Lead induced injury on in vitro cultured rat fibroblasts

Summary The mechanism for lead induced cellular injury was studied on in vitro cultured rat fibroblasts, treated with lead in a dose that caused demonstrable cellular alterations within a couple of days. The changes were studied by means of methods for the histochemical demonstration of heavy metals and lysosomal membrane latency.The lead, added to the cultivation medium, was quickly incorporated into presumed secondary lysosomes of the fibroblasts and caused regressive cellular changes such as cytoplasmic vacuolization, nuclear pycnosis and eventually cell death. The lead exposure resulted in reduced lysosomal membrane latency and signs of enzyme and lead leakage to the cytoplasm.The cell damage might be mediated through lysosomal membrane alteration resulting in reduced latency and presumably leakage of lytic enzymes.Supported by the Swedish Medical Research Council (grant no 12 X-2037).     

Heavy metal localization and age related accumulation in the rat nervous system

Summary A modified sulfide-silver method was used to demonstrate tissue bound heavy metals in the rat brain at various ages. An accumulation of sulfide-silver positive material was found to accompany aging, indicating heavy metal accumulation. This was verified by quantitative analysis using atomic absorption spectrophotometry. Iron appears to be the most important heavy metal. Besides differences between various ages, regional variations in heavy metal contents could constantly be shown. The heavy metals appear, at least in part, to be located in lysosomes. A heavy metal influence on the lysosomal membrane permeability is discussed.Supported by the Swedish Medical Research Council grant No. 12X-2037.     

Chemical and physical effects on the shape and growth of the diatomBiddulphia sinensis Greville in batch cultures: A contribution to bioindication in plankton ecology

Summary Different influences, like changes in salinity and temperature, the effects of heavy metals like copper and mercury, and the influences of nitrate and silicate deficiencies on the shape, generation time, and yield of the marine plankton diatomBiddulphia sinensis were tested in batch cultures. Those cells which show multiple or missing setae are classified as seta-aberrant, teratological forms. Sudden changes in salinity and temperature (30–15%. S; 12–20°C) lead to no more than 5% of aberrant cells (control cultures below 1%). Heavy metals (copper and mercury) cause a significant increase of the generation time, a distinct decrease of the cell yield, and (in the case of copper) up to 50% of seta-aberrant cells. The cells die at concentrations of copper above 10^–5M.l^–1, though EDTA was added as chelant. At concentrations below 10^–5M.l^–1 of copper, addition of EDTA as well as an increase of temperature from 12 to 20°C cause a reduction of toxicity. During batch growth the cells adapt to mercury but not to copper. At mercury concentrations above 10^–8M.l^–1 the cells die, even if EDTA is added. Unlike in copper-treated medium, EDTA does not reduce the toxicity of mercury. However, a detoxicating effect (20 instead of 12°C) can be observed due to higher temperature. Silicate deficiency, like the influence of copper and mercury leads to an elongation of the pervalvar axes. Particularly at a temperature of 20°C bended pervalvar axes and sometimes lateral evaginations of the frustule will be formed; this does not happen under the influence of copper and mercury. Multiple setae never appear under silicate deficiency. Under nitrate deficiency more than 10% of the cells are seta-aberrant. The suitability ofBiddulphia sinensis for test experiments in shipboard cultures is shown. Water samples were tested on the lack of nitrate, silicate, and phosphate. The percentage of seta-aberrant cells, the generation times, and the yields were determined. Simultaneous lack of phosphate and nitrate causes up to 30% of teratological cells. The seta-aberrant cells always appear during the exponential growth phase.     

Pb-induced cellular defense system in the root meristematic cells of <Emphasis Type="Italic">Allium sativum</Emphasis> L

Background  

Electron microscopy (EM) techniques enable identification of the main accumulations of lead (Pb) in cells and cellular organelles and observations of changes in cell ultrastructure. Although there is extensive literature relating to studies on the influence of heavy metals on plants, Pb tolerance strategies of plants have not yet been fully explained. Allium sativum L. is a potential plant for absorption and accumulation of heavy metals. In previous investigations the effects of different concentrations (10^-5 to 10^-3 M) of Pb were investigated in A. sativum, indicating a significant inhibitory effect on shoot and root growth at 10^-3 to 10^-4 M Pb. In the present study, we used EM and cytochemistry to investigate ultrastructural alterations, identify the synthesis and distribution of cysteine-rich proteins induced by Pb and explain the possible mechanisms of the Pb-induced cellular defense system in A. sativum.     

Blockade of heavy metals accumulation in Chlorella vulgaris cells by 24-epibrassinolide

This study was conducted on the influence of 24-epibrassinolide (24-epiBL) mixed with varying concentrations of heavy metals (copper, lead, cadmium, zinc) upon the growth and accumulation of these heavy metals in the cell of the alga Chlorella vulgaris Beijerinck (Chlorophyceae). Heavy metals at the concentration of 10^–3 M, alone or mixed with 24-epiBL, showed a lethal effect on C. vulgaris. At metal concentrations of 10^–6–10^–4 M, a combination with 24-epiBL appeared to have a stronger stimulatory effect on a number of cells than a single metal (a stronger inhibitory effect). 24-EpiBL at the concentration of 10^–8 M in combination with heavy metals (in the range 10^–6–10^–4 M) blocked metal accumulation in algal cells. 24-EpiBL has an anti-stress effect on C. vulgaris contaminated by heavy metals. The inhibitory effect on metal accumulation of 24-epiBL mixed with different heavy metals was arranged in the following order: zinc > cadmium > lead > copper. This process is correlated with the stimulation of growth of C. vulgaris. The stimulatory effect of 24-epiBL mixed with heavy metals leading to an increased pH in the medium (5.28–6.20) was significantly higher than the impact due to the increased acidity in the medium due to metals alone (pH 3.10–5.85). Lower pH increased the toxicity of heavy metals in C. vulgaris cells.     

Nickel toxicity inPseudomonas tabaci: Single cell and bulk sample analysis of bacteria cultured at high cation levels

Summary The growth ofPseudomonas tabaci in nutrient medium is partially inhibited in the presence of 10^–3 M added nickel (threshold toxic concentration), with complete inhibition at 10^–2 M nickel—but no effect at 10^–4 and 10^–5 M. Toxic levels of nickel affect both cell division and cell viability.Spectrophotometric determination of intracellular levels of nickel at different external concentrations showed that the highest internal values occurred with cells cultured in 10^–4M (non-toxic) nickel medium rather than in 10^–3 (toxic) medium—suggesting that nickel toxicity does not primarily relate to internal concentration.X-ray microanalysis, carried out on whole bacterial cells, showed that toxic levels of nickel in the external medium resulted in a range of ionic changes in the cell, including a decrease in the level of K (K efflux) and an increase in the levels of Mn, Fe, Ni, and Cu (transition metal cation influx). Other changes induced by nickel toxicity included an increase in the level of soluble S (with a decrease in insoluble S), an increased cell dry mass, and a conspicuous plasmolysis—which was observed both in whole cells and in ultrathin sections.The results obtained support a primary toxic effect of nickel at the cell surface—possibly directly affecting the transport activity of the plasmalemma. The resulting changes, particularly involving the influx of a range of cations, may lead to secondary toxic activities affecting the whole metabolism, leading to plasmolysis and inhibition of division.     

Effects of heavy metals on growth and ultrastructure ofChara vulgaris

Summary The toxicity of some heavy metals to the common macrophytic freshwater algaChara vulgaris was studied under laboratory conditions. For experiments, apical tips of algae containing two internodes were cultivated for fourteen days in the presence of various concentrations of cadmium, mercury or lead (as triethyl lead or lead nitrate). Fifty percent growth inhibition occurred with concentrations of 8.5×10^–8 M (9.5 ppb) cadmium, 7.5×10^–7M (150ppb) mercury, 1.6×10^–6 M (330ppb) organic lead or 4× 10^–5 M (8000 ppb) inorganic lead. Sublethal concentrations of these metals caused alterations in the fine structure of internodal cells which turned out to be at least partly metal-specific or in the case of lead, the effects depended on whether the lead was ionic or organically bound. Cadmium and inorganic lead induced disorders of cell wall microfibrils which resulted in local wall protuberances. Mercury affected the chloroplasts which mostly showed considerably increased grana stacks. In addition, mercury caused a dilation of the endoplasmic reticulum and of the mitochondrial tubuli. Organic lead damaged the membrane system of chloroplasts; sheet- or tubule-like thylakoids were disarranged and showed whorl-like structures. At higher concentrations of organic lead, tubular invaginations of the plasmalemma (charasomes) disappeared. The fine structure of nuclei was not altered by any of the metals.     

Pituitary monoamines of the cat with special reference to the presence of an unidentified monoamine-like substance in the adenohypophysis

Summary In the pituitary gland of the cat, dopamine (M.V. 0.78 g/g), noradrenaline (M.V. 0.29 g/g) and 5-HT (M.V. 0.94 g/g) have been found. With the histochemical fluorescence method, a rich system of delicate fluorescent varicose fibres, often provided with irregular swellings or droplets, was observed in the neural lobe and pars intermedia. Microspectrofluorimetrically, these fibre structures exhibit the spectral characteristics of catecholamines. Most cells in the pars intermedia and a large number of cells in pars distalis show a yellowish fluorescence, with microspectrofluorimetric characteristics which differ entirely from those of the catecholamines and 5-HT. In animals treated with reserpine, the pituitary dopamine, noradrenaline, and 5-HT are largely depleted. However, the intensity and the spectral properties of the cellular fluorescence are not affected by this treatment, whereas the fluorescent fibres can no longer be seen. Thus none — or only little — of the catecholamines and 5-HT but some other monoamine-like substance is stored in the fluorescent cells of the adenohypophysis. Preliminary studies suggest that this substance is closely related to or perhaps identical with tryptamine.Supported by grants from the Swedish Medical Research Council (No B 68-12X-712-03 B and B 68-14X-56-04 B), the Association for the Aid of Crippled Children, New York, and by the Faculty of Medicine, University of Lund, Sweden.     

Complexation of Uranium by Cells and S-Layer Sheets of Bacillus sphaericus JG-A12

Bacillus sphaericus JG-A12 is a natural isolate recovered from a uranium mining waste pile near the town of Johanngeorgenstadt in Saxony, Germany. The cells of this strain are enveloped by a highly ordered crystalline proteinaceous surface layer (S-layer) possessing an ability to bind uranium and other heavy metals. Purified and recrystallized S-layer proteins were shown to be phosphorylated by phosphoprotein-specific staining, inductive coupled plasma mass spectrometry analysis, and a colorimetric method. We used extended X-ray absorption fine-structure (EXAFS) spectroscopy to determine the structural parameters of the uranium complexes formed by purified and recrystallized S-layer sheets of B. sphaericus JG-A12. In addition, we investigated the complexation of uranium by the vegetative bacterial cells. The EXAFS analysis demonstrated that in all samples studied, the U(VI) is coordinated to carboxyl groups in a bidentate fashion with an average distance between the U atom and the C atom of 2.88 ± 0.02 Å and to phosphate groups in a monodentate fashion with an average distance between the U atom and the P atom of 3.62 ± 0.02 Å. Transmission electron microscopy showed that the uranium accumulated by the cells of this strain is located in dense deposits at the cell surface.     

Enzymatic sulfation of cholesterol by rat gastric mucosa

A sulfotransferase which catalyzes transfer of the sulfate group from 3'-phosphoadenosine-5'phosphosulfate to cholesterol has been demonstrated in the rat gastric mucosa. The product of the reaction was characterized as cholesterol sulfate by two-dimensional thin-layer Chromatographic behavior, and gas-liquid chromatography of cholesterol after acid solvolysis. The bulk of enzyme activity was found in the cytosol fraction. Sulfation of cholesterol did not require added Mg⁺², Mn⁺², or Ca⁺², and was unaffected by ethylenedia-minetetraacetate. Triton X-100 moderately enhanced the enzyme activity. A broad pH optimum from pH 6.0–9.0 was exhibited with a maximum at pH 7.0–7.5. The apparent Km for PAPS was 0.8 × 10^?6M. The possible function of cholesterol sulfate in gastric mucosa is discussed.     

Molecular rearrangements of thylakoids after heavy metal poisoning, as seen by Fourier transform infrared (FTIR) and electron spin resonance (ESR) spectroscopy

The specific effects exerted by different heavy metals on both the function and the structure of the photosynthetic apparatus were addressed. The functional analysis performed via the fluorescence induction kinetics revealed that the applied toxic heavy metals can be classified into two groups: Cd and Ni had no significant effect on the photosynthetic electron transport, while Cu, Pb and Zn strongly inhibited the Photosystem II (PS II) activity, as evidenced by the dramatic decreases in both the variable (F_v) and the maximal (F_m) fluorescence. The structural effects of the heavy metal ions on the thylakoid membranes were considered in three relations: (1) lipids, (2) proteins — studied by Fourier transform infrared (FTIR) spectroscopy, and (3) lipid—protein interactions — investigated by electron spin resonance (ESR) spectroscopy using spin-labeled probe molecules. The studied heavy metal ions had only a non-specific rigidifying effect on the thylakoid lipids. As regards proteins, Cd and Ni had no effect on the course of their heat denaturation. The heat denaturation of the proteins was accompanied by a decrease in the -helix content (1656 cm^-1), a parallel increase in the disordered segments (1651 cm^-1), a decrease in the intramolecular -sheet (1636 cm^-1) content and the concomitant appearance of an intermolecular -structure (1621 cm^-1). In contrast with Cd and Ni, Cu and Zn blocked the appearance of the intermolecular -structure. Pb represented an intermediate case. It seems that these heavy metals alter the native membrane structure in such a way that heat-induced aggregation becomes more limited. The ESR data revealed that certain heavy metals also affect the lipid—protein interactions. While Cd and Ni had hardly any effect on the solvation fraction of thylakoid lipids, Cu, Pb and Zn increased the fraction of lipids solvating the proteins. On the basis of the FTIR and ESR data, it seems that Cu, Pb, and Zn increase the surfaces available for lipid—protein interactions by dissociating membrane protein complexes, and that these lipidated proteins have a smaller chance to aggregate upon heat denaturation. The data presented here indicate that the damaging effects of poisonous heavy metals are element-specific, Cu, Pb and Zn interact directly with the thylakoid membranes of the photosynthetic apparatus, while Cd and Ni interfere rather with other metabolic processes of plants.     

A comparative study of the effect of various detergents on the structure and function of sarcoplasmic reticulum vesicles

Summary The effects produced by the detergents Triton X-100, sodium dodecylsulphate and sodium cholate on sarcoplasmic reticulum vesicles have been comparatively studied. In all cases, maximal effects are found 5 min after detergent addition. Triton X-100 and SDS are approximately ten times more effective than cholate in protein and phospholipid solubilization. Both Triton X-100 and SDS maintain Ca⁺⁺ accumulation in SR vesicles at detergent concentrations below 10^–3 M; higher concentrations cause a strong inhibition. On the other hand, cholate produces a gradual inhibition of Ca⁺⁺ accumulation in the concentration range between 10^–4 M and 2.5 × 10^–2 M. Triton X-100 and SDS produce a gradual solubilization of the specific Ca⁺⁺-ATPase activity up to a 10^–3 M detergent concentration, above which a strong inactivation occurs, while the enzyme solubilization increases with the presence of cholate in the whole concentration range under study. The different behaviour of sodium cholate, when compared to SDS or Triton X-100, is discussed in relation to the surfactant molecular structures. The possibility of membrane lysis and reassembly in the presence of some detergents is also considered.Abbreviations SR sarcoplasmic reticulum - SDS sodium dodecylsulphate - DTT dithiothreitol - EGTA ethyleneglycoltetraacetate - PEP phosphoenolpyruvate     

Effects of dexamethasone on metallothionein induction by Zn, Cu, and Cd in Chang liver cells

Metallothioneins (MTs) were induced in Chang liver cells by the metals, Zn, Cu and Cd, and the glucocorticoid hormone, dexamethasone. When 116 microM Zn, 32 microM Cu and 18 microM Cd, and 10(-7) M dexamethasone, respectively, were administered for 9 h, MTs induced by each inducer in the cells reached maximum levels. The maximum accumulation of MT level induced by dexamethasone was the lowest of the four inducers investigated; the levels induced by Zn, Cu and Cd were 4.7, 1.2 and 1.5 times of that induced by dexamethasone. When dexamethasone was added to the cells together with the heavy metals (Zn, Cu and Cd), dexamethasone had an additive effect on the maximum MT accumulations induced by heavy metals as compared to when induction was conducted using one of heavy metals alone or by dexamethasone alone. However, dexamethasone did almost not effect the metal accumulations in the cells, although the maximum MT levels induced by heavy metal increased by dexamethasone. These results suggest that the process of MT induction by heavy metals and that by dexamethasone are independent of one another. When dexamethasone was added to the cells together with a high concentration of Cu (32 microM) induced the maximum MT accumulation, Cu transport into the cells decreased by 20-40% of that into non-treated cells, which was statistically significant.     

In-vitro auxin binding to particulate cell fractions from corn coleoptiles

Summary When low concentrations (e.g. 10^-6 M) of labelled 3-indoleacetic acid (¹⁴C-IAA) or -naphthaleneacetic acid (¹⁴C-NAA) are added in vitro to homogenates of corn coleoptiles, radioactivity is reversibly bound to pelletable particles. From the saturation kinetics of the binding it is possible to estimate an apparent K _M between 10^-6 M and 10^-5 M and a concentration of specific sites of 10^-7–10^-6 M per tissue volume.The binding is auxin-specific. Among many compounds tested, only auxins and such auxin analogues that are known to interact directly with auxin in transport and/or growth were found to interfere with this binding. For instance, the growth-active d-dichlorophenoxyisopropionic acid at 10^-4 M inhibits ¹⁴C-NAA binding more than the less active l-isomer.The auxin-binding fractions are practically free of DNA and cytochrome-C oxidase and contain binding sites for 1-naphthylphthalamic acid. The results are discussed in context with the hyothesis—derived mainly from physiological data—that auxin receptors are localized at the plasma membrane.     

Biosynthesis and structure of membrane and secretory immunoglobulins

Summary Almost all of the body's extracellular immunoglobulin (Ig) is derived from Ig-secreting plasma cells of lymphoid tissues. The secreted material is a heterogeneous mixture of different classes and specificities. Lymphoid tissues also contain a large number of essentially non-secretory cells — B lymphocytes — which bear Ig firmly associated with their plasma membranes. Ig molecules thus exist in two functionally different forms, as membrane-bound antigen receptors on the surface of B lymphocytes on the one hand, and as humoral secreted Ig antibodies on the other. On B cells, membrane-bound heavy chains have an apparent mol. wt. slightly larger than that of secreted heavy chains from plasma cells. Membrane-bound but not secreted heavy chains bind detergents, thus suggesting the presence of a hydrophobic region in membrane-bound heavy chains, which is absent in secreted heavy chains. Most investigations have dealt with immunoglobulin M. The two types of IgM heavy chains differ at their carboxy termini. Recent investigations at the nucleic acid level demonstrate that membrane-associated µ chains contain a 41-residue hydrophobic tail adjacent to the last constant domain, whereas secretory µ chains contain a 20-residue hydrophilic tail. At the present time, evidence is accumulating that all membrane-bound Ig heavy chain classes may contain similar hydrophobic structures necessary for anchorage of the molecules into the lipid bilayer.     

Heat shock protein HSP90 and its association with the cytoskeleton: a morphological study.

To investigate the cellular localization of the 90-kilodalton heat shock protein (HSP90) and its interaction with the cytoskeleton, we performed single- and double-staining immunofluorescence microscopy of cytoskeletal proteins and HSP90 in the absence and presence of cytoskeletal inhibitors. As a model, we used a human endometrial adenocarcinoma cell line (Ishikawa cells), which expresses HSP90. We confirmed the recently reported colocalization of HSP90 with microtubules. However, Ishikawa cells treated with 10(-5) M of the antimicrotubule agents colchicine or triethyl lead showed residual filamentous structures stained with anti-HSP90 antibodies, while no microtubules were visualized with anti-tubulin antibodies. In the presence of 10(-5) M cytochalasin B, the microfilament staining of the cells disappeared, while residual filamentous structures were labeled with anti-HSP90 antibodies. Furthermore, Ishikawa cells treated with 10(-5) M triethyl lead and stained with anti-HSP90 antibodies demonstrated residual filamentous structures, clearly different from those of reorganized vimentin intermediate filaments. Conversely, similar reorganized morphology of filamentous structures stained with both anti-HSP90 and anti-cytokeratins antibodies were observed when Ishikawa cells were treated with 2 x 10(-5) M cytochalasin B and 2 x 10(-5) M colchicine. HSP90 was also present in Ishikawa cell preparations of the Triton X-100 insoluble cytoskeleton. In addition, Triton-insoluble cytoskeleton treated with 10(-5). M triethyl lead and double stained with anti-HSP90 and anti-vimentin antibodies demonstrated clearly different filamentous patterns, when exposed on the same photographic plaque.(ABSTRACT TRUNCATED AT 250 WORDS)     

On the histochemical demonstration of N-acetyl-β-galactosaminidase

Summary Aging neurons accumulate lipofuscin pigment granules which appear to be secondary lysosomes of the residual body variety. The biological significance of the residual bodies is debated. They were here studied with the aim of testing a hypothesis that the membranes surrounding these granules might be more vulnerable than the membranes around younger types of lysosomes.For this purpose large motor neurons of young and old rats were compared with respect to lysosomal membrane latency, using a modified Bitensky lysosomal lability test. Utilizing successively increasing incubation times, the lysosomes of old neurons, in particular the residual bodies in polar aggregates of old neurons—presumed to represent lipofuscin pigment granules—were found to have a clearly reduced latency in comparison with lysosomes of young neurons.These findings support the notion that the residual bodies are more fragile than younger lysosomes.Supported by the Swedish Medical Research Council (Project No. 12X-2037).     

Cadmium Resistance Screening in Nitrilotriacetate-Buffered Minimal Media

Media used to determine the MICs of heavy metals for bacteria are unreliable because organic components in the media bind or chelate most of the metal being studied. To define specific metal activity in media and to maintain metal activity at a constant level, HEPES-MES [N-2-hydroxyethylpiperazine-N′ -2-ethanesulfonic acid−2-(N-morpholine)ethanesulfonic acid] salts medium with arabinose medium was modified, and the modified medium was used to examine the MIC of cadmium for Rhizobium fredii USDA 201. Arabinose-HEPES-MES was modified by addition of the chelator nitrilotriacetate to buffer the supply of free Cd²⁺ ion to maintain a constant Cd activity and by the use of only MES to buffer pH (buffered arabinose-MES medium [BAM]). Ca and Mg were supplied at the normal levels for soil solutions, and other trace elements were supplied at the levels required for normal growth of plants. The concentration of free Cd²⁺ ion was calculated by using the computer program GEOCHEM-PC with a corrected data base. The Cd MIC in BAM was 14.0 μM, while that in a tryptone-yeast extract medium was 107 μM. The results indicate that substantial free Cd²⁺ is removed from solution in most standard media, resulting in falsely high MICs. The new BAM medium allows for the precise determination of MICs, thus avoiding the uncertainties associated with other media.     

Effects of aplysiatoxin and debromoaplysiatoxin on growth and differentiation of normal human bronchial epithelial cells

The effects of aplysiatoxin and debromoaplysiatoxin on the clonal growth rate, cross-linked envelope formation and plasminogen activator secretion of normal human bronchial epithelial cells were studied. Neither compound was mitogenic over a wide range of concentrations (10^–13 to 10^–7M). Both aplysiatoxin and debromoaplysiatoxin inhibited clonal growth rate with 50% inhibitory concentrations of 3 × 10^–11M and 10^–10M, respectively. Both compounds induced the formation of cross-linked envelopes and increased plasminogen activator secretion with equal potency. These data are similar to those previously obtained with 12-0-tetradecanoylphorbol-13-acetate and teleocidin B and suggest that aplysiatoxin and debromoaplysiatoxin induce terminal squamous differentiation in normal human bronchial epithelial cells.Abbreviations TPA 12-0-tetradecanoylphorbol-13-acetate - NHBE Normal Human bronchial epithelial - ID₅₀ 50% inhibitory concentration (dose) - PA Plasminogen activator - CLE Cross-linked envelope - LHC Laboratory of Human Carcinogenesis     

Effects of two mitosis inhibitors (Colchicine and Vinblastine) on the distribution and axonal transport of noradrenaline storage particles,studied by fluorescence and electron microscopy

Summary The lumbar sympathetic ganglia and the interganglionic interconnecting nerves of untreated rats and rats treated with Colchicine (COL) or Vinblastine (VIN) were studied with the help of the Falck-Hillarp fluorescence technique and electron microscopy. Both in untreated and drug treated rats there was a good correlation between the distribution of noradrenaline (NA) specific fluorescence and granular vesicles supporting the previous view that the granular vesicles represent the main intraneuronal NA storage sites. The granular vesicles were present both in the cell bodies—mainly in the peripheral part of the cytoplasm— and in the axons of untreated rats. After local application of COL or VIN on the ganglia there was a marked increase in fluorescence intensity and number of granular vesicles in many cell bodies. Often increased number of granular vesicles were found in the neighbourhood of the Golgi apparatus, in which region only few such vesicles are found in untreated rats. In some cell bodies high numbers of granular vesicles could be found all over the cytoplasm.When applied locally to axons the mitosis inhibitors caused a marked accumulation of fluorescence and granular vesicles—and other cell organelles like mitochondria and tubules of the endoplasmic reticulum-proximal to the site of application.A prominent feature both in cell bodies and axons of drug treated rats were large bundles of neurofilaments running through the cytoplasm. In the axons these filaments were often localized to the central part of the axon and surrounded by vesicles and tubules. Microtubules, on the other hand, which are rather numerous in cell bodies and axons of untreated rats seemed to be reduced in number after COL or VIN treatment, especially in those axons in which large amounts of subcellular organelles had accumulated.The present findings are discussed with respect to intraneuronal transport of NA and possible mechanisms behind this transport. It is suggested that the accumulation of fluorescence and granular vesicles after application of mitosis inhibitors is due to an interruption of the centrifugal transport of NA granules. The increased numbers of granular vesicles in the neighbourhood of the Golgi apparatus suggest that granular vesicles are produced in this part of the cytoplasm. This does not exclude a local formation of granular vesicles in other parts of the neuron. Furthermore, the possibility is discussed that the interruption of the transport is related to the increased number of neurofilaments and a possible decrease or disarrangement of microtubules. This discussion is based on previous suggestions that microtubules are involved in intracellular transport mechanisms and on recent findings that COL and VIN bind to proteins specific for microtubules.This study has been supported by grants from the Swedish Medical Research Council (B70-14X-2887-01; B71-14X-2887-02A; B71-14P-3262-01 A; B70-14X-2207-04; B71-14X-2207-05A; K70-40P-3045-01A), from Magnus Bergwalls Foundation, from Wilhelm and Martina Lundgrens Foundation, from the Medical Faculty, University of Göteborg.For generous supply of vinblastine (Velbe^®) we thank Eli Lilly Ltd.The skilful technical assistance of Mrs Kirsten Collin, Mrs Waldraut Hiort and Mr Pär-Anders Larsson is gratefully acknowledged.