Extracellular matrix vesicle distribution in primary mineralization two weeks after injury to rat tibial bone (ultrastructural tissue morphometry)

Primary mineralization on the 14th day of bone healing served as a model to study the distribution of extracellular matrix vesicles by means of transmission electron microscopy combined with computerized morphometry. Vesicles were traced on electron micrographs and classified according to diameter, distance from the calcified front, and type. The different types were determined as follows: electron-lucent vesicles ("empty"), vesicles with amorphous contents ("amorphic"), vesicles containing crystalline depositions ("crystal"), and vesicles with crystals and ruptured membranes ("rupture"). The majority of the vesicles measured between 0.02 and 0.07 micron and were located at a distance of less than 3 micron from the calcified front. They were distributed according to "empty", "amorphic", "crystal" and "rupture" type in concentrations of 10%, 31%, 51% and 8%, respectively. The diameters of the "rupture" vesicles were significantly larger than those of the "empty" and "amorphic" types. The sequence of their location, starting at the calcified front, ran as follows: "rupture", "crystal", "amorphic" and "empty", with the "rupture" type proximate to the front. According to the working hypothesis on calcification via extracellular matrix vesicles, it is thought that the "empty" vesicles are secreted by the cell and that subsequently amorphous Ca and Pi accumulate intravesicularly to form a hydroxyapatite crystal which, in turn, brings about rupture of the vesicle's membrane. The results of the present study support this theory and, additionally, show that the maturation process is accompanied by an increase of the vesicular diameter and by its approximation to the calcifying front.     

The association of amorphous mineral deposits with the plasma membrane of pre- and young odontoblasts and their relationship to the origin of dentinal matrix vesicles in rat incisor teeth.

Young and preodontoblasts and matrix vesicles which occur in the presecretory region of incisor teeth of growing rats were examined in stained and unstained ultrathin sections in order to characterize sites involved in the initial mineralization of dentin. Common to pre- and young odontoblasts in the presecretory region were hemispherical membrane-associated amorphous densities, measuring 5-35 nm in diameter after fixation in glutaraldehyde-osmium tetroxide or glutaraldehyde only. Amorphous densities were associated also with the limiting membranes of some vesicles in the extracellular matrix. Other vesicles in the extracellular matrix contained needle-like crystalline deposits typical of dentinal matrix vesicles. Fully differentiated odontoblasts in more incisal regions of the tooth lacked plasma membrane-associated amorphous densities. Neither amorphous nor crystalline densities were associated with any other cellular or subcellular structures in cells of the presecretory region. Flotation of ultrathin sections on solutions of EDTA or EGTA removed the amorphous densities from the plasma membranes, suggesting that the amorphous densities are calcium-containing mineral deposits. Amorphous deposits were associated with the membrane of vesicular structures protruding from the surfaces of pre- and young odontoblasts, suggesting that vesicles found in the extracellular matrix arise by budding from the plasma membranes of pre- and young odontoblasts. The occurrence of amorphous mineral deposits in association with the limiting membrane of some vesicles in the extracellular matrix, and the occurrence of needle-like mineral crystals within other matrix vesicles, suggest that an amorphous-to-crystalline phase transformation of mineral takes place within the matrix vesicle. The results of this study suggest that calcium-binding sites associated with plasma membranes of pre- and young odontoblasts act as nucleating centers for primary mineral deposition in tooth dentin.     

Correlative transmission and scanning electron microscopy of the initial mineralization of healing alveolar bone in rats

Primary mineralization in healing sockets after extraction of molar teeth was studied in rats. The observations obtained by scanning electron microscopy were correlated by transmission electron microscopy. The process is characterized by abundance of extracellular matrix vesicles distributed between the forming cells and the calcifying fronts. The occurrence of osmiophilic material and solitary hydroxyapatitte crystals within the vesicles was followed by accumulation of hydroxyapatite crystals, disappearance of the vesicular membrane and formation of calcospherites that conglomerate into calcified fronts. The process described here in bone healing is essentially similar to primary mineralization in other normal and pathological calcified tissues.     

Vesicle aggregation in model systems of supersaturated bile: relation to crystal nucleation and lipid composition of the vesicular phase

The presence of small vesicles composed of phospholipid and cholesterol has recently been demonstrated in super-saturated model and in dilute native human biles by several groups using differing methods. Among compositional factors shown to favor spontaneous vesicle formation and prolong the cholesterol monohydrate nucleation time in model bile systems are dilution, a raised cholesterol saturation index (CSI), and a low bile salt/phospholipid ratio. Time-lapse video-enhanced microscopy of a series of model bile systems representing systematically designed variations in the above factors revealed strong evidence for an essential linkage between antecedent vesicle aggregation and subsequent crystal nucleation. Stability of vesicles was inversely related to their degree of cholesterol saturation, i.e., the greater the degree of vesicular cholesterol saturation, the less their stability (metastability). Instability of vesicles was reflected by their early aggregation followed by rapid cholesterol crystal nucleation. The lowest degree of vesicular cholesterol saturation was found in dilute systems which also exhibited the greatest metastability despite a high degree of cholesterol solubility (raised CSI). Conversely, the more concentrated and least metastable systems exhibited both rapid vesicle aggregation and rapid onset of crystal nucleation. These systems, while influenced by the other compositional factors, were found to have a high degree of vesicular cholesterol saturation, i.e., cholesterol/phospholipid molar ratio = 2.0. An additional finding was the extreme variability in the proportionate distribution of total solution cholesterol distributed to the vesicular phase, i.e., from zero to as high as 37%. Higher solute concentration, raised bile salt/lecithin ratio, and raised CSI were interactive and almost equally capable of increasing the proportionate amount of cholesterol in the vesicular phase. In conclusion, lipid compositional differences in model bile systems drastically affect the cholesterol saturation of spontaneously formed phospholipid-cholesterol vesicles. This effect, in turn, exerts a potent influence upon the metastability of vesicles, subsequently affecting the cholesterol crystal nucleation time.     

Experimental calcification in rat submandibular gland.

The present study describes the phenomenon of calciphylaxis, rapid calcification due to treatment with sensitizer dihydrotachysterol (DHT) and challenging agent 5-hydroxytryptamine (5-HT) in the rat submandibular gland (SMG) in terms of light and electron microscopy, and histochemistry. For biophysical analysis of the calcified bodies, X-ray microanalysis (XMA) and X-ray powdered diffraction methods were used. The calcified lesions in the salivary glands were histologically divided into 3 types: type 1, calcification of basal membranes in duct-like structures; type 2, granular calcified materials with remarkable necrotic changes in cell, containing 3 kinds of small vesicular structures observed in electron microscopy; and type 3, von Kossa's positive structures containing needle-like crystalline and electron-dense amorphous materials. Con A and UEA-1 lectin staining reactions were strong in the type 1 and 2 lesions. These findings suggest that the calcification matrix may contain mannose, fucose and glucose. The X-ray microanalysis of calcified materials revealed the magnesium whitelockite pattern, the type 3 displayed high quantities of Ca, P, and Mg ions comparing with the type 1 and 2, and the X-ray diffraction showed the hydroxyapatite pattern. We suggest that the above changes may be categorized as dystrophic calcification due to necrotic alterations brought about by the hypercalcaemic condition.     

Functional aspects of lysosomal organelles in blood platelets

Summary Biochemical and ultrastructural findings from intact and fractionated pig and human blood platelets indicate the presence of different types of alpha granules. An electron dense type can be distinguished from a lighter one with a fibrillar matrix. The major part of the lysosomal hydrolases beta-glucuronidase and cathepsin is associated with the lighter granules. Most of the acid phosphatase appears in the vesicle fraction. The ultracytochemical investigation shows the acid phosphatase bound to both types of granules or vesicles. This enzyme is visible in the narrow spaces between the outer membranes of aggregated thrombocytes. Furthermore, there can be seen the transformation of alpha granules into vesicles and vesicles, which contain acid phosphatase releasing their contents into the extracellular medium. Thus the significance of the vesicle system for the release of substances out of the platelet seems to be proved. From this point of view the different types could be considered as alpha granules in different functional stages.Supported by Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg.     

Neuroglandular synapses in the pharynx of the sea anemone Aiptasia pallida (Cnidaria, Anthozoa)

Scanning and transmission electron microscopy of the pharynx of the sea anemone Aiptasia pallida revealed a heavily ciliated epidermis and two types of gland cells not known previously to be innervated. By tracing serial cross sections of the pharynx, we located and characterized two types of neuroglandular synapses (i.e., those having clear vesicles and those with dense-cored vesicles). The diameters of the vesicles at each synapse were averaged; clear vesicles ranged from 70 to 103 nm in diameter and were observed at synapses to both mucous and zymogenic gland cells. Dense-cored vesicles ranged from 53 to 85 nm in diameter and were observed at synapses to two mucous gland cells. One mucous gland cell had three neuroglandular synapses, one with clear vesicles and two with dense-cored vesicles. The occurrence of either clear or dense-cored vesicles at neuroglandular synapses suggests that at least two types of neurotransmitter substances control the secretion of mucus in the sea anemone pharynx. To date, only clear vesicles have been observed at a neurozymogenic gland cell synapse in the pharynx. No evidence of immunoreactivity to phenylethanolamine-N-methyl transferase was observed at neuroglandular synapses, suggesting that adrenaline is not a transmitter in the pharynx of A. pallida.     

Role of calcium-phosphate deposition in vascular smooth muscle cell calcification

In this work we are studying whether calcium phosphate deposition (CPD) during vascular calcification is a passive or a cell-mediated mechanism. Passive CPD was studied in fixed vascular smooth muscle cells (VSMC), which calcify faster than live cells in the presence of 1.8 mM Ca2(+) and 2 mM P(i). CPD seems to be a cell-independent process that depends on the concentration of calcium, phosphate, and hydroxyl ions, but not on Ca × P(i) concentration products, given that deposition is obtained with 2 × 2 and 4 × 1 Ca × P(i) mM2 but not with 2 × 1 or 1 × 4 Ca × P(i) mM2. Incubation with 4 mM P(i) without CPD (i.e., plus 1 mM Ca) does not induce osteogene expression. Increased expression of bone markers such as Bmp2 and Cbfa1 is only observed concomitantly with CPD. Hydroxyapatite is the only crystalline phase in both lysed and live cells. Lysed cell deposits are highly crystalline, whereas live cell deposits still contain large amounts of amorphous calcium. High-resolution transmission electron microscopy revealed a nanostructure of rounded crystallites of 5-10 nm oriented at random in lysed cells, which is compatible with spontaneous precipitation. The nanostructure in live cells consisted of long fiber crystals, 10-nm thick, embedded in an amorphous matrix. This structure indicates an active role of cells in the process of hydroxyapatite crystallization. In conclusion, our data suggest that CPD is a passive phenomenon, which triggers the osteogenic changes that are involved in the formation of a well organized, calcified crystalline structure.     

Glutamate, a neurotransmitter--and so much more. A synopsis of Wierzba III

It appears almost incredible that the first indications that glutamate excites brain tissue were obtained during the second half of the 20th century, that vesicles containing glutamate were demonstrated in glutamatergic neurons less than 25 years ago, and that glutamate was not accepted as the major excitatory transmitter until about the same time. During this span of time it has also become realized that glutamate is so much more than a conventional neurotransmitter: (1) astrocytes express vesicles accumulating glutamate by vesicular transporters akin to the vesicular glutamate transporters in glutamatergic neurons, and they release glutamate by exocytosis; (2) a series of metabolic processes in astrocytes (glutamate uptake, glutamine synthetase activity, glutamine release) are involved in neuronal reutilization of transmitter glutamate; (3) glutamine may also be utilized for synthesis of GABA, the major inhibitory transmitter; (4) de novo synthesis of glutamate accounts for 20% of cerebral glucose metabolism, all of which initially occurs in astrocytes, and at steady state a corresponding amount of glutamate is oxidatively degraded, mainly or exclusively in astrocytes; (5) tissue contents of glutamate/glutamine increase during enhanced glutamatergic activity, i.e., astrocytic de novo synthesis exceeds astrocytic metabolic degradation of glutamate.     

Changes in Sympathetic Nerve Terminals in the Heart of Cold-Exposed Rats

Abstract: Changes in sympathetic nerve terminals of the heart after varying periods of exposure of rats to 4°C were investigated. Two indices were used for changes in the number of noradrenaline storage vesicles, i.e., vesicular dopamine β-hydroxylase (DBH) activity and noradrenaline storage capacity. The latter was obtained after uptake of [³H]noradrenaline; endogenous content, uptake of exogenous noradrenaline, and degree of saturation of the vesicles were calculated using the specific activity of the [³H]noradrenaline. As a measure of tyrosine hydroxylase activity, whole ventricular noradrenaline, dopamine, and dihydroxyphenylacetic acid content were used. After 4 h of cold exposure there was an increase in vesicular endogenous noradrenaline content, uptake, storage capacity, and DBH activity as well as a large increase in whole ventricular dopamine. After 6 h in the cold, vesicular endogenous noradrenaline content, storage capacity, and DBH activity were decreased. The results suggest that during cold exposure there is an initial increase followed by a decrease in the number of functional vesicles in the nerve terminal, which could explain the fluctuations in the rate of noradrenaline release.     

Accurate separation of vesicles, micelles and cholesterol crystals in supersaturated model biles by ultracentrifugation, ultrafiltration and dialysis.

Gel filtration with bile salts at intermixed micellar/vesicular concentrations (IMC) in the eluant has been proposed to isolate vesicles and micelles from supersaturated model biles, but the presence of vesicular aggregates makes this method unreliable. We have now validated a new method for isolation of various phases. First, aggregated vesicles and - if present - cholesterol crystals are pelleted by short ultracentrifugation. Cholesterol contained in crystals and vesicular aggregates can be quantitated from the difference of cholesterol contents in the pellets before and after bile salt-induced solubilization of the vesicular aggregates. Micelles are then isolated by ultrafiltration of the supernatant through a highly selective 300 kDa filter and unilamellar vesicles by dialysis against buffer containing bile salts at IMC values. Lipids contained in unilamellar vesicles are also estimated by subtraction of lipid contents in filtered micelles from lipid contents in (unilamellar vesicle+micelle containing) supernatant ('subtraction method'). 'Ultrafiltration-dialysis' and 'subtraction' methods yielded identical lipid solubilization in unilamellar vesicles and identical vesicular cholesterol/phospholipid ratios. In contrast, gel filtration yielded much more lipids in micelles and less in unilamellar vesicles, with much higher vesicular cholesterol/phospholipid ratios. When vesicles obtained by dialysis were analyzed by gel filtration, vesicular cholesterol/phospholipid ratios increased strongly, despite correct IMC values for bile salts in the eluant. Subsequent extraction of column material showed significant amounts of lipids. In conclusion, gel filtration may underestimate vesicular lipids and overestimate vesicular cholesterol/phospholipid ratios, supposedly because of lipids remaining attached to the column. Combined ultracentrifugation-ultrafiltration-dialysis should be considered state-of-the-art methodology for quantification of cholesterol carriers in model biles.     

Rats that differentially respond to cocaine differ in their dopaminergic storage capacity of the nucleus accumbens

Cocaine (COC) inhibits the re-uptake of dopamine. However, the dopamine response to COC also depends on dopamine inside storage vesicles. The aim of this study was to investigate whether rats that differentially respond to COC differ in their dopaminergic storage capacity of the nucleus accumbens. Total and vesicular levels of accumbal dopamine as well as accumbal vesicular monoamine transporter-2 levels were established in high (HR) and low responders (LR) to novelty rats. Moreover, the effects of reserpine (RES) on the COC-induced increase of extracellular accumbal dopamine were investigated. HR displayed higher accumbal levels of total and vesicular dopamine than LR. Moreover, HR displayed more accumbal vesicular monoamine transporters-2 than LR. COC increased extracellular accumbal dopamine more strongly in HR than in LR. A low dose of RES prevented the COC-induced increase of accumbal dopamine in LR, but not in HR. A higher dose of RES was required to inhibit the COC-induced increase of accumbal dopamine in HR. These data demonstrate that HR were marked by a larger accumbal dopaminergic storage pool than LR. It is hypothesized that HR are more sensitive to COC than LR, because COC can release more dopamine from accumbal storage vesicles in HR than in LR.     

Ultrastructural pathology of the compound eye and optic neuropiles of the retinal degeneration mutant (w rdgB KS222) Drosophila melanogaster

Summary Lanthanum, applied to the outside of the fixed sciatic nerve of Rana pipiens, did not enter the endoneurium, but was halted by functionally tight junctions at the inner layers of the perineurium. This component of the bloodnerve barrier consists of several concentric layers of cells interspersed with an extracellular matrix of amorphous ground substance, collagen fibrils, and fine filaments. Numerous vesicular profiles are closely associated with the surface membranes of all the cells. The application of lanthanum to fixed tissue revealed that these profiles are attached to the cell surface by narrow necks, and open to the extracellular space. The attenuated cells are filled by the vesicular structures, which often appear to overlap. Stereoscopic electron microscopy showed that these vesicles did not fuse with each other or with the apposing cell surface to form transcellular channels. Channel formation does not appear to contribute significantly to the permeability of any of the perineurial layers.     

A Role of Vesicular Transduction of Intercellular Signals in Cancer Development

Export of biologically active compounds is essential for any living cell. Transport of bioactive molecules through a cellular membrane can be active, or passive, or vesicular. In the past decade, vesicular transduction of intercellular signals has attracted great interest in the scientific community. An extremely important role of the vesicle transduction has been established for almost all processes in a living body. Not only profiles of protein and RNA expression in a cell, but also its secretome change during various pathologies, including cancer development. The enhanced secretion of vesicles by transformed cells is one important factor in creating a special microenvironment that favors tumor progression. At present, a role of exosomes has been demonstrated for such important processes as an epithelial-mesenchymal transition, angiogenesis, metastatic niche formation, chemotherapeutic resistance, and interaction with the immune system. The special biological role of the extracellular vesicles and their basic differences depend on their molecular composition. Therefore, special protein and lipid markers are responsible for a vesicular targeted delivery with information due to the preferable interaction with cells of a definite type. The exosomes of cancer cells can facilitate apoptosis or growth of neighboring malignant cells depending on the exosome composition. These and other special features of the extracellular vesicles make studies of their composition and role especially interesting and attract significant attention from researchers. Despite the rapid progress in this field, there are still many unresolved problems, such as a search for specific markers which allow identification of different types of vesicles or vesicles secreted by distinct cells, as well as screening of vesicular markers of cancers and other diseases that are associated with disorders in a functioning immune system. This review is mainly focused on the role of intercellular vesicular transport of bioorganic molecules in cancer progression. We believe that a successful treatment of oncological diseases is impossible without an understanding of the intercellular communication of both cancer cells between each other and with other systems of an organism and with a concept of an active participation of the cell-secreted vesicles in this process.     

Leaky vesicle fusion induced by phosphatidylinositol-specific phospholipase C: observation of mixing of vesicular inner monolayers

Large unilamellar vesicles containing phosphatidylinositol (PI), neutral phospholipids, and cholesterol are induced to fuse by the catalytic activity of phosphatidylinositol-specific phospholipase C (PI-PLC). PI cleavage by PI-PLC is followed by vesicle aggregation, intervesicular lipid mixing, and mixing of vesicular aqueous contents. An average of 2-3 vesicles merge into a large one in the fusion process. Vesicle fusion is accompanied by leakage of vesicular contents. A novel method has been developed to monitor mixing of lipids located in the inner monolayers of the vesicles involved in fusion. Using this method, the mixing of inner monolayer lipids and that of vesicular aqueous contents are seen to occur simultaneously, thus giving rise to the fusion pore. Kinetic studies show, for fusing vesicles, second-order dependence of lipid mixing on diacylglycerol concentration in the bilayer. Varying proportions of PI in the liposomal formulation lead to different physical effects of PI-PLC. Specifically, 30-40 mol % PI lead to vesicle fusion, while with 5-10 mol % PI only hemifusion is detected, i.e., mixing of outer monolayer lipids without mixing of aqueous contents. However, when diacylglycerol is included in the bilayers containing 5 mol % PI, PI-PLC activity leads to complete fusion.     

The effect of corticosteroids on microvillous vesicles from the small intestine of adult rats: an automated, morphometric analysis

The hypothesis that the corticosterone-induced enhancement of D-glucose uptake by brush-border membrane vesicles from the small intestine of adult rats is caused by an alteration in vesicle volume was tested by means of structural morphometric computerized analysis. By using a Ca2+ precipitation method, microvillous membrane vesicles were prepared from different groups of adult rats: normal controls; animals sacrificed 8 days after adrenalectomy; sham-operated pair-fed controls; animals treated with corticosterone (1 or 10 mg x 100 g b.wt.-1 daily i.p. for 8 days); corticosterone vehicle-treated controls. The vesicles were processed by conventional electron microscopy techniques. Morphometric computerized analysis was carried out on electron micrographs at a 20,000 x magnification, after selecting by an interactive method the vesicles showing certain morphologic features. The morphometric parameters determined for the vesicles from each experimental group were diameter, shape factor (minimal to maximal diameter ratio), surface area and volume. The following main findings were obtained: the vesicles were consistently spherical, with the glycocalyx on the outer side; sham-operation and pair-feeding with adrenalectomized rats, as well as corticosterone-vehicle injection, significantly reduced vesicular volume as compared to normal controls; adrenalectomy significantly decreased vesicular volume as compared to pair-feeding; excess of corticosteroids caused opposite changes (i.e. a significant increase of vesicle volumes as compared to vehicle injection), which were apparently dose-dependent. A comparison with D-glucose vesicular transport data previously obtained under the same experimental conditions revealed the existence of a positive relationship between vesicular volume and D-glucose half-filling time (t 1/2). A positive relationship between vesicular volume and D-glucose vesicular space was found for all experimental conditions except vehicle injection. As a whole, the morphometric results were consistent with functional findings, supporting our previous hypothesis that the corticosterone-induced increase in D-glucose transport by small intestinal microvillous vesicles is due, at least in part, to enlargement of the vesicle.     

Proteomic analysis of extracellular vesicles derived from Mycobacterium tuberculosis

The release of extracellular vesicles, also known as outer membrane vesicles, membrane vesicles, exosomes, and microvesicles, is an evolutionarily conserved phenomenon from bacteria to eukaryotes. It has been reported that Mycobacterium tuberculosis releases extracellular vesicles harboring immunologically active molecules, and these extracellular vesicles have been suggested to be applicable in vaccine development and biomarker discovery. However, the comprehensive proteomic analysis has not been performed for M. tuberculosis extracellular vesicles. In this study, we identified a total of 287 vesicular proteins by four LC‐MS/MS analyses with high confidence. In addition, we identified several vesicular proteins associated with the virulence of M. tuberculosis. This comprehensive proteome profile will help elucidate the pathogenic mechanism of M. tuberculosis. The data have been deposited to the ProteomeXchange with identifier PXD001160 ( http://proteomecentral.proteomexchange.org/dataset/PXD001160 ).     

Immunoisolation and partial characterization of endothelial plasmalemmal vesicles (caveolae).

Plasmalemmal vesicles (PVs) or caveolae are plasma membrane invaginations and associated vesicles of regular size and shape found in most mammalian cell types. They are particularly numerous in the continuous endothelium of certain microvascular beds (e.g., heart, lung, and muscles) in which they have been identified as transcytotic vesicular carriers. Their chemistry and function have been extensively studied in the last years by various means, including several attempts to isolate them by cell fractionation from different cell types. The methods so far used rely on nonspecific physical parameters of the caveolae and their membrane (e.g., size-specific gravity and solubility in detergents) which do not rule out contamination from other membrane sources, especially the plasmalemma proper. We report here a different method for the isolation of PVs from plasmalemmal fragments obtained by a silica-coating procedure from the rat lung vasculature. The method includes sonication and flotation of a mixed vesicle fraction, as the first step, followed by specific immunoisolation of PVs on anticaveolin-coated magnetic microspheres, as the second step. The mixed vesicle fraction, is thereby resolved into a bound subfraction (B), which consists primarily of PVs or caveolae, and a nonbound subfraction (NB) enriched in vesicles derived from the plasmalemma proper. The results so far obtained indicate that some specific endothelial membrane proteins (e.g., thrombomodulin, functional thrombin receptor) are distributed about evenly between the B and NB subfractions, whereas others are restricted to the NB subfraction (e.g., angiotensin converting enzyme, podocalyxin). Glycoproteins distribute unevenly between the two subfractions and antigens involved in signal transduction [e.g., annexin II, protein kinase C alpha, the G alpha subunits of heterotrimeric G proteins (alpha s, alpha q, alpha i2, alpha i3), small GTP-binding proteins, endothelial nitric oxide synthase, and nonreceptor protein kinase c-src] are concentrated in the NB (plasmalemma proper-enriched) subfraction rather than in the caveolae of the B subfraction. Additional work should show whether discrepancies between our findings and those already recorded in the literature represent inadequate fractionation techniques or are accounted for by chemical differentiation of caveolae from one cell type to another.     

Recycled Synaptic Vesicles Contain Vesicle but Not Plasma Membrane Marker, Newly Synthesized Acetylcholine, and a Sample of Extracellular Medium

To monitor the fate of the synaptic vesicle membrane compartment, synaptic vesicles were isolated under varying experimental conditions from blocks of perfused Torpedo electric organ. In accordance with previous results, after low-frequency stimulation (0.1 Hz, 1,800 pulses) of perfused blocks of electric organ, a population of vesicles (VP2 type) can be separated by density gradient centrifugation and chromatography on porous glass beads that is denser and smaller than resting vesicles (VP1 type). By simultaneous application of fluorescein isothiocyanate-dextran as extracellular volume marker and [3H]acetate as precursor of vesicular acetylcholine, and by identifying the vesicular membrane compartment with an antibody against the synaptic vesicle transmembrane glycoprotein SV2, we can show that the membrane compartment of part of the synaptic vesicles becomes recycled during the stimulation period. It then contains both newly synthesized acetylcholine and a sample of extracellular medium. Recycled vesicles have not incorporated the presynaptic plasma membrane marker acetylcholinesterase. Cisternae or vacuoles are presumably not involved in vesicle recycling. After a subsequent period of recovery (18 h), all vesicular membrane compartments behave like VP1 vesicles on subcellular fractionation and still retain both volume markers. Our results imply that on low-frequency stimulation, synaptic vesicles are directly recycled, equilibrating their luminal contents with the extracellular medium and retaining their membrane identity and capability to accumulate acetylcholine.