Effect of ergot alkaloids on 3H-flunitrazepam binding to mouse brain GABAA receptors

In vitro effects of dihydroergotoxine, dihydroergosine, dihydroergotamine, alpha-dihydroergocriptine (ergot alkaloids), diazepam, methyl-beta-Carboline-3-carboxilate (beta-CCM), flumazenil (benzodiazepines), gamma-amino butyric acid (GABA) and thiopental (barbiturate) were studied on mouse brain (cerebrum minus cerebral cortex) benzodiazepine binding sites labeled with 3H-flunitrazepam. Specific, high affinity (affinity constant, Kd = 57.7 8.6 nM) binding sites for 3H-flunitrazepam on mouse brain membranes were identified. All benzodiazepine drugs inhibited 3H-flunitrazepam binding with nanomolar potencies. In contrast to benzodiazepines, all ergot drugs, GABA and thiopental produced an enhancement of 3H-flunitrazepam binding to its binding site at the GABAA receptor of the mouse brain. The rank order of potency was: neurotransmitter (GABA) > dihydroergotoxine > thiopental > alpha-dihydroergocriptine > dihydroergosine > dihydroergotamine. The results suggest that dihydrogenated ergot derivatives do not bind to the brain benzodiazepine binding sites labeled with 3H-flunitrazepam. However, an enhancement of 3H-flunitrazepam binding by all ergot drugs tested, clearly identifies an allosteric interaction with the benzodiazepine binding sites of GABAA receptors.     

Downregulation and subcellular redistribution of the γ-aminobutyric acidA receptor induced by tunicamycin in cultured brain neurons

The significance of N-linked glycosylation and oligosaccharide processing was examined for the expression of γ-aminobutyric acid_A receptor (GABA_AR) in cultured neurons derived from chick embryo brains. Incubation of cultures with 5 μg/ml of tunicamycin for 24 h blocked the binding of ³H-flunitrazepam and ³H-muscimol, probes for the benzodiazepine and GABA sites on the receptor, by about 20% and 28%, respectively. The loss of ligand binding was due to a reduction in the number of binding sites with no significant changes in receptor affinity. Light microscopic immunocytochemistry also revealed that the treatment reduced approximately 13% of the intensity of GABA_AR immunoreactivity in the neuronal somata. Furthermore, the fraction of intracellular receptors was decreased to 24% from 34% of control in the presence of the agent, as revealed by trypsinization of cells in situ followed by ³H-flunitrazepam binding. The molecular weight of the receptor subunit protein was lowered around 0.5 kDa after tunicamycin treatment, in accordance with that following N-glycosidase F digestion, indicating the blockade of N-linked glycosylation of GABA_AR by tunicamycin. Moreover, intense inhibitions of 91% and 44%, respectively, were detected to the general galactosylation and mannosylation in the tunicamycin-treated cells, whereas the protein synthesis was hindered by 13%, through assaying the incorporation of ³H-sugars and ³H-leucine. Nevertheless, treatment with castanospermine or swainsonine (10 μg/ml, 24 h), inhibitors to maturation of oligosaccharides, failed to produce significant changes in the ligand binding. In addition, in situ hybridization analysis showed that these three inhibitors did not perturb the mRNA of GABA_AR α₁-subunit. The data suggest that tunicamycin causes the downregulation and subcellular redistribution of GABA_AR by producing irregularly glycosylated receptors and modifying their localization. Both galactosylation and mannosylation during the process of N-linked glycosylation may be important for the functional expression and intracellular transport of GABA_AR. J. Cell. Biochem. 70:38–48, 1998. © 1998 Wiley-Liss, Inc.     

Agonist-Dependent Internalization of γ-Aminobutyric AcidA/Benzodiazepine Receptors in Chick Cortical Neurons

An impermeant benzodiazepine receptor ligand was prepared by derivatization of the aminobenzodiazepine 1012-S with 4-sulfophenylisothiocyanate. The resulting N-(4-sulfophenyl)-thiocarbamoyl derivative of 1012-S (SPTC-1012S) was purified by reverse-phase HPLC, and the predicted structure was verified by mass spectrometry. The apparent affinity of SPTC-1012S (IC50 = 9.8 +/- 2.9 nM) for displacement of [3H]flunitrazepam from intact chick cortical neurons was similar to that of 1012-S (IC50 = 4.0 +/- 0.3 nM). However, at concentrations from 0.1 to 10 microM, 1012-S was consistently more efficacious than SPTC-1012S, a finding indicating that 6-8% of the benzodiazepine receptor pool was not accessible to the impermeant compound. This inaccessible pool was eliminated by permeabilization of the cells with saponin or Triton X-100, a result suggesting that approximately 7% of neuronal benzodiazepine receptors are intracellular. Acute treatment (1-4 h at 37 degrees C) of neurons with 100 microM gamma-aminobutyric acid (GABA) or 100 nM clonazepam had little effect on the level of [3H]flunitrazepam binding but increased the proportion of intracellular receptors by 61 and 74%, respectively, compared with untreated controls. Similar treatment with 1 mM GABA increased the level of intracellular sites by 154-176%. The effect of GABA on receptor internalization was blocked by cotreatment with the GABAA receptor antagonist R 5135. The results suggest that SPTC-1012S can be used as a probe to study the internalization of the GABAA/benzodiazepine receptor complex under normal conditions or following acute or chronic treatment with agonists.     

Effects of monensin on the synthesis, transport, and intracellular degradation of proteoglycans in rat ovarian granulosa cells in culture

Rat ovarian granulosa cells, isolated from immature female rats 48 h after stimulation with 5 IU of pregnant mare's serum gonadotropin, were maintained in culture. The effects of monensin, a monovalent cationic ionophore, on various aspects of proteoglycan metabolism were studied by metabolically labeling cultures with [35S]sulfate, [3H]glucosamine, or [3H]glucose. Monensin inhibited post-translational modification of both heparan sulfate (HS) proteoglycans and dermatan sulfate (DS) proteoglycans, resulting in decreased synthesis of completed proteoglycans [( 35S]sulfate incorporation decreased to 10% of control by 30 microM monensin, with an ED50 approximately 1 microM). Proteoglycans synthesized in the presence of monensin showed undersulfation of both DS and HS glycosaminoglycans and altered N-linked and O-linked oligosaccharides, suggesting that the processing of all sugar moieties is closely associated. Monensin caused a decrease in the endogenous sugar supply to the UDP-N-acetylhexosamine pool as indicated by an increased 3H incorporation into DS chains [( 3H]glucosamine as precursor) in spite of the decrease in glycosaminoglycan synthesis. Monensin reduced and delayed transport of both secretory and membrane-associated proteoglycans from the Golgi complex to the cell surface. It took 2-4 min for newly labeled proteoglycans to reach the main transport process inhibited by monensin. Monensin at 30 microM did not prevent internalization of cell surface 35S-labeled proteoglycans but almost completely inhibited their intracellular degradation to free [35S]sulfate (ED50 approximately 1 microM), resulting in intracellular accumulation of both DS and HS proteoglycans. Pulse-chase experiments demonstrated that one of the intracellular degradation pathways involving proteolysis of both DS and HS proteoglycans and limited endoglycosidic cleavage of HS continued to operate in the presence of monensin. These results suggest that the intracellular degradation of proteoglycans involve both acidic and nonacidic compartments with monensin inhibiting those processes that normally occur in such acidic compartments as endosomes or lysosomes by raising their pH.     

Monoclonal Antibodies Recognizing the Benzodiazepine Receptor of Chick Embryo Brain

Abstract

The benzodiazepine receptor (BZDR) of the embryonic chick brain contained three subunit proteins with molecular weights of 48-kilodalton (KD), 50-KD and 51-KD at a pI of 5.6, as demonstrated by two-dimensional gel electrophoresis and fluorography of the ³H-flunitrazepam (FNZ)-photolabeled receptor. Monoclonal antibodies (mAB) against the receptor were produced by using the spleen cells of one mouse immunized with the three subunit proteins extracted from SDS-PAGE gels. When the radioligand-labeled membranes were subjected to two-dimensional gel electrophoresis followed by immunoblotting using the mAB 2C3, both 50-KD and 51-KD bands with a pI of 5.6 were immunoreactive and radioactive. Thus, the mAB 2C3 recognized a common epitope on the 50-KD and 51-KD subunits of the BZDR. In addition, the mAB 2C3 was used with immunocytochemistry to determine the distribution of the receptor in the chick embryo brain. The BZDR immunoreactivity was observed among various brain areas, including hippocampus, optic tectum and cerebellum. The reaction product was localized in the neuronal membranes and cytoplasm. Certain neurons in the culture derived from embryonic chick brains were also immunoreactive as detected by immunocytochemical staining.     

Effect of monensin on ricin and fluid phase transport in polarized MDCK cells.

The effect of monensin on endocytosis, transcytosis, recycling and transport to the Golgi apparatus in filter-grown Madin-Darby canine kidney (MDCK) cells was investigated using 125I-labeled ricin as a marker for membrane transport, and horseradish peroxidase (HRP) as a marker for fluid phase transport. Monensin (10 microM) stimulated transcytosis of both markers about 3-fold in the basolateral to apical direction. Transcytosis of HRP in the opposite direction, apical to basolateral, was reduced to approximately 50% of the control by monensin, whereas that of ricin was slightly increased. Recycling of markers endocytosed from the apical surface was reduced in the presence of monensin and there was an increased accumulation of both ricin and HRP in the cells. Transport of ricin to the Golgi apparatus increased to the same extent as the increase in intracellular accumulation. No change in recycling or accumulation was observed with monensin when the markers were added basolaterally, but transport of ricin to the Golgi apparatus increased almost 3-fold. Our results indicate that basolateral to apical transcytosis is increased in the absence of low endosomal pH, and they suggest that apical to basolateral transcytosis of a membrane-bound marker (ricin) is affected by monensin differently from that of a fluid phase marker (HRP).     

Golgi apparatus cisternae of monensin-treated cells accumulate in the cytoplasm of liver slices

Protein transport via the endoplasmic reticulum Golgi apparatus-cell surface export route was blocked when slices (6-15 cells thick) of livers of 10-day-old rats were incubated with 1 microM monensin. Production of secretory vesicles by Golgi apparatus was reduced or eliminated and, in their place, swollen cisternae accumulated in the cytoplasm at the trans Golgi apparatus face. The swelling response was restricted to the six external cell layers of the liver slices, and the number of cells showing the response was little increased by either a greater concentration of monensin or by longer times of incubation. When monensin was added post-chase to the slices, flux of radioactive proteins to the cell surface was inhibited by about 80% as determined from standard pulse-chase analyses with isolated cell fractions. Radioactive proteins accumulated in both endoplasmic reticulum and Golgi apparatus and in a fraction that may contain monensin-blocked Golgi apparatus cisternae released from the stack. The latter fraction was characterized by galactosyltransferase/thiamine pyrophosphatase ratios similar to those of Golgi apparatus from control slices. The use of monensin with the tissue slice system may provide an opportunity for the cells to accumulate monensin-blocked Golgi apparatus cisternae in sufficient quantities to permit their isolation and purification by conventional cell fractionation methods.     

Independent benzodiazepine and beta-carboline binding sites in the brain of aggressive and anxious-defensive mice

Distribution of specific 3H-flunitrazepam and 3H-beta-carboline-3-carboxylate binding sites in the brain regions of aggressive and timid-defensive mice was investigated before and after subchronic injection of diazepam (5 mg/kg). The absence of differences between the affinity and concentration of 3H-flunitrazepam binding sites in diencephalon and brain cortex in aggressive and defensive mice may be explained by general benzodiazepine receptor reaction on isolation and agonistic interaction stress. Significant predominance of 3H-beta-carboline-3-carboxylate binding sites in the brain cortex, as compared to the concentration of 3H-flunitrazepam binding sites suggests the presence of specific binding sites for beta-carbolines, which have specific distribution in the brain.     

Acute effects of insulin on surface insulin receptors in isolated hepatocytes. Evidence for a recycling pathway

Isolated rat hepatocytes were incubated for 1 h at 37 degrees C with 10 nM insulin. Following washout of insulin, cells were incubated with [125I] monoiodoinsulin at 15 degrees C to assess surface insulin binding. Preincubation with 10 nM insulin did not cause a decrease in insulin binding. Scatchard analysis confirmed that insulin receptor number remained constant. In the presence of 200 microM chloroquine or 25 microM monensin, surface insulin binding after preincubation with 10 nM insulin fell to 81.1 +/- 1.2% or 39.0 +/- 2.7% of control, respectively. It is suggested that the maintenance of insulin receptor number following acute insulin treatment in vitro is due to an insulin receptor recycling pathway, possibly involving lysosomes and/or the Golgi apparatus.     

Effect of drugs and temperature on biosynthesis and transport of glycosphingolipids in cultured neurotumor cells

Neuroblastoma and glioma cells were grown in the presence of [³H]galactose, and the incorporation of ³H into gangliosides and the transport of newly synthesized gangliosides to the cell surface were examined under different experimental conditions. A variety of drugs, including inhibitors of protein synthesis and energy metabolism, modulators of the cytoskeleton and the ionophore monensin, had no effect on the transport of newly synthesized GD1a in neuroblastoma cells. Only low temperature effectively blocked translocation to the plasma membrane. Monensin, however, had marked effects on the biosynthesis of gangliosides and neutral glycosphingolipids. Whereas incorporation of ³H into complex glycosphingolipids was reduced, labeling of glucosylceramide was increased in cells exposed to monensin. In addition, biosynthesis of the latter glycolipid was less susceptible to low temperatures than that of more complex ones. Previous studies have implicated the Golgi apparatus as the predominant site of glycosylation of gangliosides. As monensin has been reported to interfere with the Golgi apparatus, our results indicate that glucosylceramide may be synthesized at a site that is separate from the site where further glycosylation occurs. Once synthesis of a ganglioside is completed, transport of the molecule to the cell surface proceeds under conditions of cytoskeletal disruption, energy depletion and ionic inbalance, but not low temperature.     

Functional reconstitution of the bovine brain GABAA receptor from solubilized components

The GABAA/benzodiazepine receptor has been solubilized from membrane preparations of bovine cerebral cortex and has been reconstituted, in a functionally active form, into phospholipid vesicles. In preliminary experiments, the receptor was labeled with the photoactive benzodiazepine [3H]flunitrazepam prior to solubilization. A peptide of apparent molecular weight 53,500 was specifically labeled by this method, and this was used as a marker for the receptor during the reconstitution procedures. The labeled protein was solubilized with approximately 40% efficiency by 1% beta-octyl glucoside. Reconstitution was achieved by mixing the solubilized proteins with a 4:1 mixture of soybean asolectin and bovine brain phospholipids, followed by chromatography on Sephadex G-50-80 to remove detergent. The incorporation of the GABAA receptor into membrane vesicles has been verified by sucrose gradient centrifugation in which the [3H]-flunitrazepam-labeled peptide comigrated with [14C]phosphatidylcholine used as a lipid marker. Vesicles prepared without labeled markers retained the ability to bind both [3H]flunitrazepam and the GABA analogue [3H]muscimol. Furthermore, the binding parameters were very similar to those measured using native membrane preparations. A novel fluorescence technique has been used to measure chloride transport mediated by the GABAA receptor in reconstituted vesicles. Chloride influx was rapidly stimulated in the presence of micromolar concentrations of muscimol and was blocked by preincubation of the membranes with muscimol (desensitization). Flux was also blocked by pretreatment with the competitive GABAA receptor blocker bicuculline or with the noncompetitive GABAA receptor antagonist picrotoxin.     

Evidence that all newly synthesized proteins destined for fast axonal transport pass through the Golgi apparatus

Effects of the sodium ionophore, monensin, were examined on the passage from neuronal cell body to axon of materials undergoing fast intracellular transport. In vitro exposure of bullfrog dorsal root ganglia to concentrations of drug less than 1.0 micron led to a dose-dependent depression in the amount of fast-transported [3H]leucine- or [3H]glycerol-labeled material appearing in the nerve trunk. Incorporation of either precursor was unaffected. Exposure of a desheathed nerve trunk to similar concentrations of monensin, while ganglia were incubated in drug-free medium, had no effect on transport. With [3H]fucose as precursor, fast transport of labeled glycoproteins was depressed to the same extent as with [3H]leucine; synthesis, again, was unaffected. By contrast, with [3H]galactose as precursor, an apparent reduction in transport of labeled glycoproteins was accounted for by a marked depression in incorporation. The inference from these findings, that monensin acts to block fast transport at the level of the Golgi apparatus, was supported by ultrastructural examination of the drug-treated neurons. An extensive and selective disruption of Golgi saccules was observed, accompanied by an accumulation of clumped smooth membranous cisternae. Quantitative analyses of 48 individual fast-transported protein species, after separation by two-dimensional gel electrophoresis, revealed that monensin depresses all proteins to a similar extent. These results indicate that passage through the Golgi apparatus is an obligatory step in the intracellular routing of materials destined for fast axonal transport.     

Characterization with Antibodies of the γ-Aminobutyric AcidA/Benzodiazepine Receptor Complex During Development of the Rat Brain

The postnatal development of the gamma-aminobutyric acidA/benzodiazepine receptor (GABAR/BZDR) complex of the rat brain has been investigated using the monoclonal antibody 62-3G1 and the polyclonal rabbit antiserum A, specific for the 57,000 and 51,000 Mr receptor subunits, respectively. Both GABAR and BZDR binding activities co-precipitated during all postnatal ages. Adult rats showed a main 51,000 Mr[3H]flunitrazepam photoaffinity-labeled peptide, whereas newborn rats showed several photolabeled peptides of higher Mr. All the photolabeled peptides could be immunoprecipitated with each antibody regardless of the age of the rats. These results suggest that the physical coupling between the GABAR and the BZDR is already present in newborn animals and it is maintained afterwards during development. Glycosidase and peptidase treatments of the immunoprecipitated GABAR/BZDR complex indicated that all the [3H]flunitrazepam-photolabeled subunits are different peptides, although they seem to conserve a high degree of homology. In addition to the age-dependent heterogeneity, the results also suggest that for each age, there is heterogeneity in the subunit composition of the GABAR/BZDR complex.     

Transport and processing of beta-hexosaminidase in normal and mucolipidosis-II cultured fibroblasts. Effect of monensin and nigericin.

The univalent-cation ionophores monensin (4.0 microM) and nigericin (0.5 microM) inhibited the abnormal excretion of beta-hexosaminidase from mucolipidosis-II cultured fibroblasts by 62 and 76% respectively, with a corresponding intracellular accumulation of the enzyme. As shown by lectin binding, the enzyme which accumulated in monensin-treated cells did not contain galactose residues, whereas the corresponding enzyme from nigericin-treated cells was galactosylated. The results suggest that monensin acts at an early point in the process of hydrolase glycosylation, and nigericin acts later, both presumably within the Golgi region, allowing the accumulation of different glycosylated forms of the enzyme. The intra- and extra-cellular distribution of beta-hexosaminidase in ionophore-treated normal cells was essentially unchanged, whereas concanavalin A precipitability of excreted enzyme was increased and its ability to be taken up by deficient fibroblasts was decreased. The bivalent-cation ionophore A23187 (1 microM) reduced beta-hexosaminidase excretion from mucolipidosis-II cells by 82% and by 96% when used with EGTA (1 mM). However, there was no intracellular accumulation of enzyme, suggesting that the effect of this ionophore was restricted to the inhibition of synthesis. It therefore appears that the actual transport of beta-hexosaminidase in mucolipidosis-II cells is affected by univalent-cation ionophores in a selective manner. These findings suggest that individual ionophores could be used to identify the sites of hydrolase oligosaccharide processing in the Golgi region by causing intermediate glycosylated forms of the transported hydrolase to accumulate in a specific Golgi compartment preceding the blocking site of the ionophore.     

Effects of Inhibitors of Protein Synthesis and Intracellular Transport on the γ-Aminobutyric Acid Agonist-Induced Functional Differentiation of Cultured Cerebellar Granule Cells

The effect of inhibitors of protein synthesis (actinomycin D, cycloheximide), proteases (leupeptin), and intracellular transport (colchicine, monensin) on the gamma-aminobutyric acid (GABA) agonist [4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP)]-induced changes in morphological differentiation and GABA receptor expression was investigated in cultured cerebellar granule cells. After 4 days in culture the neurons were exposed to the inhibitors for 6 h in the simultaneous presence of THIP. Subsequently, cultures were either fixed for electron microscopic examination or used for preparation of membranes for [3H]GABA binding assays. In some experiments the functional activity of the newly induced low-affinity GABA receptors was assessed by investigation of the ability of GABA to inhibit neurotransmitter release from the neurons. These experiments were performed to differentiate between an intracellular and a plasma membrane localization of the receptors. In all experiments cultures treated with THIP alone served as controls. The inhibitors of protein synthesis totally abolished the ability of THIP to induce low-affinity GABA receptors. In contrast, the inhibitors of intracellular transport as well as the protease inhibitor did not affect this parameter. However, studies of effects of GABA on transmitter release from monensin-treated cultures showed that transmitter release could not be inhibited by GABA in these cells in spite of the presence of low-affinity GABA receptors in the membrane preparations. This indicates that the low-affinity receptors were not located in the plasma membrane. This is in good agreement with the corresponding morphological findings, that monensin treatment led to an intense vacuolization of the Golgi apparatus, thereby preventing intracellular transport of the newly synthesized GABA receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modification of γ-Aminobutyric AcidA Receptor Binding and Function by N-Ethoxycarbonyl-2-Ethoxy-1,2-Dihydroquinoline In Vitro and In Vivo: Effects of Aging

The irreversible protein-modifying reagent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) was used to investigate binding site characteristics on the gamma-aminobutyric acidA (GABAA) receptor complex. In vitro, preincubation with EEDQ led to a concentration-dependent decrease in receptor number for benzodiazepine, t-butylbicyclophosphorothionate (TBPS), and GABA binding sites in cerebral cortex. The effect was maximal at the highest concentration of EEDQ used (10(-4) M) and was greatest for the benzodiazepine site. Pretreatment of membranes with the benzodiazepine antagonist Ro 15-1788, 1 or 10 microM, or the agonist lorazepam, 10 microM, largely prevented the effects of EEDQ. Scatchard analysis indicated no effect of EEDQ, 10(-4) M, on apparent affinity, but a decrease in receptor density for each site. Administration of EEDQ to mice, 12.5 mg/kg i.p., led to a substantial (55-65%) decrease in number of benzodiazepine binding sites in cortex after 4 h. Slightly smaller changes were observed for TBPS and GABA binding. No changes were observed in apparent affinity at any site. Prior administration of Ro 15-1788, 5 mg/kg, prevented the effect of EEDQ on benzodiazepine binding. Density of benzodiazepine binding sites gradually recovered over time, and receptor density returned to control values by 96 h after EEDQ injection. Number of binding sites in cortex for TBPS and GABA also increased over time after EEDQ. Benzodiazepine sites in cerebellum were decreased proportionally to cortex after EEDQ, and increased over a similar time course. Function of the GABAA receptor in chloride uptake in cortex was markedly reduced (65%) by EEDQ.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amoxapine inhibition of GABA-stimulated chloride conductance: investigations of potential sites of activity

Amoxapine inhibits GABA-stimulated chloride conductance by acting on the GABAA-receptor chloride-ionophore complex which can be studied using membrane vesicles prepared from rat cerebral cortex. Amoxapine produces a right shift in the GABA concentration-response curve for the stimulation of 36Cl- uptake into these vesicles with no apparent change in the maximum response. Schild analysis of these data gave a pA2 value of 5.52 with a slope of 0.79. Amoxapine inhibits the binding of the GABAA receptor selective antagonist [3H]SR 95531 with an IC50 value of 3.45 microM and a pseudo Hill coefficient of 0.83. In contrast, 10 microM amoxapine inhibits [3H]flunitrazepam binding by less than 25% while the benzodiazepine antagonist Ro 15-1788 reduces the amoxapine inhibition of GABA-stimulated chloride conductance only at high concentrations. These data suggest that amoxapine does not inhibit chloride conductance by acting as a benzodiazepine inverse agonist and either acts directly on the GABAA receptor as an antagonist or blocks GABA activity at a site closely coupled to it. The ability of amoxapine to inhibit GABA-stimulated chloride conductance is a likely explanation for its proconvulsant activity observed at high doses.     

Effects of Monensin and Colchicine on Myelin Galactolipids

Monensin and colchicine have been used in a variety of systems to disrupt functioning of the Golgi apparatus and transport of Golgi-derived vesicles to the plasma membrane. In this study the effects of monensin and colchicine on the synthesis of cerebroside and sulfatide and their appearance in myelin were examined to determine whether these myelin components are processed through the Golgi apparatus. Brain slices from rats 17 days old were incubated with [3H]galactose and [35S]-sulfate to label cerebroside and sulfatide. Myelin was isolated on sucrose density gradients. Fractions highly enriched in cerebroside and sulfatide were prepared from homogenates and myelin fractions by lipid extraction, alkaline methanolysis, and in some cases TLC. Monensin at 0.1 microM had no significant effect on synthesis of these galactolipids as measured by incorporation of [3H]-galactose into cerebroside or [35S]sulfate into sulfatide in homogenates. However, appearance of [35S]sulfatide in the myelin fraction was reduced to 49% of control, while appearance of [3H]cerebroside was not significantly reduced. Colchicine from 1 mM to 0.1 microM had effects similar to monensin, that is, appearance of [35S]sulfatide in myelin was depressed, but again [3H]cerebroside was not affected. Incorporation of [35S]sulfate into sulfatide in homogenate was 93% of control, while appearance of [35S]sulfatide in the myelin fraction was depressed to 58% of control. The inhibition of appearance of sulfatide in myelin by colchicine and monensin is consistent with the view that sulfation of cerebroside occurs in the Golgi and that sulfatide is transported via Golgi-derived vesicles to the forming myelin membrane.(ABSTRACT TRUNCATED AT 250 WORDS)     

Abnormal laminin secretion from parietal endoderm-like F9 cells in the presence of monensin

We studied the effects of monensin on post-translational modification and intracellular transport of precursors of laminin subunits in parietal endoderm-like F9 cells. At concentrations higher than 0.1 microM, monensin inhibited the processing of high-mannose type precursors for all three subunits and caused their cytoplasmic accumulation. Furthermore, the secretion of mature subunits of laminin was inhibited. Instead, polypeptides with similar molecular weights to those of intracellular precursors were secreted. These polypeptides were immunologically related to laminin subunits and were sensitive to digestion with beta-N-acetylglucosaminidase H (Endo H). This indicated that Golgi complexes of the cells can transport the precursors of laminin subunits even with their terminal glycosylation inactivated by monensin. Tunicamycin induced the accumulation of unglycosylated precursors and strongly reduced their secretion into the medium.     

Effect of monensin on receptor recycling during continuous endocytosis of asialoorosomucoid

The binding of asialoglycoproteins to their liver cell receptor results in internalization of the ligand-receptor complex. These complexes rapidly appear in intracellular compartments termed endosomes whose acidification results in ligand-receptor dissociation. Ligand and receptor subsequently segregate: ligand is transported to lysosomes and is degraded while receptor recycles to the cell surface. The proton ionophore monensin prevents acidification of endosomes and reversibly inhibits this acid-dependent dissociation of ligand from receptor. The present study determined the effect of monensin treatment of short-term cultured rat hepatocytes on cell-surface-receptor content, determined both by their binding activity and immunologically, following continuous endocytosis of asialoorosomucoid. Inclusion of 5 microM monensin in the incubation medium reduced the number of immunologically detectable cell-surface receptors by 20% in the absence of ligand. During continuous endocytosis of asialoorosomucoid, inclusion of monensin resulted in a 30-40% reduction of cell-surface receptor detectable either by ligand binding or immunologically. These results suggest that the reduced liver-cell-surface content of receptor in monensin is due to intracellular trapping of ligand-receptor complexes. The reduction of surface receptor during monensin incubation in the absence of ligand suggests that "constitutive recycling" of plasma membrane components also requires intracellular acidification.