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PubMed2002

The biogenesis and functions of exosomes.

Exosomes are membrane vesicles with a diameter of 40-100 nm that are secreted by many cell types into the extracellular milieu. They correspond to the internal vesicles of an endosomal compartment, the multivesicular body and are released upon exocytic fusion of this organelle with the plasma membrane. Intracellularly, they are formed by inward budding of the endosomal membrane in a process that sequesters particular proteins and lipids. The unique composition of exosomes may confer specific functions on them upon secretion. Although their physiological role in vivo is far from being unraveled, it is apparent that they function in a multitude of processes, including intercellular communication during the immune response. Exosomes may have evolved early in the evolution of multicellular organisms and also seem to be important for tissue developmental processes.

Antigen-Presenting Cells↗

PubMed1988

Ultrastructural discrimination of lipid droplets and vesicles in atherosclerosis: value of osmium-thiocarbohydrazide-osmium and tannic acid-paraphenylenediamine techniques.

Electron microscopy of atherosclerotic arterial tissue commonly fails to distinguish lipid vesicles from droplets, especially when these are found in the extracellular space. The distinction is important, because vesicular or membranous lipid is composed of phospholipid and unesterified cholesterol, whereas neutral lipid in droplet form implies the presence of cholesteryl ester in atherosclerosis. A new procedure with sequential tannic acid and p-phenylenediamine treatments of osmicated tissue (TA-PDA) allows reliable ultrastructural discrimination of lipid vesicles and droplets. The multilamellar character of many vesicles is revealed. Extracellular droplets are found to possess many surface pits associated with membranous blebs. Pitting of droplets is especially evident after the use of an alternative tissue processing technique, the osmium-thiocarbohydrazide-osmium (OTO) sequence applied en bloc. The two complementary techniques will prove useful for electron microscopic studies of atherosclerotic and other lipid-rich tissues.

Adult↗

PubMed2004

Hydrogen ions control synaptic vesicle ion channel activity in Torpedo electromotor neurones.

During exocytosis the synaptic vesicle fuses with the surface membrane and undergoes a pH jump. When the synaptic vesicle is inside the presynaptic nerve terminal its internal pH is about 5.5 and after fusion, the inside of the vesicle comes in contact with the extracellular medium with a pH of about 7.25. We examined the effect of such pH jump on the opening of the non-specific ion channel in the synaptic vesicle membrane, in the context of the post-fusion hypothesis of transmitter release control. The vesicles were isolated from Torpedo ocellata electromotor neurones. The pH dependence of the opening of the non-specific ion channel was examined using the fused vesicle-attached configuration of the patch clamp technique. The rate of opening depends on both pH and voltage. Increasing the pH from 5.5 to 7.25 activated dramatically the non-specific ion channel of the vesicle membrane. The single channel conductance did not change significantly with the alteration in the pH, and neither did the mean channel open time. These results support the hypothesis that during partial fusion of the vesicle with the surface membrane, ion channels in the vesicle membrane open, admit ions and thus help in the ion exchange process mechanism, leading to the release of the transmitter from the intravesicular ion exchange matrix. This process may have also a pathophysiological significance in conditions of altered pH.

Animals↗

PubMed1996

Mu-opioid receptor is located on the plasma membrane of dendrites that receive asymmetric synapses from axon terminals containing leucine-enkephalin in the rat nucleus locus coeruleus.

We have recently shown, by using immunoelectron microscopy, that the mu-opioid receptor (mu OR) is prominently distributed within noradrenergic perikarya and dendrites of the nucleus locus coeruleus (LC), many of which receive excitatory-type (i.e., asymmetric) synaptic contacts from unlabeled axon terminals. To characterize further the neurotransmitter present in these afferent terminals, we examined in the present study the ultrastructural localization of an antipeptide sequence unique to the mu OR in sections that were also dually labeled for the opioid peptide leucine-enkephalin (L-ENK). Immunogold-silver labeling for mu OR was localized to extrasynaptic portions of the plasma membranes of perikarya and dendrites. The mu OR-labeled dendrites were usually postsynaptic to axon terminals containing heterogeneous types of synaptic vesicles and forming asymmetric synaptic specializations characteristic of excitatory-type synapses. The majority of these were immunolabeled for the endogenous opioid peptide L-ENK. Some mu OR-labeled dendrites received synaptic contacts from unlabeled axon terminals in fields containing L-ENK immunoreactivity. In such cases, the mu OR-labeled dendrites were in proximity to L-ENK axon terminals that contained intense peroxidase labeling within large dense core vesicles along the perimeter of the axoplasm. These results indicate that L-ENK may be released by exocytosis from the dense core vesicles and diffuse within the extracellular space to reach mu OR sites on the postsynaptic dendrite or dendrites of other neighboring neurons. The present study also reveals that unlabeled terminals apposed to mu OR-labeled dendrites may contain other opioid peptides, such as methionine-enkephalin. These data demonstrate several sites where endogenous opioid peptides may interact with mu OR receptive sites in the LC and may provide an anatomical substrate for the LC's involvement in mechanisms of opiate dependence and withdrawal.

Animals↗

PubMed1996

Recent advances in the ultrastructural assessment of osteoclastic resorptive functions.

Ultrastructural enzyme and immunocytochemical studies have made great contributions to clarifying intriguing questions as to the actual role of osteoclastic ruffled borders in bone resorption. In the present study, vacuolar-type H(+)-ATPase and cysteine-proteinase (cathepsin) were localized in osteoclasts by means of light and electron microscopic immunocytochemistry. The specific immunoreactivity of vacuolar-type H(+)-ATPase was detected along the ruffled border membranes, associated pale vacuoles, and cisterns of the rough-surfaced endoplasmic reticulum of osteoclasts. Anti-cathepsin B immunoreaction occurred in Golgi vesicles, lysosomes, pale vesicles and vacuoles, and the extracellular canals of ruffled borders of osteoclasts. The resorbing bone surfaces were also immunoreactive for anti-cathepsin B. In a coculture system of osteoclasts with devitalized dentine slices, a specific H(+)-ATPase inhibitor (bafilomycin A1) markedly reduced both demineralized areas and resorption lacuna formation on the dentine slices. On the other hand, the cathepsin inhibitor, E-64, inhibited only resorption lacuna formation but had no effect on demineralization of the dentine slices. These results suggest that H(+)-ATPase and cathepsins in osteoclasts are involved, respectively, in the extracellular solubilization of apatite crystals and subsequent degradation of bone matrix and that the ruffled border-clear zone complex of osteoclasts is the main site of cell-matrix interactions during bone resorption processes.

Animals↗

PubMed1987

Myocardial capillaries in the fetal and the neonatal rat: a morphometric analysis of the maturing myocardial capillary bed.

Developing myocardial capillaries from 16-day-gestation fetus to adult undergo several morphological changes including a thinning of the lateral extensions of the capillary endothelial cells, the formation of a basal lamina, and an increase in the number of plasmalemmal vesicles. A decrease in the extracellular space, an increase in the number of capillaries, and a decrease in the capillary diameter were also observed during the developmental period. In view of these ultrastructural changes, a morphometric analysis was made on the developing myocardial wall to demonstrate specific quantitative changes. The volumes which were occupied by capillary endothelial cells, capillary lumina, extracellular space, and myocardial myocytes within a reference volume of myocardium were measured; and we found that 8% of the reference myocardial volume was occupied by capillary endothelial cells, 85% was occupied by myocardial myocytes, 4% was occupied by capillary lumina, and, except for a significant change in extracellular space at 16 days gestation, 3% was occupied by extracellular space. Each volume ratio was found to be nearly constant throughout the studied period. In contrast to this constancy in the volume ratios, other parameters which were measured demonstrated significant changes during the developmental period studied. These overall changes include a 135% increase in capillary density, a 63% increase in luminal surface area of capillary endothelial cells, a 24% decrease in capillary diameter, a 12% decrease in diffusion distance, and a 35% decrease in the diameter of the erythrocyte population.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1976

Effect of cholecystokinin-pancreozymin (CCK-PZ) on glycoprotein secretion from mouse gallbladder epithelium: an ultrastructural and cytochemical study.

Structural changes in the gallbladder epithelial cells of the mouse were studied following in vivo and in vitro stimulation of the gallbladder with the gastrointestinal hormone cholecystokinin-pancreozymin (CCK-PZ). Signs of increased secretory activity were observed within the first 2-3 min after hormone administration. At the ultrastructural level, best visualized with the PA-CrA-silver technique, granule discharge was observed, as was an overall increase in size of the granules. After prolonged in vitro incubation or repeated in vivo stimulation, there was an almost total depletion of secretory granules. This phenomenon is accompanied by an enhanced uptake of extracellular thorium dioxide by endocytotic vesicles at the apical cell surface. An exocytosis-endocytosis coupling mechanism may be important for membrane conservation in the gallbladder epithelial cells. The findings establish that the hormone CCK-PZ stimulates the secretion of glycoproteins from the mouse gallbladder epithelium.

Animals↗

PubMed1976

Acid phosphatase localization in the fungus Whetzelinia sclerotiorum.

Acid phosphatase was localized by light and electron microscopy in chains of vacuoles in hyphal tip cells of Whetzelinia sclerotiorum. The enzyme was present in these vacuoles whether or not conditions favored extracellular acid phosphatase secretion. Apical vesicles, microbodies, Woronin bodies, and lipid bodies did not contain acid phosphatase. The implications regarding terminology of organelles in filamentous fungi are discussed with special reference to the fungal spherosome concept.

Acid Phosphatase↗

PubMed1982

Immunoelectron microscopic localization of albumin in smooth and striated muscle tissues of rat.

Localization of serum albumin in the striated and smooth muscles of rat was studied by an improved immunocytochemical method. Diaphragm, ventricular myocardium, and smooth muscle of stomach were examined. In all of these tissues, albumin was found in the interstitial space and small subsarcolemmal caveolae and vesicles. In addition, the transverse tubular system of the striated muscle stained positive for albumin. The subsarcolemmal vesicles containing albumin did not show any evidence of fusion with lysosomes. Furthermore, in smooth muscle, most of these vesicles were open to the extracellular space. These results demonstrate that albumin in smooth and striated muscle is confined to the extracellular space suggesting that substances such as fatty acids which are carried by albumin are split from it and taken up at the level of the plasma membrane.

Animals↗

PubMed1998

Glutamate release inhibitors: a critical assessment of their action mechanism.

A number of important experimental data do not support the widespread hypothesis that Na(+)-channels block is cerebroprotective, essentially because it reduces presynaptic glutamate release: (i) the inhibition of exocytosis by these compounds is not specific to glutamate; (ii) aspartate efflux produced by various stimuli was also reduced, but aspartate cannot be released by exocytosis because it is not concentrated within presynaptic vesicles; and (iii) glutamate accumulated extracellularly during ischaemic or traumatic insult to the CNS is mainly of cytosolic origin. As an alternative, we propose that use-dependent Na(+)-channel blockers enhance the resistance of nerve cells to insults, primarily by decreasing their energy demand, and that reduced efflux of glutamate and other compounds is a consequence of attenuated cellular stress.

Animals↗

PubMed2001

Regulation of plant NR activity by reversible phosphorylation, 14-3-3 proteins and proteolysis.

This review highlights progress in dissecting how plant nitrate reductase (NR) activity is regulated by Ca2+, protein kinases, protein kinase kinases, protein phosphatases, 14-3-3 proteins and protease(s). The signalling components that regulate NR have also been discovered to target other enzymes of metabolism, vesicle trafficking and cellular signalling. Extracellular sugars exert a major impact on the 14-3-3-binding status and stability of many target proteins, including NR in plants, whereas other stimuli affect the regulation of some targets and not others. We thus begin to see how selective or global switches in cellular behaviour are triggered by regulatory networks in response to different environmental stimuli. Surprisingly, the question of how changes in NR activity actually affect the rate of nitrate assimilation is turning out to be a tough problem.

14-3-3 Proteins↗

PubMed1988

SCH 28080 is a lumenally acting, K+-site inhibitor of the gastric (H+ + K+)-ATPase.

SCH 28080 (2-methyl-8-(phenylmethoxy)imidazo[1,2-a] pyridine-3-acetonitrile) is an effective inhibitor of acid secretion in vivo and is a reversible, K+-competitive inhibitor of the gastric (H+ + K+)-ATPase in vitro. The actions of SCH 28080 have been studied on gastric vesicle preparations containing the (H+ + K+)-ATPase. At pH 7, inhibition was competitive with respect to K+ for both ATPase (Ki = 24 nM) and pNPPase (Ki = 275 nM) activities. A close analogue of SCH 28080 (methylated in the 1-N position), that was not expected to cross membranes freely, inhibited ATPase and pNPPase activity less effectively in intact vesicle preparations, where the lumenal (extracellular) face of the membrane was not directly accessible. This suggested that SCH 28080 inhibited both enzyme activities at a lumenal site on the enzyme. Being a protonatable weak base (pKa = 5.6), SCH 28080 would be expected to accumulate on the lumenal, acidic side of the parietal cell membrane in its protonated form. The potency of SCH 28080, relative to that of the "non-protonatable" analogue, increased at low pH, commensurate with the proportion of SCH 28080 in the protonated form. Thus the accumulating protonated form was the active inhibitory species. SCH 28080 (50 nM) blocked the rapid, K+-stimulated dephosphorylation of the catalytic phosphoenzyme intermediate of the (H+ + K+)-ATPase at room temperature. At 4 degrees, higher concentrations of the inhibitor were required, suggesting that the rate of inhibitor binding was slow at low temperatures.

4-Nitrophenylphosphatase↗

PubMed1981

Subcellular distribution of calcium in zona fasciculata cells of the rat adrenal cortex.

Zona fasciculata cells from the adrenal cortex of female Sprague-Dawley rats were fixed by immersion in potassium pyroantimonate-osmium tetroxide and potassium pyroantimonate-glutaraldehyde to study the distribution of calcium. Potassium pyroantimonate-osmium tetroxide treatment gave reproducible patterns of electron-opaque precipitate, whereas inconsistent deposits of reaction product were seen after potassium pyroantimonate-glutaraldehyde fixation. Nuclei showed sparse precipitate over heterochromatin and dense aggregates over areas of nucleoli surrounded by portions of the nucleolar-dense component. Two major cytoplasmic sites of precipitate were identified: mitochondria and vesicles of smooth endoplasmic reticulum. Most of the intramitochondrial precipitate was localized to the intracristal space. Precipitate was also seen in vesicles of Golgi apparatus. The extracellular space was filled with closely packed electron-opaque particles. Observation of tissues treated with control fixative saturated with EGTA showed little if any reaction, confirming that calcium was the primary cation precipitated by potassium pyroantimonate. Our results provide a method suitable for accurate localization of calcium in adrenocortical cells.

Adrenal Cortex↗

PubMed1991

Dopamine metabolism alterations in a manganese-treated pheochromocytoma cell line (PC12).

By monitoring dopamine metabolism in rat pheochromocytoma derived PC12 cell cultures during extended treatment with manganese chloride, we assessed the functional changes occurring in a dopaminergic system during the development of manganese-induced damage. Besides eliciting a specific toxic effect on PC12 cells, manganese induced the complete disappearance of extracellular (free) but not intracellular (vesicle stored) dopamine and its metabolite 3,4-dihydroxyphenylacetic acid. This effect was observed also using low manganese concentrations (1 microM) and mainly occurred by non-enzymatic catechol oxidation since it was still evident in a cell free medium and it was fully prevented by ascorbic acid but not by reduced glutathione. The possibility of a mere "non-biological" action was ruled out by the observation of an irreversible effect of manganese as manifested by the cells' apparent inability to release dopamine or 3,4-dihydroxyphenylacetic acid into the culture medium even after complete manganese removal (post-manganese incubation). That a free radical mechanism was not involved in the genesis of this irreversible effect was shown by the fact that neither ascorbic acid, catalase, superoxide dismutase nor glutathione-peroxidase were able to prevent the decrease in catecholamine levels in the "post-manganese" incubation medium when added at the same time as the manganese. The results establish this PC12 cell system as an in vitro model for studying interactions between manganese and catechols and provide a detailed description of the nature of the neurochemical alterations that this heavy metal can induce in a dopaminergic system.

Adrenal Gland Neoplasms↗

PubMed2005

In vitro chondrocyte differentiation using costochondral chondrocytes as a source of primary rat chondrocyte cultures: an improved isolation and cryopreservation method.

INTRODUCTION: Isolating and culturing primary chondrocytes such that they retain their cell type and differentiate to a hypertrophic state is central to many investigations of skeletal growth and its regulation. The ability to store frozen chondrocytes has additional scientific and tissue engineering interest. Previous work has produced approaches of varying yield and complexity but does not permit frozen storage of cells for subsequent differentiation in culture. Investigations of growth plate dysplasias secondary to defective osteoclastogenesis in rodent models of osteopetrosis led us to adapt and modify a culture method and to cryopreserve neonatal rat costochondral chondrocytes. METHODS: Chondrocytes were isolated from dissected ribs of 3-day-old rat pups by collagenase, hyaluronidase, and trypsin serial digestions. This was done either immediately or after the isolation was interrupted following an initial protease treatment to allow the chondrocytes, still in partially digested rib rudiments, to be frozen and later thawed for culture. Cells were plated in flat-bottom wells and allowed to adhere and grow under different conditions. Choice of media permitted cells to be maintained or induced to differentiate. Cell growth was monitored, as was expression of several relevant genes: collagen types II and X; osteocalcin, Sox9, adipocyte FABP, MyoD, aggrecan, and others. Mineralization was measured by alizarin red binding, and cultures were examined by light, fluorescence, and electron microscopy. RESULTS: Cells retained their chondrocyte phenotype and ability to differentiate and mineralize the collagen-rich extracellular matrix even after freezing-thawing. RT-PCR showed retention of chondrocyte-specific gene expression, including aggrecan and collagen II. The cells had a flattened, "proliferating zone" appearance initially, and by 2 weeks post-confluence, exhibited swelling and other salient features of hypertrophic cells seen in vivo. Collagen fibrils were abundant in the extracellular matrix, along with matrix vesicles. The switch to collagen type X as marker for hypertrophy was not rigidly temporally regulated as happens in vivo, but its expression increased during hypertrophic differentiation. CONCLUSIONS: This method should prove valuable as a means of studying chondrocyte regulation and has the advantages of simpler initial dissection, yields of a purer chondrocyte population, and the ability to stockpile frozen raw material for subsequent studies.

Animals↗

PubMed1999

Inducible nitric oxide synthase colocalizes with signs of lipid oxidation/peroxidation in human atherosclerotic plaques.

OBJECTIVE: Advanced human atherosclerotic plaques are characterized by the abundant presence of the autofluorescent non-soluble lipid pigment ceroid, consisting of oxidized lipoproteins. The aim of the present study was to examine the topographical and cellular distribution of inducible nitric oxide synthase (iNOS or NOS II) within different stages of atherosclerosis and its colocalization with ceroid deposits and nitrotyrosine. METHODS AND RESULTS: Different stages of atherosclerosis were studied by immunohistochemistry on whole-mount longitudinal sections of carotid endarterectomy specimens. In the adaptive intimal thickening the predominant cell type were smooth muscle cells. The fatty streaks contained both smooth muscle cells and macrophages with an extremely low NOS II immunoreactivity. The advanced atherosclerotic plaques however, showed a very dense infiltration by macrophages, of which a subpopulation expressed NOS II as a vesicular immunoreactivity in their cytoplasm. These were mainly present around the necrotic core, in association with ceroid accumulation and nitrotyrosine. Fluorescence quenching microscopy showed the presence of NOS II on autofluorescent ceroid vesicles in the macrophages. Large extracellular ceroid granules were not NOS II immunoreactive. NOS II mRNA was detected by RT-PCR and the protein by Western blot in the plaque tissue but not in mammary arteries used as controls. CONCLUSION: Ceroid, nitrotyrosine and NOS II colocalized in late stages of atherosclerosis and were found around the necrotic core in the plaque. This could suggest that NOS II expression in macrophages is involved in oxidation and peroxidation of lipids, leading to ceroid formation.

Aged↗

PubMed1998

Analysis of rates of receptor-mediated endocytosis and exocytosis of a fluorescent hapten-protein conjugate in murine macrophage: implications for antigen processing.

A novel fluorescent hapten-protein conjugate was constructed to monitor the events required for CD 4+ lymphocyte recognition of antigenic proteins. Previous studies utilizing the probe demonstrated that the hapten-protein was localized to an acidic endocytic compartment within the macrophage and that the hapten-protein was sensitive to multiple intracellular events including enzymatic degradation, acidification, and disulfide bond reduction. More importantly, recent experiments indicated that efficient internalization of the probe was dependent upon specific recognition of the hapten. Therefore, the present report addressed the effect of receptor-mediated endocytosis upon the processing of the hapten-protein within murine peritoneal macrophage. These studies determined that the rate of endocytosis was significantly faster than the rate of exocytosis. Specifically, the rate of exocytosis was estimated to be 3.4 x 10(4)s-1 based on a unimolecular rate constant. Although at higher concentrations, a slightly slower rate was observed (1.9 x 10(4)s-1). This study also represented one of the first efforts to measure the intracellular concentration effect typically associated with receptor-mediated endocytosis. Experiments involving a radioactively labeled hapten-protein conjugate revealed that the probe was at 100-fold higher concentration within the endocytic vesicles when compared to the extracellular media. The intracellular mechanism involved in this phenomenon was discussed as well as the implications of these findings upon MHC II-peptide binding.

Animals↗

PubMed1998

Tissue reaction of hydroxyapatite sol to rat molar pulp.

Hydroxyapatite sol was prepared by dispersing unheated hydroxyapatite microcrystals into distilled water. A suspension of hydroxyapatite heated at 800 degrees C (HAp-800 degrees C) in distilled water was used as a comparative material. These were characterized by X-ray diffractometry, SEM and electroconductivity measurements. 0.001 ml of each material was injected into the upper first molar pulp of rats. After 7-14 days post-operatively, the group with hydroxyapatite sol showed slight inflammation in the pulp cavities and a highly calcified tubular dentine bridge was observed. Osteodentine had developed and adhered to the surface of the hydroxyapatite microcrystals. Matrix vesicles were observed in the extracellular matrix between collagen fibrils and electron-dense granules. After 28 days, the inflammation completely disappeared and tubular dentine was observed below the osteodentine. Acid phosphates (ACPase) activity was detected along all biosynthesizing pathways in odontoblast-like cells. Localized dehydrogenate (LDH) activity indicated phagocytosis of hydroxyapatite microcrystals by odontoblasts. In the group with HAp-800 degrees C after 7-14 days post-operatively, the inflammation was moderate. After 28 days, relatively fewer calcified dentine tubules were observed. ACPase activity was detected only along the membranes bounding cytoplasmatic vacuoles. It was concluded that hydroxyapatite sol is a safe biomaterial for use as dental pulp and induces early formation of dentine-bridge more so than HAp-800 degrees C.

Journal Article↗