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PubMed1984

Effects of tunicamycin on thin-section and freeze-fracture images of microvilli of the duodenal epithelial cells of the mouse.

The effect of tunicamycin, which is known to inhibit the synthesis of N-linked glycoprotein, on the duodenal absorptive epithelial cell of the mouse was studied in thin-section as well as freeze-fracture images. In tunicamycin-treated animals, the apical part of the epithelial cell was almost negative to the PAS reaction. Moreover, microvilli of the epithelial cell became shorter, larger in diameter, and fewer in number in tunicamycin-treated mice. In addition, freeze-fracture images revealed that the population density of membrane particles of the microvillus membrane was lowered by tunicamycin treatment. These results may indicate that the inhibition of synthesis of N-linked glycoprotein causes a decrease of membrane supply from the Golgi apparatus to the apical plasma membrane.

Animals↗

PubMed1980

Alterations in the distribution of glycoproteins in epithelial cells of murine colon after injection of tunicamycin.

Glycoproteins are associated with several structures of colonic absorptive cells of the mouse. These include the cell coat, Golgi apparatus and vesicles that transport the glycoproteins from the apparatus to the cell surface (Michaels and Leblond 1976). In many in vitro systems, the antibiotic tunicamycin inhibits the glycosylation of asparagine residues yielding carbohydrate-poor glycoproteins. In the present in vivo study, tunicamycin was injected into mice. The murine colonic epithelial cells were prepared routinely for electron microscopy and cytochemistry. Cells from the experimental and control animals were similar morphologically. However, staining by the periodic acid-chromic acid-silver methenamine technique, revealed differences in the distribution of glycoproteins. In animals that received the higher dosages of tunicamycin there was a substantial reduction in silver staining in both the Golgi apparatus and the vesicles of colonic epithelial cells compared to these structures in cells of identically treated control tissues, whereas the staining over the cell coat was not significantly altered. Possible explanations for the staining of the cell coat in the treated animals were provided in the text. This report demonstrates the feasibility of using tunicamycin in vivo and detection of the change obtained by the silver methenamine method.

Animals↗

PubMed1992

Tunicamycin, puromycin and brefeldin A influence the subcellular distribution of neuropeptides in hypothalamic magnocellular neurones of rat.

Magnocellular neurones in the supraoptic nuclei of normal Long Evans and homozygous Brattleboro rats were examined electron-microscopically after intracisternal injections of tunicamycin, puromycin, or brefeldin A. Moderate (50 micrograms) or high (200 micrograms) doses of tunicamycin caused the formation of electron-dense filamentous accretions in the endoplasmic reticulum (ER) cisterns of vasopressin neurones, but only the high dose of tunicamycin also caused accretions to form in the ER of some oxytocin neurones. Immunogold labelling of ultrathin sections from tunicamycin-treated rats revealed that, in about 5% of vasopressin neurones, the accretions could be immunogold-labelled for vasopressin and its associated neurophysin. However, in the majority of vasopressin neurones, the sections required trypsinisation before immunolabelling of the accretions could be detected. Small accretions in the ER of oxytocin neurones did not label for oxytocin or its neurophysin without prior trypsinisation, whereas larger accretions in other oxytocin cells could be labelled without prior trypsin treatment. Administration of puromycin resulted in the formation of small ER accretions in both vasopressin and oxytocin neurones. These accretions were immunolabelled with antisera, respectively, to vasopressin and oxytocin, but neurophysin-immunoreactivity was in most cases absent and was not revealed by treatment with trypsin, suggesting that neurophysin-immunoreactive epitopes were absent from truncated peptides forming the accretions. Brefeldin A caused dilatation of ER cisterns and disruption of the Golgi apparatus in both oxytocin and vasopressin neurones, but did not cause accretions to form in the ER.

Animals↗

PubMed1994

Tunicamycin inhibits prostaglandin F2 alpha receptor-mediated phosphoinositide hydrolysis in cultured rat astrocytes.

Effect of tunicamycin, an inhibitor of N-linked glycosylation, on prostaglandin (PG) F2 alpha-stimulated phosphoinositide (PI) hydrolysis was examined in cultured rat astrocytes. Pretreatment of cultured astrocytes with tunicamycin (25-250 ng/ml) inhibited subsequent PGF2 alpha (1 microM)-stimulated PI-hydrolysis in concentration- and time-dependent manners. The inhibition completely recovered after removal of tunicamycin and re-incubation for 12 h. Tunicamycin pretreatment (100 ng/ml for 12 h) significantly blocked [35S]methionine incorporation into cultured astrocytes, but cell viability was not affected under the condition. Inhibitors of processing of N-linked sugar chains such as bromoconduritol, 1-deoxymannojirimycin, and swainsonine had no effect on PI response to PGF2 alpha. These observations suggest that PGF2 alpha receptor is N-linked glycosylated.

Animals↗

PubMed1994

Transport of phosphatidylcholine to Xenopus photoreceptor rod outer segments in the presence of tunicamycin.

Study of the dynamics of membrane protein and phospholipid transport from the inner to the outer segment of vertebrate photoreceptors has shown an interesting dissociation of the two components under a number of experimental treatments which inhibit protein synthesis or transport. Under conditions which block the addition of opsin to outer segments, various lipids continue to be synthesized and transported to the outer segment in the presence of monensin, puromycin, brefeldin A, tunicamycin and several general metabolic inhibitors. In the current study, isolated retinas from adult Xenopus laevis were incubated with or without 20 micrograms mg-1 of tunicamycin in total darkness or light for 2-12 h in the presence of [3H]choline to study the dependence of phosphatidylcholine synthesis and transport on protein transport to the outer segment. Phosphatidylcholine is a major bulk lipid of outer segments, comprising close to one half of the phospholipid of outer segment phospholipids, and blocking choline uptake in retinas is known to cause photoreceptor degeneration. Biochemical analysis demonstrates that tunicamycin does not block the synthesis of phosphatidylcholine in photoreceptor inner segments or transport of radiolabelled phosphatidylcholine to outer segments during 6 h incubations with [3H]choline in light or total darkness. Light and electron microscopic autoradiography and morphometric analysis show that [3H]choline radiolabelled phospholipid does not accumulate in a band of newly formed basal discs in the outer segment or in the tubulo-vesicular structures which accumulate in the intersegmental space of tunicamycin-treated retinas. We conclude that transport of phosphatidylcholine can occur independently of opsin transport to the outer segment but whether this represents two separable components of a single pathway or involves two distinct routes of transport to the outer segment is still unresolved.

Animals↗

PubMed1982

Glycoprotein requirement for neurite outgrowth in goldfish retina explants: effects of tunicamycin.

The role of glycoproteins in neurite outgrowth in vitro has been examined using the protein glycosylation inhibitor, tunicamycin. The concentration-dependent inhibitory effects of tunicamycin on neuritic outgrowth from goldfish retinal explants closely paralleled its effects on the dolichol pathway of glycoprotein biosynthesis. The neurite membrane assembled in the presence of an intermediate dose of tunicamycin (5 micrograms/ml) was deficient in carbohydrate, as indicated by a reduced capacity to bind the lectins Concanavalin A, wheat germ agglutinin and ricin. These results suggest that tunicamycin may prove useful in assessing the role of cell surface carbohydrate in neuronal recognition phenomena.

Animals↗

PubMed1989

Developmental changes in the open time and conductance of acetylcholine receptors in aneural cultured Xenopus myocytes treated with cycloheximide or tunicamycin.

Aneural cultures of Xenopus myocytes were treated with cycloheximide or tunicamycin in order to determine the influence of continued protein synthesis and insertion of glycosylated acetylcholine (ACh) receptor proteins on developmental changes in ACh-activated single-channel events. The developmental stage of the cultures was estimated from the stage of intact embryos maintained at the same temperature as the cultures. Single-channel recordings (5 x 10(-7) M ACh) from untreated cultures revealed primarily low conductance (27.4 +/- 0.6 pS) events for the first 14 h in culture (temperature = 23-25 degrees C) and a second high conductance class of events (42.5 +/- 0.9 pS) at later developmental stages (after Stage 28). Treatment with cycloheximide (at Stage 28) or tunicamycin (at Stage 22) produced significantly (P less than 0.01) fewer high conductance events at later stages (Stages 31-47), but had no effect on the conductance or open time of the low conductance event. A significant decrease (Chi square, P less than 0.05) in ACh sensitivity was observed at Stage 27 in myocytes treated with tunicamycin at Stage 22. The results strongly suggest that the low and high conductance events represent two distinct receptor molecules synthesized at different developmental stages. The tunicamycin results also suggest that a developmental reduction in the mean open time of low conductance events in untreated cultures does not depend upon the continued insertion of newly synthesized receptors.

Acetylcholine↗

PubMed1985

Correlation of tunicamycin-sensitive surface glycoproteins from Trypanosoma cruzi with parasite interiorization into mammalian cells.

Trypomastigote forms of Trypanosoma cruzi lose infectivity to cultured mammalian cells when exposed to tunicamycin. Upon reincubation into fresh medium, parasites recover their full penetration capacity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [35S]methionine-labeled polypeptides showed that tunicamycin-treated parasites present several components with altered electrophoretic mobility when compared with controls. Immunoprecipitation with rabbit hyperimmune and human chagasic sera indicated that the surface antigens of approximate molecular masses of 175-180, 120-125, 90-95 and 85 kDa are not encountered in tunicamycin-treated trypomastigotes. By affinity chromatography on wheat germ agglutinin-Sepharose, it was observed that the trypomastigote-specific 85 kDa glycoprotein (Tc-85) is affected by the drug. The other affected components are glycoproteins with affinity for concanavalin A. The results suggest that tunicamycin-sensitive surface glycoproteins from T. cruzi are involved in the parasite interiorization into mammalian cells.

Animals↗

PubMed1991

Tunicamycin-resistant variants from five species of Leishmania contain amplified DNA in extrachromosomal circles of different sizes with a transcriptionally active homologous region.

Twelve independent variants were selected from five species of Leishmania for resistance to tunicamycin by exposure of cultured promastigotes to increasing concentrations of this antibiotic, an inhibitor of the microsomal N-acetylglucosamine-1-phosphate transferase in the dolichol pathway of N-glycosylation. All variants obtained from all species, as found previously with Leishmania amazonensis, contain amplified chromosomal DNA exclusively as extrachromosomal circles. These circular amplicons hybridize with amplified DNAs cloned previously from tunicamycin-resistant Leishmania amazonensis, but not with those from Leishmania resistant to other drugs. The amplicons from tunicamycin-resistant cells vary with different species in size from 30 to 70 kb, but all share a homologous region of 20 kb. Multiple independent transcripts are overexpressed from this region. Elevation of the microsomal glycosyltransferase activity is demonstrated in these variants from representative species. The results thus provide further evidence that this enzyme is overexpressed due to amplification of the gene in these cells. The consistent observation of this event in all cases studied also suggests that this is the predominant, if not the only mechanism of tunicamycin resistance in Leishmania.

Animals↗

PubMed1988

The neurotoxicity of tunicamycin.

The neurotoxicity of tunicamycin, an inhibitor of glycosylation of glycoproteins and a potential anti-tumour agent, was investigated by injection of the chemical directly into the lateral ventricle of the brain of rats. A delayed neurological syndrome developed due to anoxic necrosis of brain cells and spongy degeneration of the white matter. In many brains the lesion had the characteristic features of an infarct. Since the predominant lesion was vascular, the action of tunicamycin on blood vessels was investigated in vitro using rabbit aortic rings. In this system the normal relaxation response to acetylcholine in aortic rings pre-contracted with noradrenaline was progressively inhibited as exposure of the ring to tunicamycin increased from 2 h to 5 h. This relaxation response depends on the release of endothelium-derived relaxing factor from endothelial cells. Hence it is proposed that the important primary target in tunicamycin neurotoxicity is endothelial cells of the brain microvessels.

Animals↗

PubMed1994

Tunicamycin alters channel gating characteristics of junctional and extrajunctional acetylcholine receptors expressed in Xenopus oocytes.

The acute effects of tunicamycin on mouse junctional and extrajunctional acetylcholine receptors (AChRs) expressed in Xenopus oocytes, distinct from its ability to block N-glycosylation of the protein, was examined at the single channel level. Tunicamycin reduced the frequency of ACh-activated single channel openings and prolonged the mean channel open times of AChRs in a dose-dependent manner, without affecting inward conductances. Continuous application of ACh to both junctional and extrajunctional AChR channels very slowly decreased the number of channel opening events, and tunicamycin accelerated this process. These results suggest that tunicamycin alters channel gating kinetics and accelerates transition towards a desensitized state.

Animals↗

PubMed1980

Effect of tunicamycin on the turnover of epidermal growth factor receptors in cultured calf aorta smooth muscle cells. Comparison with IMR-90 human lung fibroblasts.

Treatment of cultured calf aorta smooth muscle cells with tunicamycin, a potent inhibitor of dolichol-mediated glycosylation, resulted in progressive loss of receptors for epidermal growth factor with 50% of receptors lost after 6 h. Receptor half-life was also 6 h with cycloheximide treatment but was 12 h with either actinomycin D or camptothesin treatment. The epidermal growth factor-induced processing (internalization and/or degradation) of residual receptors remaining after tunicamycin treatment appeared to be unaltered. 50% decrease in 125I-labeled epidermal growth factor binding was observed also with IMR-90 fibroblasts upon 6 h treatment with tunicamycin, although these cells were less sensitive to inhibition by tunicamycin of glycosylation and protein synthesis.

Animals↗

PubMed1988

Visualization of glycoproteins after tunicamycin and monensin treatment of herpes simplex virus infected cells.

The effects of tunicamycin and monensin on the morphogenesis of herpes simplex virus type 1 and on the ultrastructure and function of host cell membranes was investigated by conventional technics of electron microscopy and cytochemical localization of glycoproteins with thiocarbohydrazide-SO2. Infected RS 537 rabbit fibroblasts were treated with tunicamycin, which inhibits the glycosylation of many glycoproteins, or monensin, which inhibits the transport of proteins to the cell surface, and were compared with untreated infected cells. Tunicamycin treatment almost entirely suppresses the perinuclear envelopment of viral capsids, induces the nuclear export of unusually numerous naked viral capsids, and prevents the proliferation of the Golgi apparatus. On the other hand, perinuclear envelopment of viral capsids still occurs following a monensin treatment; however, enveloped viral capsids are not released into the extracellular space; in addition this treatment induces the proliferation of the rough endoplasmic reticulum (RER). The number of structures stained for glycoproteins in tunicamycin-treated cells is markedly lower than that in nontreated infected cells, whereas an unusual additional staining of the entire outer nuclear membrane and of the RER occurs following monensin treatment.

Animals↗

PubMed2006

Novel potential of tunicamycin as an activator of the aryl hydrocarbon receptor -- dioxin responsive element signaling pathway.

Tunicamycin is a well-known inhibitor of protein glycosylation and used as an inducer of endoplasmic reticulum (ER) stress. We found that tunicamycin induced expression of cytochrome P450 1A1 in a dose-dependent manner. Like dioxin, the transcriptional induction was associated with dose-dependent activation of the dioxin responsive element (DRE). This effect was independent of inhibition of protein glycosylation or induction of ER stress. Pharmacological and genetic inhibition of the aryl hydrocarbon receptor (AhR) significantly attenuated activation of DRE by tunicamycin. These results elucidated the novel potential of tunicamycin as an activator of the AhR -- DRE signaling pathway.

Animals↗

PubMed2004

Protective effect of nicotine on tunicamycin-induced apoptosis of PC12h cells.

Nicotine has been reported to have neuroprotective effects. The present study deals with the neuroprotective effect of nicotine on the tunicamycin-induced apoptosis of PC12h cells. Treatment of PC12h cells with tunicamycin causes endoplasmic reticulum stress leading to apoptosis. Nicotine dose-dependently prevented the tunicamycin-induced apoptosis. Hoechst 33258 staining demonstrated the protective effect of nicotine against tunicamycin-induced apoptosis. Treatment with nicotinic acetylcholine receptor (nAChR) and L-type voltage-sensitive calcium channel (L-VSCC) antagonists prevented the nicotine-induced protective effect. A phosphatidylinositol 3-kinase (PI3-K) inhibitor had no influence on the nicotine-induced neuroprotective effect. These results show that the neuroprotective effect of nicotine occurs through nAChRs including the alpha 7 subtype and L-VSCC in PC12h cells and not through the PI3-K/Akt pathway.

Animals↗

PubMed2003

Tunicamycin inhibits NMDA and AMPA receptor responses independently of N-glycosylation.

In a whole-cell patch-clamp configuration, currents through N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor channels were monitored in cultured rat hippocampal neurons, and those currents were depressed to 25 and 28% of basal levels, respectively, by 3-min treatment with tunicamycin (10 microM), an inhibitor of protein N-glycosylation. Tunicamycin (10 microM) reduced amplitude of population spikes elicited in the dentate gyrus of rat hippocampal slices, reaching 78% of basal levels 60 min after the beginning of treatment, and long-term potentiation (LTP) of the perforant path was never induced in the presence of tunicamycin. Tunicamycin, thus, appears to serve as a modulator for NMDA and AMPA receptors, regardless of N-glycosylation, thereby inhibiting neurotransmission and LTP in the dentate gyrus.

Animals↗

PubMed1999

Inhibition of N-linked glycosylation of the human type 1alpha metabotropic glutamate receptor by tunicamycin: effects on cell-surface receptor expression and function.

The potential role of N-linked glycosylation of the human type 1alpha metabotropic glutamate (mGlu1alpha) receptor was studied in a recombinant, inducible expression system, where receptor expression was induced in the absence and presence of tunicamycin. In the absence of tunicamycin the mGlu1alpha receptor appeared to be expressed, at least in part, as a dimer consisting of monomers of approx. 145 and 160 KDa relative molecular mass (Mr). In the presence of tunicamycin only a single monomeric protein could be detected approximating the Mr predicted for the human mGlu1alpha receptor based on its primary amino acid sequence (130 KDa). Exposure to tunicamycin during receptor induction did not appear to affect the cell surface expression of the mGlu1alpha receptor as determined immunocytochemically or using a cell-surface biotinylation strategy, but reduced agonist-stimulated phosphoinositide hydrolysis by approximately 50% compared to control cell populations. Our data suggest that non-N-glycosylated human mGlu1alpha receptors can traffic to the cell surface and activate phospholipase C.

Animals↗

PubMed1994

Effect of tunicamycin on metabolism of unconjugated N-glycans in relation to regulation of tomato fruit ripening.

The unconjugated N-glycans (UNGs) Man3(Xyl)GlcNAc(Fuc)GlcNAc and Man5GlcNAc influence tomato (Lycopersicon esculentum cv 'Rutgers') fruit ripening at nanomolar concentrations. In addition, we have shown a delaying effect on ripening of tomato pericarp discs by 10 ng per g fresh weight Man5GlcNAc, one of 10 UNGs purified from mature green tomato pericarp. In the present study, oligomannosidic UNGs from tomato fruit exhibited activity similar to Man5GlcNAc. However, complex UNGs isolated from tomato pericarp did not show the same inhibitory effect as Man3(Xyl)GlcNAc(Fuc)GlcNAc, a complex N-glycan not detected in tomato. The metabolism of UNGs was also studied by metabolic labeling with 14C-glucosamine, revealing that the amount of oligomannosidic and complex type UNGs may change inversely during ripening. Both 14C-labelled UNGs disappeared after treatment with tunicamycin, an inhibitor of N-glycosylation. There was a correlation between the appearance of UNGs and recovery of ripening after tunicamycin treatment. Furthermore, application of 10 nM Man5GlcNAc with 40 microM tunicamycin prevented the delaying effect of tunicamycin. Our results indicate that endogenous unconjugated Man5GlcNAc may have a vital role in triggering and modulating tomato fruit ripening.

Carbohydrate Sequence↗