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PubMed1991

Extracellular Mg2+ induces a loss of microvilli, membrane retrieval, and the subsequent cortical reaction, in the oocyte of the prawn Palaemon serratus.

Surface changes induced by sea water were analyzed in the ovulated oocyte of the prawn Palaemon serratus. They depended on the presence of external Mg2+ but not on external Ca2+ alone. Increasing external Mg2+ from 0 mM to 30 mM stimulated first a progressive disappearance of preexisting microvilli, which was over within 30 min of incubation. This is correlated with membrane removal via internalization of coated vesicles, ascertained by observations of endocytosis of an extracellular fluid-phase marker and by measurement of a diminution in membrane capacitance (Cm). Thirty-five minutes after sea water contact, the prawn oocyte underwent a cortical reaction independent of fertilization. It consists in a heavy exocytosis of ring-shaped elements, leading to the deposition of a thick capsule, and requiring a threshold Mg2+ concentration of greater than or equal to 10 mM and at least a 3-min incubation with Mg2+. Concurrently, the values of the membrane capacitance (Cm) and conductance (Gm) increased about 2 and 10 times their initial values, respectively. The calcium ionophore ionomycin, added to Mg(2+)-free artificial sea water, stimulated the cortical reaction with requirement of external Ca2+. Other divalent cations (Mn2+, Zn2+, Co2+, Ni2+, Cd2+) instead of Mg2+, induced the cortical reaction, but Ba2+, Sr2+, and La3+ did not. When eggs are fertilized, the cortical reaction takes place in two steps, the first being a discrete exocytosis of a foamy material and the second always involving ring-shaped elements.

Animals↗
PubMed1983

Intracellular and extracellular localization of fibronectin in primary cultures of aortic smooth muscle cells using immunoperoxidase cytochemistry in electron microscopy.

This report describes the intracellular and extracellular localization of fibronectin at the ultrastructural level in primary cultures of aortic smooth muscle cells. Fibronectin was present in all the cisternae of the rough endoplasmic reticulum except the perinuclear cisterna, in large vesicles associated with the trans side of the Golgi complex, and in single large vesicles in the cytoplasm often associated with microtubules. The extracellular microfibrils were heavily stained. In sections parallel to the plane of growth bundles of extracellular microfibrils in continuity with arrays of intracellular microfilaments were observed (fibronexus). The basement membrane around the aortic smooth muscle cells was discontinuous and diffusely stained. The results indicate that fibronectin is localized in the cytoplasmic membranous apparatus of protein synthesis, processing, and secretion. The lack of reaction product in the flat cisternae of the Golgi complex let suggest either that fibronection may not be present in significant amounts within the flattened cisternae or that the method is insufficient in detecting the glycoprotein in this subcompartment off the Golgi complex.

Animals↗
PubMed1991

Vacuoles and vesicles in the rat junctional epithelium: a study with serial ultrathin sections.

To investigate whether the electron-lucent structures resembling vesicles and vacuoles in the rat molar junctional epithelium (JE) are in fact intracellular or extracellular, a study using serial ultrathin sections was carried out. In one series of experiments, the animals were not treated before the tissues were conventionally fixed; in another, anesthetized animals were administered horseradish peroxidase 20 min before the tissues were fixed. A large number of electron-lucent structures resembling vesicles and vacuoles were detected in both the peripheral and central cytoplasm of the JE localized at enamel and connective tissue sites. These were 70 to 800 nm in diameter and had a lucency similar to that of the extracellular space in untreated specimens fixed with conventional fixative. Serial ultrathin sectioning revealed that the electron-lucent structures gradually became part of the extracellular space in the following sections. There were also found in the middle portion of the cytoplasm in specimens pretreated with horseradish peroxidase. Numerous vacuole-like structures containing peroxidase-positive materials were found to be contiguous with the extracellular space. A small number of vesicles, also containing peroxidase-positive materials, did not appear in the previous or following sections. These results indicate that almost all electron-lucent structures resembling vesicles and vacuoles in the JE are located at the end of a long infolding, and are still in contact with the extracellular space.

Animals↗
PubMed1978

Serial synapses in Aplysia.

Serial synapses occur between small profiles in the neuropil of Aplysia abdominal ganglion. Material was fixed in phosphate buffered OsO4, embedded in epon, and sections were stained with uranyl acetate and lead citrate. A class of synapses had the following characteristics: (1) synaptic vesicles clustered against the presynaptic membrane, (2) a widened extracellular space of about 20 nm containing electron-dense material, (3) straightening of the pre- and postsynaptic membranes, and (4) no postsynaptic membrane specialization. Some density between the presynaptic membrane and the adjacent synaptic vesicles was occasionally observed. Synapses occurred between small profiles in the neuropil (typical profile diameters were 1--3 micrometer). In this sample of approximately 100 synapses, four serial synapses were identified. The serial synaptic profiles were all small. In addition to the finding of serial synapses, 40% of the postsynaptic profiles contained vesicles similar to the synaptic vesicles seen in presynaptic profiles. Serial synapses may be the anatomical substrate of presynaptic inhibition and facilitation and of dishabituation.

Animals↗
PubMed1986

Effect of amino acid levels on matrix vesicle formation by epiphyseal growth plate chondrocytes in primary culture.

The effect of varying the amino acid concentrations of the culture medium on matrix vesicle formation was studied in primary cultures of chicken epiphyseal growth plate chondrocytes grown in Dulbecco's modified Eagle's medium (DME) supplemented with 10% fetal bovine serum (FBS). Decreasing the levels of free amino acids in the culture medium to levels of one-half, one quarter, and one eighth of the values normally present in DME caused a progressive decline in matrix vesicle (MV) formation. Increasing the level in the culture medium of those amino acids that are enriched in extracellular fluid (ECF) of growth plate cartilage significantly increased formation of matrix vesicles (MV), as assayed by the alkaline phosphatase (AP) activities present in high-speed sediments from spent culture media. However, adjusting the levels of all amino acids to match those of the ECF produced the greatest stimulation of MV formation. Of the amino acids that are notably enriched in ECF, glutamate (GLU), alanine (ALA), serine (SER), asparagine (ASN), and taurine (TAU) individually enhanced MV production, whereas proline (PRO), glycine (GLY), and aspartate (ASP) had essentially no effect. The simple combination of ECF levels of ALA and GLU resulted in a stimulation of MV formation equal to that observed when the eight aforementioned amino acids were elevated to ECF levels. Other combinations of ASP and GLY, or of TAU, SER, and ASN showed some stimulation, but at a lower level. Increasing the amino acid concentrations, alone or in combination, also increased the levels of cellular AP, and to a lesser extent cellular protein. While increases in cellular AP were generally correlated with increased formation of AP-rich MV, this was not uniformly true. These results indicate that in addition to hormones and growth factors, nutritional factors such as the levels of amino acids are also critical for normal phenotypic expression, growth, and matrix formation by epiphyseal chondrocytes.

Amino Acids↗
PubMed1990

Ultrastructure of the electrotonic and chemical components of the lateral-to-motor and medial-to-motor synapses in crayfish nerve cord.

Lateral-to-motor and medial-to-motor synapses in crayfish nerve cords are composed of an electrical and a chemical component. The presynaptic terminals showed localized clusters of synaptic vesicles, electron-dense areas, coated pits, and coated vesicles. In thin sections, active zones were defined by electron-dense regions where synaptic vesicles attached and, in freeze-fracture replicas, by clusters of intramembrane particles localized in bands with vesicle openings on the sides of these bands. The cytoplasmic surface of the postsynaptic membrane opposite the active zones was coated with electron-dense material that in freeze-fracture replicas was seen as an increase in intramembrane particles located in the external leaflet (EF-face). This specialization of the postsynaptic membrane may correspond to the neurochemical receptor. Also, pre- and postsynaptic membranes were separated by a wider extracellular gap than those of adjacent nonsynaptic regions and electrical synapses or gap junctions. Synaptic vesicles were located exclusively at the synaptic regions by means of a cytoskeleton that was different for the electrical and the chemical components. The vesicles associated with the electrical component were anchored to a cytoskeleton composed of a beaded layer of densities located parallel to the membrane. This cytoskeleton maintained the synaptic vesicles separated from the presynaptic membrane by a distance of 13 +/- 2 nm. The synaptic vesicles associated with the chemical component were anchored to electron-dense regions formed by filaments arranged in bundles, anchored to the presynaptic membrane. Vesicles lined both sides positioned to discharge their contents into the extracellular space and to replace the discharged vesicles.

Animals↗
PubMed2000

Vesicle-associated urokinase plasminogen activator promotes invasion in prostate cancer cell lines.

The ability of a cell to modify the extracellular matrix is important in several pathophysiological alterations including tumorigenesis. Cell transformation is accompanied by changes in the surrounding stroma as a result of the action of specific proteases such as the urokinase plasminogen activator (uPA), which has been associated with invasive potential in many tumor types. In this study, we analyzed the release of vesicle-associated uPA by the aggressive prostatic carcinoma cell line PC3 and the implications of this release for the invasive behaviour of prostatic tumor cells. Zymography and Western blot analysis revealed the presence of vesicle-associated uPA in the high-molecular weight form. Vesicles adhered to and degraded both collagen IV and reconstituted basal membrane (Matrigel), and plasminogen enhanced the degradation in a dose-dependent manner. Addition of membrane vesicles shed by PC3 cells to cultures of the poorly invasive prostate cancer cell line LnCaP enhanced the adhesive and invasive capabilities of the latter, suggesting a mechanism involving substrate recognition and degradation. Together, these findings indicate that membrane vesicles can promote tumor invasion and point to the important role of vesicle-associated uPA in the extracellular compartment.

3T3 Cells↗
PubMed1979

Characterization of the extracellular lipase of Bacillus subtilis and its relationship to a membrane-bound lipase found in a mutant strain.

Bacillus subtilis CMK33 is a mutant that is more osmotically fragile than the wild type when it is converted to the protoplast form. The protoplasts of this mutant contain a membrane-bound lipase, which is not found in protoplasts of the wild type. Hydrolysis of the membrane lipid of mutant protoplasts by the lipase is the cause of their fragility. A protein found in the wild type organism specifically inhibits the lipase (Kent, C., and Lennarz, W. J. (1972) Proc. Natl. Acad. Sci. U. S. A. 69, 2793-2797). This paper reports that cultures of both mutant and wild type cells contain an extracellular lipase which accumulates during the logarithmic phase of growth. The extracellular activity appears to be induced by a component of the growth medium. The membrane-bound lipase of the mutant has been partially purified and its properties have been compared to those of the extracellular lipase of the wild type. Their properties and sensitivity to the wild type inhibitor are similar, which suggests that the two molecules are closely related. The subcellular location of the lipase in the mutant has been investigated and compared to the location of the membrane-bound portion of the lipase inhibitor in the wild type. The lipase is located almost exclusively in the cytoplasmic membrane and not in mesosomal vesicles. In contrast, the lipase inhibitor is located in both types of membranes and is more concentrated in mesosomal vesicles. Under appropriate conditions, the appearance of new extracellular lipase activity in mutant cultures is paralleled by the loss of an equivalent amount of lipase activity from protoplasts prepared from the cells. This suggests that the membrane-bound lipase may be an intermediate in the secretion of the extracellular lipase. Because of the mutation in B. subtilis CMK33, which results in the absence of the lipase inhibitor, this intermediate can be found in protoplasts of the mutant, although it is not detectable in the wild type. Consequently, the mutant may be useful in studies of the mechanism of secretion of exoenzymes by Bacilli.

Bacillus subtilis↗
PubMedDate not supplied

System dynamics of subcellular transport.

In pharmacokinetic experiments, interpretations often hinge on treating cells as a "black box": a single, lumped compartment or boundary. Here, a combinatorial library of fluorescent small molecules was used to visualize subcellular transport pathways in living cells, using a kinetic, high content imaging system to monitor spatiotemporal variations of intracellular probe distribution. Most probes accumulate in cytoplasmic vesicles and probe kinetics conform to a nested, two-compartment dynamical system. At steady state, probes preferentially partition from the extracellular medium to the cytosol, and from the cytosol to cytoplasmic vesicles, with hydrophobic molecules favoring sequestration. Altogether, these results point to a general organizing principle underlying the system dynamics of subcellular, small molecule transport. In addition to plasma membrane permeability, subcellular transport phenomena can determine the active concentration of small molecules in the cytosol and the efflux of small molecules from cells. Fundamentally, direct observation of intracellular probe distribution challenges the simple boundary model of classical pharmacokinetics, which considers cells as static permeability barriers.

4-Chloro-7-nitrobenzofurazan↗
PubMed2003

Evidence for chelatable zinc in the extracellular space of the hippocampus, but little evidence for synaptic release of Zn.

Zinc colocalizes with glutamate in the synaptic vesicles of certain glutamatergic vesicles in the mammalian brain. Here, I introduce a method for detecting Zn in the extracellular space of brain slices and another method for detecting the passage of Zn out of the slice. In both cases, the fluorimetric Zn probe FluoZin-3 is used in conjunction with a slow Zn chelator, Ca-EDTA, to reduce background fluorescence. In addition, a new Zn chelator, ethylenediiminodi-2-pentanedioic acid, with little affinity for Ca or Mg is introduced. These tools are then used to show that little Zn (approximately 2 nm) is released during the course of synaptic transmission into the extracellular space. However, when hippocampal slices are subjected to a high potassium stimulus (50 mM) combined with an increase in osmolarity, Zn is externalized in the Timm's-stained areas (approximately 6 nm). This stimulus also leads to even greater Zn elevations in area CA1 that is only weakly stained by the Timm's method. Nevertheless, even under these conditions, little if any Zn makes its way out of the slices. I present evidence for a layer of Zn in the extracellular space that maps onto the Timm's stained region of the hippocampus. This Zn veneer appears to be loosely associated with molecules in the extracellular space and may be the raison d'être for vesicular Zn.

Animals↗
PubMed1996

Comparative internalization and recycling of different amphotericin B formulations by a macrophage-like cell line.

The amount of amphotericin B (AmB) associated with cultured murine macrophage-like J774 cells, after incubation with various AmB lipid formulations, was determined by absorption spectroscopy. Large, negatively charged, AmB-containing, multilamellar vesicles and small cholesteryl sulphate-AmB complexes both enhanced the amount of AmB associated with J774 cells at 37 degrees C (up to 500-fold the extracellular concentration). In contrast, AmB-containing, small, negatively charged vesicles (AmBisome), positively charged, oligolamellar vesicles and mixed micelles showed a lower association of the antibiotic with cells, compared with AmB added from a solution in dimethylsulphoxide or Fungizone. Experiments performed at 40 degrees C showed a large reduction of AmB uptake for AmB preparations and AmB added from a solution in dimethysulphoxide or Fungizone, suggesting a high percentage of internalization of the antibiotic. Experiments in the presence of cytochalasin B resulted in a decrease of AmB uptake mainly for the preparations of large diameter, suggesting that these formulations were taken up by phagocytosis. A comparative study with Chinese hamster ovary cells, a model of non-phagocytic cells, showed a reduction in the take up of AmB. This reduction was always more marked when AmB was incorporated in lipid formulations. On the other hand, accumulation of the antibiotic in J774 cells was shown to be followed by its release from the cells in an unbound form, the extent of release depending on the type of vector used. The results suggest that in some cases macrophages can be considered as reservoirs of antibiotic, releasing free AmB in the medium.

Amphotericin B↗
PubMed1982

Presence of matrix vesicles in the trabecular meshwork of glaucomatous eyes.

In the last few years matrix vesicles (M.V.) have been found in the vessel wall in various diseases and are considered to be related with pathological changes of the extracellular fibers of the tissue (atypical collagen etc.). Since the trabecular meshwork, at least the cribriform region, may be looked upon as a part of a vessel wall, it was an obvious conclusion to look for M.V. even in this area. Lysosomal and non-lysosomal M.V. were found in 138 trabeculectomy-specimens of various kinds of glaucoma, located for the most part within the cribriform or juxta-canalicular region of the meshwork. In ten cases small, electron-dense M.V. were found which ultrahistochemically showed a positive reaction for acid phosphatase. This proves that these vesicles are indeed extracellular lysosomes.

Glaucoma↗
PubMed2002

Breakdown of the blood-brain barrier and neuropathological changes induced by Phoneutria nigriventer spider venom.

The blood-brain barrier (BBB) is responsible for selective flux of substances between blood and brain. The selective permeability of the BBB is crucial for the maintenance of the brain microenvironment homeostasis, and alterations in the barrier may be involved in many pathophysiological processes. Phoneutria nigriventer armed spider venom produces excitatory signals and symptoms in humans, and its recognized neurotoxic action suggests a potential ability to alter BBB permeability. The aim of the present study was to investigate the capacity of P. nigriventer venom (PNV) in promoting BBB breakdown in adult rats. After intravenous injection of 850 micro g/kg of the whole venom, BBB lesions were evaluated after 18 h to 9 days by ultrastructural methods using the extracellular tracer lanthanum nitrate. Clinical signs and symptoms of rats showed acute neurotoxicity, with some of the animals presenting convulsions, but which were clinically resolved by 12 h post-envenoming. The results showed that PNV is able to increase BBB permeability, particularly in the hippocampus. Changes were first detected in arterioles and post-capillary venules 18 h to 5 days after venom inoculation. The increased permeation of the extracellular tracer peaked on day 1, representing about 42% of the examined vessels (P<0.01). This appeared to occur by both transendothelial and intercellular routes, i.e., by pinocytic transport and through interendothelial junctions. Concomitantly, the surrounding tissue showed vasogenic edema and swollen astrocytic processes, without inflammatory infiltrates. The peak of the edema occurrence was observed on day 3, in about 60% of the vessels (P<0.001). Enhanced capillary permeability was observed on day 9, and affected 36% of all capillaries (P<0.05). The affected capillaries were characterized by increased number of pinocytotic vesicles, which, in addition, were filled with the extracellular tracer, but without visible transport through the interendothelial pathway. This study demonstrates that systemic PNV inoculation induces BBB breakdown through trans- and paracellular routes. It is concluded that BBB breakdown is an event not associated with the acute neurotoxicity exhibited by the rats.

Animals↗
PubMed1977

Sequential uptake of horseradish peroxidase by lymphoid follicle epithelium of Peyer's patches in the normal unobstructed mouse intestine: an ultrastructural study.

Membranous epithelial or "M" cells in lymphoid follicle epithelium of Peyer's patches extend between the adjacent columnar cells, forming a membrane separating lymphocytes in the epithelial cell layer from the intestinal lumen. They lack developed microvilli, glycocalyx, or terminal web but contain numerous vesicles. Transport of particles from ligated intestinal loops by such cells without evidence of further uptake of these particles by lymphocytes has been described by others. Horseradish peroxidase (HRP), an enzyme known to function orally and parenterally as a foreign protein antigen, was injected into the unligated intestines of fasted healthy 2-month-old white Swiss mice to determine if HRP would be absorbed in detectable quantities by M cells, and whether subsequent uptake of HRP by lymphocytes could be documented. At intervals from 1 to 60 min the most distal ileal Peyer's patch was fixed, removed, reacted with H2O2-3,3'-diaminobenzidine and examined by light and electron microscopy for HRP reaction product. At 1 min HRP adhered to surfaces of columnar cells and M cells and extended down into surface pits in the M cell. After 5 min HRP was found in vesicles of M cells but not in columnar cells. At 1 hr HRP was detected in the extracellular space between M cells and their enfolded lymphocytes, as well as in vesicles within these lymphocytes. Transport of intestinal luminal material by M cells with subsequent uptake by lymphocytes provides a specific route for antigen uptake into the intestinal lymphoid system.

Animals↗
PubMed2000

Secretory responses of intact glomus cells in thin slices of rat carotid body to hypoxia and tetraethylammonium.

We have developed a thin-slice preparation of whole rat carotid body that allows us to perform patch-clamp recording of membrane ionic currents and to monitor catecholamine secretion by amperometry in single glomus cells under direct visual control. In normoxic conditions (P(O(2)) approximately 140 mmHg; 1 mmHg = 133 Pa), most glomus cells did not have measurable secretory activity, but exposure to hypoxia (P(O(2)) approximately 20 mmHg) elicited the appearance of a large number of spike-like exocytotic events. This neurosecretory response to hypoxia was fully reversible and required extracellular Ca(2+) influx. The average charge of single quantal events was 46 +/- 25 fC (n = 218), which yields an estimate of approximately 140,000 catecholamine molecules per vesicle. Addition of tetraethylammonium (TEA; 2-5 mM) to the extracellular solution induced in most (>95%) cells tested (n = 32) a secretory response similar to that elicited by low P(O(2)). Cells nonresponsive to hypoxia but activated by exposure to high external K(+) were also stimulated by TEA. A secretory response similar to the responses to hypoxia and TEA was also observed after treatment of the cells with iberiotoxin to block selectively Ca(2+)- and voltage-activated maxi-K(+) channels. Our data further show that membrane ion channels are critically involved in sensory transduction in the carotid body. We also show that in intact glomus cells inhibition of voltage-dependent K(+) channels can contribute to initiation of the secretory response to low P(O(2)).

Animals↗
PubMedDate not supplied

Responses of rat pleural mesothelia to increased intrathoracic pressure.

Direct measurements of pleural fluid hydrostatic and colloid osmotic pressures after infusion of saline, bovine serum albumin, or silicone into the pleural space were coupled with ultrastructural morphometric analyses to assess the response of pleural mesothelial cells to hydrothorax. Increases of hydrostatic pressure, either independent or in combination with decreases of osmotic pressure, served to increase the number of plasmalemmal vesicles in mesothelial cells of both the visceral and parietal pleurae. These results support the hypothesis that an increase in vesicle numerical density represents a response to elevations of extracellular fluid pressures. Fluid resorption from the pleural space with subsequent accumulation within the visceral pleural interstitium was also associated with the formation of invaginations of the mesothelial basal plasmalemma. That the invaginations were not observed in the absence of interstitial fluid accumulation supports the concept that basal surface invaginations represent distortions of mesothelial cell membranes in response to pressure differentials across the plasmalemma. The results of this study are most consistent with the interpretation that increased numbers of plasmalemmal vesicles and invaginations of the basal plasmalemma represent adaptive conformational mechanisms of pleural mesothelial cells to prevent monolayer disruption by elevated extracellular fluid pressures.

Adaptation, Physiological↗
PubMed1983

Thin-section, tracer, and freeze-fracture study of the smooth-ended maturation ameloblasts in rat incisors.

The morphology and functional roles of the smooth-ended maturation ameloblasts (SAs) of rat incisors were examined by means of routine thin sections, tracer experiments, and freeze-fracture replication. SAs possessed two sets of junctional complexes consisting of tight junctions (fasiae occludentes) and gap junctions at the proximal and distal ends. Neither the proximal nor the distal junctional complex formed a complete barrier around the cell; intravenously injected horseradish peroxidase (HRP) reached the developing enamel surface through SAs extracellular spaces. SA supranuclear cytoplasm included such various cytoplasmic vesicles as the multivesicular body (MVB), large and vacuoles, dense body, and coated vesicles. The HRP that reached the enamel surface was incorporated into some coated vesicles and small vacuoles via coated pits and membrane invaginations of the distal cell surface of SA. Then, in the process of time, it migrated into the MVB and large endocytic vacuoles. These results indicate that the SA layer forms an extracellular transfer route for metabolites between papillary layer and the enamel surface and that SAs absorb exogenous protein.

Ameloblasts↗