Accumulation of H+ in Vacuoles Induced by a Marine Peptide Toxin,Theonellamide F,in Rat Embryonic 3Y1 Fibroblasts

The effects of theonellamide F, a marine bicyclic peptide, on vacuolar formation in cultured cells were studied. Theonellamide F induced large vacuoles in 6 types of mammalian cells. The vacuoles induced by theonellamide F in 3Y1 cells accumulated acridine orange, a fluorescent probe indicating the presence of an acidic organelle. Their disappearance following treatment with bafilomycin A1 suggests that these vacuoles contain vacuolar ATPase to maintain an acidic internal milieu, and this is similar to those induced by Helicobacter pylori toxin VacA. The vacuoles induced by theonellamide F were not significantly decreased in size or number by nocodazole treatment, and the localization of a small GTPase, rab7, did not always correspond to the outline of the vacuoles. These results suggest that the molecular mode of action of vacuolar formation by theonellamides may differ from that by VacA and can be considered unique.     

Effect of monensin on the Golgi apparatus of absorptive cells in the small intestine of the rat

Summary The effect of short-time treatment with the ionophore monensin, administered intraluminally at concentrations of 5 and 10 M, was studied on the Golgi apparatus of absorptive cells in the small intestine of the rat. At 2–3 min after treatment most of the Golgi stacks exhibited dilated cisternae. At 4–5 min stacked cisternae were absent; they were replaced by groups of smooth-surfaced vacuoles. Dilatation and vacuolization occurred in the entire stacks without preferential effect on any particular Golgi subcompartment.Monensin did not influence the cytochemical Golgi reaction of thiamine pyrophosphatase and acid phosphatase. The characteristic staining pattern of these two enzymes in all Golgi cisternae of absorptive cells in the proximal small intestine, and the reactivity restricted to trans cisternae in distal segments of the small intestine, were unchanged after treatment with monensin. In the distal small intestine, the cytochemical pattern allowed the monensin-induced vacuoles to be attributed to the former cisor trans-Golgi face. Further, the cytochemical results demonstrate that vacuolization is not restricted to the stacked cisternae, but includes the trans-most cisterna. The latter, usually located at some distance from the Golgi stacks, has been defined as belonging to the GERL system in several types of cells. The clear response to monensin, an agent that selectively affects the Golgi apparatus, indicates common properties between trans-most and stacked Golgi cisternae.     

The Effect of Nisin and Monensin on Ruminal Fermentations In Vitro

When mixed ruminal bacteria and alfalfa were incubated in vitro, monensin and nisin both inhibited methane production so long as the concentrations were greater than 1 μM. Monensin- and nisin-dependent methane depressions caused a decrease in the acetate to propionate ratio (4.5 to 3.0). Total volatile fatty acid production was decreased by both monensin and nisin addition at concentrations greater than 2 μM. Starch-digesting ruminal bacteria were initially inhibited by monensin and nisin, but this effect disappeared after two to four transfers. Nisin always inhibited cellulolytic bacteria, but the nisin-dependent inhibition of cellulose digestion was no greater than the inhibition caused by monensin. Monensin and nisin also inhibited amino acid degradation, and nisin was more effective than monensin in controlling the growth of Clostridium aminophilum, an obligate amino acid-fermenting ruminal bacterium that can tolerate low concentrations of monensin. Because nisin was as potent as monensin, bacteriocins such as nisin may have potential as feed additives. Received: 2 December 1996 / Accepted: 10 February 1997     

Complete inhibition of transferrin recycling by monensin in K562 cells

Monensin blocks human transferrin recycling in a dose-dependent and reversible manner in K562 cells, reaching 100% inhibition at a noncytocidal dose of 10(-5) M, whereas transferrin recycling is virtually unaffected by noncytocidal doses of chloroquine. The intracellular pathway of human transferrin in K562 cells, both in the presence and absence of 10(-5) M monensin, was localized by indirect immunofluorescence. Monensin blocks transferrin recycling by causing internalized ligand to accumulate in the perinuclear region of the cell. The effect of 10(-5) M monensin on human transferrin kinetics was quantitatively measured by radioimmunoassay and showed a positive correlation with immunofluorescent studies. Immunoelectron microscopic localization of human transferrin as it cycles through K562 cells reveals the appearance of perinuclear transferrin-positive multivesicular bodies within 3 min of internalization, with subsequent exocytic delivery of the ligand to the cell surface via transferrin-staining vesicles arising from these perinuclear structures within 5 min of internalization. Inhibition of ligand recycling with 10(-5) M monensin causes dilated transferrin-positive multivesicular bodies to accumulate within the cell with no evidence of recycling vesicles. A coordinated interaction between multivesicular bodies and the Golgi apparatus appears to be involved in the recycling of transferrin in K562 cells. Cell-surface-binding sites for transferrin were reduced by 50% with 10(-5) M monensin treatment; however, this effect was not attenuated by 80% protein synthesis inhibition with cycloheximide, supporting the idea that the transferrin receptor is also recycled through the Golgi.     

The effect of monensin on gametogenesis and zoosporogenesis in the aquatic fungus,Allomyces macrogynus

Summary Cytoplasmic cleavage in the gametangia and zoosporangia ofA. macrogynus was studied using monensin, an ionophore known to disrupt several endomembrane functions in plant and animal cells. Monensin interfered with normal gamete and zoospore formation in a dose dependent manner such that at a 20 M concentration very abnormal cells were released from the reproductive structures. It was evident that monensin's effect was most pronounced during the first 25 minutes of gametogenesis and parallels in time the onset and continuation of the cytoplasmic cleavage events. Observations using fluorescence and differential interference contrast microscopy indicated that the ionophore inhibited normal cytoplasmic cleavage resulting in the production of multinucleate cells, many of which had either no flagella or multiple flagella. Transmission electron microscopy showed that the monensin-treated gametangia had many large vacuoles which contained amorphous electron-opaque material. X-ray microprobe analysis demonstrated that the elemental composition of the large vacuoles was identical to that of the dense globular inclusions seen in untreated gametangia, and morphological analysis confirmed the relationship between these endomembrane structures. Thus this swollen endomembrane component probably is not involved in the cleavage process. Single endomembrane cisternae which were very common in untreated gametangia were seldom seen in monensin-treated preparations. Instead, many smaller electron-transparent vacuoles were observed. These swollen cisternae may both represent monensin-modified Golgi apparatus equivalents and/or play a critical role during the process of gametogenesis and zoosporogenesis inA. macrogynus.     

Structural changes in the vacuole and cytoskeleton are key to development of the two cytoplasmic domains supporting single-cell C<Subscript>4</Subscript> photosynthesis in <Emphasis Type="Italic">Bienertia sinuspersici</Emphasis>

Bienertia sinuspersici Akhani has an unusual mechanism of C₄ photosynthesis which occurs within individual chlorenchyma cells. To perform C₄, the mature cells have two cytoplasmic compartments consisting of a central (CCC) and a peripheral (PCC) domain containing dimorphic chloroplasts which are interconnected by cytoplasmic channels. Based on leaf development studies, young chlorenchyma cells have not developed the two cytoplasmic compartments and dimorphic chloroplasts. Fluorescent dyes which are targeted to membranes or to specific organelles were used to follow changes in cell structure and organelle distribution during formation of C₄-type chlorenchyma. Chlorenchyma cell development was divided into four stages: 1—the nucleus and chloroplasts occupy much of the cytoplasmic space and only small vacuoles are formed; 2—development of larger vacuoles, formation of a pre-CCC with some scattered chloroplasts; 3—the vacuole expands, cells have directional growth; 4—mature stage, cells have become elongated, with a distinctive CCC and PCC joined by interconnecting cytoplasmic channels. By staining vacuoles with a fluorescent dye and constructing 3D images of chloroplasts, and by microinjecting a fluorescence dye into the vacuole of living cells, it was demonstrated that the mature cell has only one vacuole, which is traversed by cytoplasmic channels connecting the CCC with the PCC. Immunofluorescent studies on isolated chlorenchyma cells treated with cytoskeleton disrupting drugs suspended in different levels of osmoticum showed that both microtubules and actin filaments are important in maintaining the cytoplasmic domains. With prolonged exposure of plants to dim light, the cytoskeleton undergoes changes and there is a dramatic shift of the CCC from the center toward the distal end of the cell.     

Monensin inhibits receptor-mediated endocytosis of asialoglycoproteins in rat hepatocytes

Isolated rat liver parenchymal cells incubated in the presence of monensin exhibited a reduced uptake of ¹²⁵I-asialofetuin (¹²⁵I-AF). Binding studies indicated that the effect was due to a rapid reduction in the number of active surface receptors for the asialoglycoprotein. Monensin had no effect on receptor internalization, but apparently interrupted the recycling of receptors back to the cell surface. Monensin also inhibited the degradation of ¹²⁵I-AF previously bound to the cells; this inhibition was probably not due to a direct effect on intralysosomal proteolysis, as no lysosomal accumulation of undegraded ligand could be demonstrated in subcellular fractionation studies by means of sucrose gradients. It is more likely that monensin inhibits transfer of the labelled ligand from endocytic vesicles to lysosomes, as indicated by the accumulation of radioactivity in the former and by the ability of monensin to prevent the normally observed time-dependent increase in the buoyant density of endocytic vesicles. Whereas the effect of monensin on binding and uptake of asialofetuin was reversible, the effect on asialofetuin degradation could not be reversed.     

Monensin interrupts the recycling of low density lipoprotein receptors in human fibroblasts

In cultured human fibroblasts, each LDL receptor mediates the internalization of approximately 100 particles of LDL every 20 hr. We provide evidence that this reutilization of LDL receptors involves the recycling of receptors into and out of the cell and that the carboxylic ionophore monensin blocks the return of the receptors to the surface. In the presence of monensin and LDL, 75% of the receptors disappeared from the cell surface within 15 min and more than 90% disappeared within 60 min. The receptors that left the surface were trapped intracellularly within perinuclear vacuoles, as visualized by indirect immunofluorescence with the use of LDL, monensin caused about 50% of the receptors to be trapped intracellularly within 15 min. The receptors that remained on the surface after monensin treatment could be trapped within the cell if LDL was added subsequently in the continued presence of monensin. Monensin did not decrease surface LDL receptors in fibroblasts from a patient (J.D.) with the internalization-defective form of familial hypercholesterolemia. In these mutant cells, LDL receptors are not localized to coated pits. The current data are interpreted to indicate that: in normal fibroblasts about 50% of surface LDL receptors absence of LDL; the remaining 50% of surface receptors can be induced to recycle by the presence of LDL; and monensin interrupts this recycling by preventing the receptor from returning to the surface, thereby causing the receptors to accumulate within the cell.     

Origin and function of the stalk-cell vacuole in Dictyostelium

Large vacuoles are characteristic of plant and fungal cells, and their origin has long attracted interest. The cellular slime mould provides a unique opportunity to study the de novo formation of vacuoles because, in its life cycle, a subset of the highly motile animal-like cells (prestalk cells) rapidly develops a single large vacuole and cellulosic cell wall to become plant-like cells (stalk cells). Here we describe the origin and process of vacuole formation using live-imaging of Dictyostelium cells expressing GFP-tagged ammonium transporter A (AmtA-GFP), which was found to reside on the membrane of stalk-cell vacuoles. We show that stalk-cell vacuoles originate from acidic vesicles and autophagosomes, which fuse to form autolysosomes. Their repeated fusion and expansion accompanied by concomitant cell wall formation enable the stalk cells to rapidly develop turgor pressure necessary to make the rigid stalk to hold the spores aloft. Contractile vacuoles, which are rich in H⁺-ATPase as in plant vacuoles, remained separate from these vacuoles. We further argue that AmtA may play an important role in the control of stalk-cell differentiation by modulating the pH of autolysosomes.     

Endocytosis,digestive vacuolar movement and exocytosis on refeeding starvedTetrahymena pyriformis GL-9

Summary Evidence is presented in support of the hypothesis that the contents of digestive vacuoles in refed starvedTetrahymena pyriformis GL-9 are egested from the cell in approximately the sequence of their order of formation. The investigations involved measurements of the rates of disappearance of digestive vacuoles from the cells and the subsequent appearance of egested globules in the surrounding medium using both cultures and individual cells. The cells were first fed peptone and latex particles for a period and then this type of vacuole formation was suppressed by the addition of excess carmine particles (or the process was repeated with the particles in reverse order). Thus two types of morphologically distinct digestive vacuoles could be produced and observed microscopically. These observations suggest that the temporal nature of the movement of the digestive vacuoles through the cell result in the temporal nature of egestion and that no selective mechanism occurs at egestion. Thus digestive vacuoles are thought to pass through the cell from cytopharynx to cytoproct in approximately the order formed and at approximately constant rate. Under conditions of excess nutrients, where the cells become filled with digestive vacuoles, they seem to be able to maintain an approximately uniform number of digestive vacuoles within themselves by maintaining approximately constant and equal rates of vacuole formation and egestion. The maximum rates of latex or carmine vacuole formation or egestion found in single cells were approximately 0.3–0.4 vacuoles per cell per minute. The results are discussed.     

Prevention of Monensin-Induced Hyperpolarization in NG108-15 Cells

The NG108-15 (neuroblastoma X glioma hybrid) cell line was used as an in vitro neuronal model to evaluate potential antagonists of the Na⁺-selective carboxylic ionophore monensin. Changes in membrane electrical characteristics induced by monensin with and without the simultaneous administration of antagonists were measured using intracellular microelectrode techniques. Bath application of monensin (3 M) produced a hyperpolarization of 35 mV. Monensin also altered the generation of action potentials in response to electrical stimulation in 14 of 24 (58%) exposed cells, as evident in a partial or complete loss of action potentials or in an alteration of action potential waveform. The antagonists used were Na⁺-K⁺ pump inhibitor ouabain (1–3 M), the Ca²⁺-dependent K⁺ channel blocker quinine (3–30 M) or drugs known to influence Ca²⁺ signaling in cells, i.e., trifluoperazine (3–10 M), verapamil (1–10 M) or chlorpromazine (3–30 M). On a molar basis, ouabain was the most and trifluoperazine the least effective of the antagonists. Quinine, verapamil and chlorpromazine all prevented the development of the hyperpolarization in an approximate concentration-dependent manner. However, none of these drugs was able to block the effects of monensin on action potentials. Indeed, high concentrations of the antagonists that were most effective in preventing the hyperpolarization accentuated impairments in action potential generation and also reduced input resistance in many cells. Thus, none of these antagonists appears suitable for transition to in vivo antidotal protection studies.     

Ultrastructural observations of maize root tips following exposure to monensin

Summary Epidermal and outer rootcap cells of maize root tips were treated with the sodium selective ionophore, monensin, and the ultrastructural changes were studied. In the presence of 10^–5 to 10^–3 M monensin, dictyosomes became distorted, cisternae separated from the stack, and secretory vesicles were released. Released secretory vesicles disappeard from the cytoplasm suggesting that their transport to, and fusion with, the plasma membrane was unaffected. Monensin did not inhibit cytoplasmic streaming of the outer rootcap cells. No new secretory vesicles were formed on the remaining dictyosomes or dictyosome fragments. In contrast to results with animal cells, swelling of plant dictyosome cisternae was observed only after fixation in glutaraldehyde-osmium tetroxide and not after fixation in potassium permanganate. Other cell components were not altered structurally by monensin. The effects of monensin on the Golgi apparatus were reversible, and dictyosomes were either repaired or new dictyosomes were formed after the root tips were removed from the monensin.Dictyosomes in epidermal cells reacted in the same manner as those in the rootcap except that numerous secretory vesicles remained in the cytoplasm, mostly in association with dictyosome fragments. Some secretory vesicles increased in size but no evidence of vesicle-vesicle fusion was noted. Cell plate formation was partially inhibited or blocked by monensin.Mention of a commercial or proprietary product in this paper does not constitute an endorsement of this product by the USDA.     

The adverse effects of HEPES,TES, and BES zwitterion buffers on the ultrastructure of cultured chick embryo epiphyseal chondrocytes

Summary Chick embryo epiphyseal chondrocytes cultured in media containing HEPES, TES, and BES zwitterion buffers, used in combination or independently, consistently developed cytoplasmic vacuoles. This cytoplasmic vacuolation was resolved when the zwitterion buffered media was replaced by media containing bicarbonate:CO₂ enriched air buffer. Vacuoles were infrequent or absent in cultures grown in bicarbonate:CO₂ enriched air. Chondrocytes with an established extracellular matrix showed less vacuolation than fibroblastlike and polygonal shaped cells that lacked such a matrix. The granular endoplasmic reticulum and Golgi dictyosomes of zwitterion buffered chondrocytes were distended and contained a flocculent amorphous material. Cytoplasmic vacuoles (0.5 to 3.0 μm diam) formed by the fusion and intracellular accumulation of Golgi vesicles and vacuoles also contained a flocculent material enhanced by ruthenium red. Membrane bound extracellular vacuoles containing ruthenium red stained proteoglycan aggregates were common in the extracellular matrix of zwitterion buffered cultures but were generally absent from bicarbonate treated cultures. Electron dense calcium deposits seemed much larger and more numerous in the presence of zwitterion buffers. It is suggested that HEPES, TES, and BES buffers, used alone or in combination, may adversely affect cell membrane systems, and thus the transport or secretory mechanisms operative in cultured chondrocytes, or both, resulting in vacuole formation and the intracellular accumulation of synthesized export material. Although the mechanism by which HEPES, TES, and BES induce these changes remains unclear, the use of zwitterion buffers in biological preparations should be treated with caution. This work forms part of a project on Connective Tissue Remodelling supported and financed by the Medical Research Council of New Zealand, of which M. H. F. is a Career Fellow.     

Monensin intercalation in liposomes: effect on cytotoxicities of ricin,Pseudomonas exotoxin A and diphtheria toxin in CHO cells

Monensin, a car☐ylic ionophore was intercalated in liposomes (liposomal monensin) and its effect on cytotoxicities of ricin, Pseudomonas exotoxin A and diphtheria toxin in CHO cells was studied. Intercalation of monensin in liposomal bilayer is found to have no effect on its stability and interaction with cells. Liposomal monensin)(1 nM) substantially enhance the cytotoxicities of ricin (62-fold) and Pseudomonas exotoxin A (11.5-fold) while it has no effect on diphtheria toxin. This observed effect is highly dependent on the liposomal lipid composition. The potentiating ability of monensin (1 nM) in neutral vesicles is significantly higher (2.2-fold) as compared to negatively charged vesicles. This ability is drastically reduced by incorporation of stearylamine in liposomes and is found to be dependent on the density of stearylamine as well as on the concentration of serum in the medium. Monensin in liposomes containing 24 mol% stearylamine has a very marginal effect on the cytotoxicity of ricin (7.5-fold) which is further reduced (1.5-fold) in the presence of 20% serum. The uptake of ¹²⁵I-gelonin from neutral vesicles is significantly higher (∼ 2.0-fold) than that from the negative vesicles. The uptake from positive vesicles is highly dependent on the concentration of stearylamine. The reduction in the lag period (30 min) of ricin action by monensin in neutral and negative vesicle is comparable with free monensin. However, monensin in positive vesicle has no effect on it. These studies have suggested that liposomes could be used as a delivery vehicle for monensin for selective elimination of tumor cells in combination with hybrid toxins.     

Immunocytochemical localization of procollagen and fibronectin in human fibroblasts: effects of the monovalent ionophore, monensin

The monovalent ionophore monensin inhibits the secretion of both procollagen and fibronectin from human fibroblasts in culture. The distribution of these proteins in control and inhibited (5 x 10(-7) M monensin) cells has been studied by immunofluorescence microscopy. In control cells, both antigens are present throughout the cytoplasm and in specific deposits in a region adjacent to the nucleus, which we identify as a Golgi zone by electron microscopy. Treatment of cells with monensin causes intracellular accumulation of procollagen and fibronectin, initially in the juxta-nuclear region and also subsequently in peripheral regions. Electron microscope studies reveal that in such cells the juxta-nuclear Golgi zone becomes filled with a new population of smooth-membraned vacuoles and that normal Golgi complexes are not found. Immunocytochemically detected procollagen and fibronectin are localized in the region of these vacuoles, whereas more peripheral deposits correspond to the dilated cisternae of rough endoplasmic reticulum, which are also caused by monensin. Procollagen and fibronectin are often codistributed in these peripheral deposits. Accumulation of exportable proteins in Golgi-related vacuoles is consistent with previous analyses of the monensin effect. The subsequent development of dilated rough endoplasmic reticulum also containing accumulated proteins may indicate that there is an additional blockade at the exit from the endoplasmic reticulum, or that the synthesized proteins exceed the capacity of the Golgi compartment and that their accumulation extends into the endoplasmic reticulum.     

Monensin inhibition of corticotropin releasing factor mediated ACTH release

Monensin is a sodium selective carboxylic ionophore that has been helpful in studying the intracellular mechanisms of protein secretion by its ability to inhibit transport of secretory proteins, particularly through the Golgi apparatus, and by its capacity to block intracellular posttranslational processing events. We studied in rat anterior pituitary cell culture the effects of monensin on: CRF stimulated ACTH release; presynthesized (stored) ACTH release; and on forskolin- (activator of adenylate cyclase) and KCl- (a membrane depolarizer which does not stimulate ACTH synthesis) induced ACTH release. Monensin inhibited CRF stimulated ACTH release in a dose-dependent fashion. The ED50 was 2.7 x 10(-8) M and maximal inhibition was 52% at 1.5 x 10(-7) M. Inhibition at 40 minutes of CRF incubation was similar to the percent inhibition noted at 1 hr 40 min and 2 hr 40 min. Monensin (1.5 x 10(-6) M) decreased the amount of ACTH release from cells incubated with cycloheximide plus CRF by 32% (p less than 0.01). Monensin individually inhibited forskolin (2 x 10(-6) M) and dibutyryl cyclic AMP (3 x 10(-3) M) mediated ACTH release in a dose-dependent fashion. The inhibition of forskolin and dibutyryl cyclic AMP mediated ACTH release by 1.5 x 10(-6) M monensin was 48% and 46% respectively. Monensin (1.5 x 10(-6) M) also reduced KCl (50 mM) stimulated ACTH release by 48%. This study demonstrates that monensin inhibits CRF mediated ACTH release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Variations in the Ability of Ruminal Gram-Negative Prevotella Species to Resist Monensin

Gram-negative, ruminal Prevotella strains (n = 15) differed greatly in their sensitivity to the feed additive monensin. Strains that were repeatedly transferred with sublethal doses tolerated more monensin than those that were unadapted, but growth experiments indicated that the sensitivity range was as great as 2000-fold. Prevotella bryantii B₁4 grew with monensin concentrations as high as 20 μM, but P. ruminicola H15a, D31d, 20-63, E40a, and D42f never initiated growth if monensin was greater than 0.01 μM. Washed cell preparations that were energized with glucose lost intracellular potassium when monensin was added, and potassium depletion could also be used as an index of monensin sensitivity. Adapted cells of P. bryantii B₁4 had a half-maximal potassium depletion constant (K _d) of 3.2 μM, but the K _d values of P. ruminicola strains H15a, D31d, 20-63, E40a, and D42f were less than 0.04 μM. Maximal potassium depletion (K _max) values range from 90% to 40%, and monensin-adapted cells always had lower K _max values than unadapted cells. A linear regression of log K _d/K _max versus percentage decrease in optical density divided by monensin concentration had an r² of 0.75, and this regression indicated that potassium depletion from washed cells closely correlated with growth inhibition. P. bryantii B₁4 had a K _d/K _max ratio that was sevenfold greater than other Prevotella strains, and this result indicated that P. bryantii may be unusual in its ability to grow with very high concentrations of monensin. Received: 13 August 1999 / Accepted: 5 October 1999     

Interaction of Cytotoxic Bicyclic Peptides, Theonellamides A and F, with Glutamate Dehydrogenase and 17β-Hydroxysteroid Dehydrogenase IV

Theonellamide A, a bicyclic peptide isolated from a Theonella sponge, was fixed on hydrazide-containing gel beads and screened for its binding proteins from rabbit liver tissues. Analysis by sodium dodecyl sulfate–polyacrylamide gel electrophoresis revealed that two major proteins of 80 kDa and 55 kDa interacted with theonellamide A. The interaction between theonellamide A and two proteins was confirmed by competition experiments in which these two proteins failed to bind to theonellamide A–conjugated gel beads in the presence of theonellamide A or F. Amino-terminal amino acid sequence analysis of peptide fragments derived from the binding proteins by lysylendopeptidase digestion demonstrated that the 80-kDa and 55-kDa proteins were 17β-hydroxysteroid dehydrogenase IV and glutamate dehydrogenase, respectively. In an in vitro assay system, amination of α-ketoglutarate by glutamate dehydrogenase was activated with theonellamide F, although this effect was weaker than that with adenosine diphosphate, a well-known activator. Received October 15, 1999; accepted January 4, 2000.     

Effect of monensin on the neuronal ultrastructure and endocytic pathway of macromolecules in cultured brain neurons

1. The endocytic pathway of horseradish peroxidase (HRP) was investigated in the perikarya of cultured neurons by electron microscopy and enzyme cytochemistry. The tracer was observed in endocytic pits and vesicles, endosomes, multivesicular bodies, and lysosomes. It took approximate 15 min for the transfer of HRP from the exterior of the cell to the lysosomes. 2. Monensin induced distension of the Golgi apparatus and formation of intracellular vacuoles. When neurons were incubated with both monensin and HRP for 30 to 120 min, the number of HRP-labeled endosomes was greater than that in the monensin-free group, whereas the reverse was seen for HRP-positive lysosomes. The formation of HRP-positive lysosomes in monensin-treated cells was blocked by 47 to 79%. 3. These results indicate that the intracellular transport of the endocytosed macromolecule is pH dependent. It is also possible that the export of lysosomal enzymes is inhibited by monensin, resulting in an accumulation of the endosomes and a reduction of the lysosomes.