after an intraventricular (i.c.v.) injection of colchicine (120 μl/20 μl saline). Sections of the PVN were hybridized using synthetic oligonucleotide probes complementary to mRNA for corticotropin-releasing hormone (CRH), neurotensin (NT), enkephalin (ENK), vasoactive intestinal polypeptide (VIP) and thyrotropin-releasing hormone (TRH). For immunohistochemistry rabbit antisera to CRH, NT, ENK, VIP and TRH were used. In situ hybridization showed a clear increase in CRH mRNA as compared to control rats after both treatments. Also NT and VIP mRNA could be seen in parvocellular neurons in reserpine and in colchicine-treated rats, whereas we so far have not been able to demonstrate these mRNAs in untreated rats. No changes in TRH mRNA could be detected after reserpine or colchicine. These results provide final evidence that subpopulations of parvocellular PVN neurons can synthesize not only CRH and ENK, but also NT and VIP, in agreement with earlier immunohistochemical results. With immunohistochemistry, after reserpine, many CRH-, but no NT- or VIP-positive neurons could be observed in the parvocellular part of the PVN. The present results demonstrate that treatment with two drugs, the monoamine depleting drug reserpine and the mitosis inhibitor colchicine, causes increased levels of mRNA for several peptides in neurons of the PVN, located almost exclusively in its parvocellular part and being part of the hypothalamo-pituitary adrenal axis.

Identification of a protein altered in mutants resistant to microtubule inhibitors as a member of the major heat shock protein (hsp70) family.

A major cellular protein (P2; approximately 70 kilodaltons) which is altered in Chinese hamster ovary (CHO) cell mutants resistant to the microtubule inhibitors colchicine and podophyllotoxin has been shown to correspond to the constitutive form of the 70-kilodalton heat shock protein (hsc70). The inference that P2 and hsc70 are the same protein is based on the following observations: (i) migration of P2 in two-dimensional polyacrylamide gels in the same position as that reported for hsc70; (ii) cross-reactivity of a monoclonal antibody which reacts with both the constitutive and induced forms of hsp70 with the P2 spot from wild-type CHO cells and with both P2 and a mutant form of P2 in a CHO cell mutant; (iii) specific reactivity of a polyclonal antibody to P2 with both the constitutive and heat-induced forms of hsp70 in human cells; (iv) identical immunofluorescent staining of dot/patchlike structures with both P2 and hsp70 antibodies in human and CHO cells; and (v) a cDNA clone for hsc70 has been isolated and sequenced from wild-type CHO cells. The in vitro transcription and translation product of this cDNA has been shown to comigrate with the P2 protein spot in two-dimensional gels, indicating their identity. The fact that there is an alteration in hsc70 in mutants resistant to antimitotic drugs suggests a role for this protein in the in vivo assembly and function of microtubules.

Identification of a Protein Altered in Mutants Resistant to Microtubule Inhibitors as a Member of the Major Heat Shock Protein (hsp70) Family

A major cellular protein (P2; approximately 70 kilodaltons) which is altered in Chinese hamster ovary (CHO) cell mutants resistant to the microtubule inhibitors colchicine and podophyllotoxin has been shown to correspond to the constitutive form of the 70-kilodalton heat shock protein (hsc70). The inference that P2 and hsc70 are the same protein is based on the following observations: (i) migration of P2 in two-dimensional polyacrylamide gels in the same position as that reported for hsc70; (ii) cross-reactivity of a monoclonal antibody which reacts with both the constitutive and induced forms of hsp70 with the P2 spot from wild-type CHO cells and with both P2 and a mutant form of P2 in a CHO cell mutant; (iii) specific reactivity of a polyclonal antibody to P2 with both the constitutive and heat-induced forms of hsp70 in human cells; (iv) identical immunofluorescent staining of dot/patchlike structures with both P2 and hsp70 antibodies in human and CHO cells; and (v) a cDNA clone for hsc70 has been isolated and sequenced from wild-type CHO cells. The in vitro transcription and translation product of this cDNA has been shown to comigrate with the P2 protein spot in two-dimensional gels, indicating their identity. The fact that there is an alteration in hsc70 in mutants resistant to antimitotic drugs suggests a role for this protein in the in vivo assembly and function of microtubules.

Biosynthesis of the 25-kDa protein in the macrogametes/zygotes of Plasmodium falciparum

Synthesis of the 25-kDa protein in the early midgut stages of Plasmodium falciparum was studied, using metabolic inhibitors (colchicine and actinomycin D) and pulse-labeling experiments. Experiments with colchicine showed that, immediately after induction of macrogametogenesis, 25-kDa protein synthesis occurs in both fertilized and nonfertilized macrogametes. The amount of 25-kDa protein synthesized increased slowly during time. Experiments with actinomycin D revealed that the slow increase of synthesis may be dependent on de novo messenger RNA synthesis.

Effect of Cholesterol Transport Inhibitors on Steroidogenesis and Plasma Membrane Cholesterol Transport in Cultured MA-10 Ley dig Tumor Cells*

These studies were directed toward understanding the cellular actions of inhibitor drugs that affect steroidogenesis and cholesterol transport. We investigated the microfilament inhibitor cytochalasin-D, the microtubule inhibitor colchicine, the calmodulin antagonist trifluoperazine, and the inhibitor of acidic vesicle function nigericin. We found that all of these compounds caused dose-dependent inhibition of progesterone synthesis in the MA-10 cells. Each compound also inhibited (Bu)2cAMP-stimulated pregnenolone synthesis, indicating that each inhibited a fundamental process required for steroidogenesis. Each compound was next evaluated for inhibitory actions on cholesterol transport to and from the plasma membrane. On the basis of inhibitor sensitivity, two different categories of cholesterol transport were defined. Transport of newly synthesized or low density lipoprotein-derived cholesterol from the cell interior to the plasma membrane was inhibitor insensitive. Plasma membrane cholesterol internalization, however, was sensitive to all of the inhibitors and did not result because of any drug effect on the acyl-coenzyme-A-cholesterol acyl transferase. Cycling of cholesteryl ester-derived cholesterol through the plasma membrane appeared to occur before its use for steroidogenesis. Thus, inhibition of plasma membrane internalization would prevent utilization of both plasma membrane cholesterol and cholesteryl ester-derived cholesterol, the two major substrate sources for steroid hormone synthesis. Consistent with this interpretation was the finding that inhibition of plasma membrane cholesterol internalization by each inhibitor paralleled the inhibitor's effect on steroidogenesis.

Effect of colchicine on the clearance of ferritin in vivo

The effect of the microtubular inhibitor colchicine, administered at either 0.036 (dose 1) or 5 mumol/kg (dose 2), on hepatic ferritin uptake was determined over a 5-h period of ferritin infusion in normal and iron-loaded rats. In the absence of colchicine, the serum ferritin concentration of normal rats was reduced after 5 h to 20 +/- 9% and in iron-loaded rats to 30 +/- 1% of the expected values, indicating ferritin clearance. After colchicine (dose 1), a similar result was observed in normal rats; however, in iron-loaded rats, an increase in serum ferritin to 77 +/- 16% of the expected value indicated a decreased ferritin clearance and/or increased ferritin release. The administration of the higher dose to normal rats also resulted in an increase in serum ferritin to 72 +/- 2% of the expected value. In iron-loaded rats, after dose 2 the ferritin concentration rose to 359 +/- 108% of the expected value, consistent with the release of endogenous ferritin. Colchicine administration had no effect on biliary ferritin in normal rats; however, in iron-loaded rats, both doses resulted in a fivefold increase in the release of biliary ferritin. The results suggest that ferritin uptake is facilitated by receptor-mediated endocytosis, which is partially inhibited by colchicine. Release of ferritin also occurs as a result of colchicine administration, and this is dependent on both the dose of colchicine and the iron status of the animal.