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PubMed1996

Prostasomes are neuroendocrine-like vesicles in human semen.

BACKGROUND: Prostasomes are prostate-derived organelles that exist extracellularly in human seminal plasma. METHODS: In this study, we have investigated and characterized human prostasomes with regard to their contents of synaptophysin, members of the chromogranin family, and some neuropeptides. RESULTS: By radioimmunoassay measurement and electron microscopy we show the presence of the neuroendocrine markers chromogranin B, neuropeptide Y, and vasoactive intestinal polypeptide in about equimolar amount in human prostasomes and chromogranin A in about 2% of that amount. To our knowledge, such a high ratio of chromogranin B to chromogranin A has never before been observed. The membrane-bound protein synaptophysin, a well-established immunocytochemical marker for neuroendocrine cells and neurones, was also detected. Hence, we show that synaptophysin could be used as a marker for intact prostasomes. CONCLUSIONS: The presence of synaptophysin has recently been shown in the serotonincontaining vesicles in platelets. A protein with a similar structure denoted granulophysin has been found in granulocytes and prostasomes. It is suggested that synaptophysin and granulophysin molecules are members of a family of proteins, maybe expressed in all cells that have regulated release of granule content. Our presented data indicate a neurotransmittor function of the prostasomes. The target cells are however not known but could be either the spermatozoa, the epithelial mucous cells of the uterus or tubas or perhaps the ovum.

Antigens, CD↗

PubMed1984

Investigation of phospholipids of the pulmonary extracellular lining by electron paramagnetic resonance. The effects of phosphatidylglycerol and unsaturated phosphatidylcholines on the fluidity of dipalmitoyl phosphatidylcholine.

The membranous structures of the pulmonary extracellular lining were removed from the lungs of rabbits by pulmonary lavage and isolated by differential centrifugation. This membranous fraction contained 93% of the total extracellular phospholipids present in lavage effluents and consisted of membranous vesicles, membrane fragments, tubular myelin and secreted lamellar bodies. The fraction was rich in phosphatidylcholine (79.4%) containing 85.2% palmitic acid in the 1-position and 57.4% palmitic acid in the 2-position. Phosphatidylglycerol was the next most abundant phospholipid, accounting for 9.4% of the total. E.p.r. spectra, obtained by using 5-doxylmethylstearate as a probe, showed that the extracellular phospholipids of the pulmonary lining were organized into structures which were much more fluid than erythrocyte-ghost membranes. The fluidity of phosphatidylcholine isolated from the membranous fraction was similar to that of the fraction itself, indicating that the minor phospholipids had very little influence on the fluidity of the major phospholipid. At physiological temperature, the fluidity of dipalmitoyl phosphatidylcholine was relatively low, but could be markedly increased by the presence of 1-palmitoyl-2-oleoyl phosphatidylcholine or phosphatidylglycerol (10%). Protein present in the extracellular phospholipid fraction did not affect the fluidity of the fraction. These studies indicate that the unsaturated phosphatidylcholines could play a major role in determining the fluidity of the important surface-tension-lowering phospholipids such as dipalmitoyl phosphatidylcholine.

Animals↗

PubMed1990

The intracellular movement and cycling of ricin.

The binding, internalization and recycling of the plant toxin ricin, was studied using electron microscopy and biochemical techniques. For the electron microscope study, ricin was visualized using a gold-labeled second antibody, in the cells of the EJ human bladder carcinoma line growing in monolayer culture. The labeled antibody/toxin complex was found to enter the cell in coated pits and to accumulate in endosomes and to a lesser extent in vesicles associated with the Golgi system. The complex recycled to the cell surface partly in uncoated vesicles, but largely in multivesicular bodies which appeared to exocytose their contents to the extracellular space. Twenty hours after the initial contact with ricin as much as 50% of the cellular label was found on the cell surface mainly associated with shed vesicles. When cells were treated with unlabeled ricin holotoxin and then after 20 h stained post-fixation, ricin molecules, partly associated with vesicles, were present on the cell surface. Biochemical studies showed that ricin was internalized by cells and then released in an intact form to the extracellular space. It was found that less than 10% of the released material had been degraded during its passage through the cells, which is in accord with the low level of label found in the lysosomal system during the morphological study.

Antitoxins↗

PubMed2001

Extracellular formation and uptake of adenosine during skeletal muscle contraction in the rat: role of adenosine transporters.

1. The existence of adenosine transporters in plasma membrane giant vesicles from rat skeletal muscles and in primary skeletal muscle cell cultures was investigated. In addition, the contribution of intracellularly or extracellularly formed adenosine to the overall extracellular adenosine concentration during muscle contraction was determined in primary skeletal muscle cell cultures. 2. In plasma membrane giant vesicles, the carrier-mediated adenosine transport demonstrated saturation kinetics with Km = 177 +/- 36 microM and Vmax = 1.9 +/- 0.2 nmol x ml(-1) x s(-1) (0.7 nmol (mg protein)(-1) x s(-1)). The existence of an adenosine transporter was further evidenced by the inhibition of the carrier-mediated adenosine transport in the presence of NBMPR (nitrobenzylthioinosine; 72% inhibition) or dipyridamol (64% inhibition; P < 0.05). 3. In primary skeletal muscle cells, the rate of extracellular adenosine accumulation was 5-fold greater (P < 0.05) with electrical stimulation than without electrical stimulation. Addition of the adenosine transporter inhibitor NBMPR led to a 57% larger (P < 0.05) rate of extracellular adenosine accumulation in the electro-stimulated muscle cells compared with control cells, demonstrating that adenosine is taken up by the skeletal muscle cells during contractions. 4. Inhibition of ecto-5'-nucleotidase with AOPCP in electro-stimulated cells resulted in a 70% lower (P < 0.05) rate of extracellular adenosine accumulation compared with control cells, indicating that adenosine to a large extent is formed in the extracellular space during contraction. 5. The present study provides evidence for the existence of an NBMPR-sensitive adenosine transporter in rat skeletal muscle. Our data furthermore demonstrate that the increase in extracellular adenosine observed during electro-stimulation of skeletal muscle is due to production of adenosine in the extracellular space of skeletal muscle and that adenosine is taken up rather than released by the skeletal muscle cells during contraction.

Adenosine↗

PubMed1996

Role of external and internal calcium on heterocarrier-mediated transmitter release.

Release-regulating heterocarriers exist on brain nerve endings. We have investigated in this study the mechanisms involved in the neurotransmitter release evoked by GABA heterocarrier activation. GABA increased the basal release of [3H]acetylcholine and [3H]noradrenaline from rat hippocampal synaptosomes and of [3H]dopamine from striatal synaptosomes. These GABA effects, insensitive to GABA receptor antagonists, were prevented by inhibiting GABA uptake but not by blocking noradrenaline, choline, or dopamine transport. Lack of extracellular Ca2+ or addition of tetrodotoxin selectively abolished the GABA-evoked release of [3H]noradrenaline, leaving unaffected that of [3H]acetylcholine or [3H]dopamine. 1,2-Bis(2-aminophenoxyl)-ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM) or vesamicol attenuated the release of [3H]acetylcholine elicited by GABA. Reserpine, but not BAPTA-AM, prevented the effect of GABA on [3H] dopamine release. Autoreceptor activation inhibited the GABA-evoked release of [3H]noradrenaline but not that of [3H]acetylcholine or [3H]dopamine. It is concluded that (a) the release of [3H]noradrenaline consequent to activation of GABA heterocarriers sited on noradrenergic terminals meets the criteria of a conventional exocytotic process, (b) the extracellular [Ca2+]-independent releases of [3H]acetylcholine and [3H]dopamine appear to occur from vesicles possibly through involvement of intraterminal Ca2+, and (c) autoreceptor activation only affects heterocarrier-mediated vesicular release linked to entry of extracellular Ca2+.

Acetylcholine↗

PubMed2002

Sequential changes in synaptic vesicle pools and endosome-like organelles during depolarization near the active zone of central nerve terminals.

During periods of high-frequency stimulation the maintenance of synaptic transmission depends on a continued supply of synaptic vesicles. Local recycling in the terminals ensures synaptic vesicle replenishment, but the intermediate steps are still a matter of debate. We analyzed changes in synaptic vesicle pools and endosome-like organelles near the active zone in central nerve terminals during depolarization at the ultrastructural level by electron microscopy. A short, 100 ms, depolarization-induced recruitment of synaptic vesicles was observed from a reserve pool to a recruited pool, within 150 nm of the active zone, and the docked pool at the active zone was increased as well. Prolonged, 15 s or 3 min, depolarization decreased the total amount of synaptic vesicles, which was accompanied by a parallel increase in size and amount of endosome-like organelles. After a period of rest, the number of endosome-like organelles decreased and the amount of synaptic vesicles was restored to control level. The endocytotic nature of part of the endosome-like organelles after 15 s and 3 min depolarization was indicated by their labeling with extracellularly added horseradish peroxidase (HRP). In addition, a small number of synaptic vesicles entrapped HRP under these conditions. After repolarization, the number of HRP-loaded endosome-like structures decreased. Simultaneously, a strong increase in amount of HRP-loaded small vesicles did occur. These results indicate that during sub-second depolarization, synaptic vesicles were rapidly recruited from the reserve pool to replenish the releasable pool, whereas prolonged depolarization (s-min) induced local endocytosis in at least two ways, i.e. either directly as vesicles or via endosome-like organelles from which synaptic vesicles were reformed.

Action Potentials↗

PubMed1997

Secretion of slime, the extracellular matrix of the plasmodium, as visualized with a fluorescent probe and its correlation with locomotion on the substratum.

Slime, the extracellular matrix of Physarum plasmodium, is secreted by the exocytosis of a vesicles that contain a slime precursor. Using an antibody raised against biochemically purified slime, we detected the intracellular localization of the slime vesicle. Slime vesicles are abundant in the advancing front of the plasmodium, as confirmed by electron microscopic observation in two different cross-sectional angles. Screening various reagents, we found that rhodamine-phosphatidylethanolamine (Rh-PE) binds specifically to slime in both its intravesicular and extracellular forms, as confirmed by immunoelectron microscopy using an antibody against fluorochrome rhodamine. The plasmodia vitally stained with Rh-PE exhibited dynamic fluorescent patterns during the course of locomotion. The fluorescence was conspicuous at the periphery of the leading pseudopods and oscillated according to the shuttle streaming that accompanied the relaxation and contraction of the periphery; it was intense in the relaxation phase when pseudopods extended, and became weak in the contraction phase when pseudopods contracted. The results collectively mean that the slime vesicles carried by the cytoplasmic streaming accumulated prior to secretion at the advancing margin of the plasmodium.

Animals↗

PubMed1983

Effects of cholesterol surface transfer on cholesterol and phosphatidylcholine synthesis in cultured rat arterial smooth muscle cells.

The purpose of this study was to examine the effects of cholesterol surface transfer between lipid vesicles and rat arterial smooth muscle cells on endogenous synthesis of cholesterol and phosphatidylcholine. Lipid vesicles containing cholesterol and egg phosphatidylcholine in different proportions were used as the extracellular lipid source. The rate of cellular cholesterol and phosphatidylcholine synthesis was determined from the [14C]acetate incorporation into these lipid classes. [3H]Cholesterol in lipid vesicles, with a cholesterol/phospholipid (C/P) mole ratio of 1:1, was rapidly transferred into rat smooth muscle cells, with a half-time of about 3.6 hours in the absence of serum proteins. Incubation of cells for 5 hours with vesicles of a high C/P mole ratio (i.e. 1.5:1) at vesicle-cholesterol concentrations above 100 micrograms/ml resulted in a marked reduction of cellular cholesterol synthesis, whereas the rate of phosphatidylcholine synthesis was increased. Cells incubated with lipid vesicles of C/P 1:2 did not show any change in cellular cholesterol or phosphatidylcholine synthesis. Incubation of cells with egg phosphatidylcholine vesicles at concentrations above 300 micrograms/ml, on the other hand, stimulated endogenous synthesis of cholesterol without affecting cellular phosphatidylcholine synthesis. The main conclusion is that cholesterol surface transfer may influence cellular lipid metabolism in the absence of mediating serum lipoproteins in a model system with cultured cells and lipid vesicles.

Acetates↗

PubMed1989

Hepatobiliary transport of the anionic organomercury compound (mersalyl) is carrier mediated.

The hepatobiliary excretion of the anionic organic mercury compound (mersalyl) was studied in the isolated perfused rat liver and in isolated rat liver plasma membrane vesicles. In the isolated perfused liver, mersalyl is immediately taken up from the perfusion medium and concentratively excreted into bile. Uptake is characterized by saturation kinetics (S)0.5 = 20 microM, Vmax = 117 nmoles/min/g liver, cooperatively of mersalyl binding sites, stimulation by extracellular sodium and temperature dependence. Uptake of mersalyl into basolateral membrane vesicles also exhibits characteristics of a carrier mediated transport: saturation kinetics (S)0.5 = 28 microM, Vmax = 1.6 nmoles/min/mg protein, dependence on extravesicular sodium, cooperativity of mersalyl binding sites, temperature dependence and transstimulation by intravesicular non-radioactive mersalyl. Uptake was inhibited by alpha-naphthylacetic acid and mercapto group reagents, indicating involvement of mercapto groups on the carrier and a binding site for carboxylic anions. Data from the isolated perfused liver and from isolated basolateral vesicles indicate that mersalyl uptake into the liver is carrier mediated. Uptake mechanism and driving forces appear analogous to those for the uptake of chemically related compounds such as taurocholic acid. Therefore it is speculated that mersalyl may be transported by carrier molecules which apparently accept numerous chemically unrelated compounds.

Animals↗

PubMed1984

In vitro precocious accumulation of calcium and matrix vesicles formation in young cartilage cells: specific effects of corticosteroids.

The present study examined the effects of various corticosteroid and noncorticosteroid hormones upon the ultrastructure of chondroprogenitor cells and chondroblasts in an organ-culture system of late fetal condylar cartilage. Corticosteroids, (triamcinolone acetonide, dexamethasone, corticosterone) at concentrations of 10(-6) - 10(-8)M stimulated markedly a precocious formation of matrix vesicles by chondroblasts. This stimulation was accompanied by a significant accretion of calcium complexes intra- and extracellularly in both the chondroprogenitor cell population and chondroblasts in vitro, as well as in the newly induced matrix vesicles. Nonglucocorticoid steroids (progesterone, estradiol, testosterone, cortexolone) did not evoke similar effects. Progesterone and testosterone, however, seemed to adversely affect the ultrastructure of the cartilage cells, whereas estradiol appeared to have a favorable effect on the morphology of cultured condylar cartilage.

Adrenal Cortex Hormones↗

PubMed1999

Evanescent-wave microscopy: a new tool to gain insight into the control of transmitter release.

Evanescent-wave excitation was used to visualize individual fluorescently labelled vesicles in an optical slice near the plasma membrane of bovine adrenal chromaffin cells. A standard upright microscope was modified to accommodate the optics used for directing a laser beam under a supracritical angle on to the glass-water interface on top of which the cells are grown. Whereas epi-illumination images appeared blurred and structureless, evanescent-wave excitation highlighted acridine orange-labelled vesicles as individual pinpoints. Three-dimensional (3D) trajectories of individual vesicles were obtained from time-resolved image stacks and used to characterize vesicles in terms of their average fluorescence F and mobility, expressed here as the 3D diffusion coefficient D(3). Based on the single-vesicle analysis, two groups of vesicles were identified. Transitions between these states were studied before and after stimulation of exocytosis by repetitive or maintained membrane depolarizations by elevated extracellular [K+]. Findings were interpreted as sequential transitions between the previously characterized pools of vesicles preceding the fusion step. The observed approach of vesicles to their docking sites was not explained in terms of free diffusion: most vesicles moved unidirectionally as if directed to their binding sites at the plasma membrane. Vesicle mobility at the membrane was low, such that the sites of docking and fusion were in close vicinity. Both the rim region and confined areas in the centre of the footprint region were the site of intense vesicle trafficking.

Acridine Orange↗

PubMed1992

Membrane recapture and early triggered secretion from the newly formed endocytotic compartment in bovine chromaffin cells.

1. Recycling of secretory vesicles in cultured bovine adrenal medullary cells was investigated. 2. Extracellular horseradish peroxidase (HRP), a fluid phase marker, was taken up into cultured adrenal medullary cells following carbamylcholine-induced secretion of catecholamine. 3. The endocytosed HRP remained compartmentalized within the cell, migrating to a low density band on a Percoll density gradient. The endocytotic compartment was distinct from the major pool of catecholamine-containing chromaffin granules, which were found at much higher densities on the Percoll gradient. 4. The chromaffin granule membrane marker dopamine beta-hydroxylase was associated with the endocytosed HRP compartment as well as with the heavier chromaffin granules. 5. A subsequent challenge of the cells with carbamylcholine triggered the release of up to forty per cent of the endocytosed HRP. 6. The time course for secretion of the fluid phase marker was similar to that for catecholamine secretion. 7. Triggered release of HRP was dependent on extracellular calcium. The dependence on the extracellular calcium concentration was similar to that of catecholamine release. 8. Release of HRP could be triggered from electropermeabilized cells by raising the intracellular Ca2+ into the micromolar range. The intracellular Ca2+ dependence of triggered HRP release was similar to that for catecholamine release. 9. HRP could be secreted as early as 5 min, and as late as 2 h after endocytosis. 10. These data provide evidence that endocytotic vesicles can rapidly re-enter the secretory cycle. Endocytosed vesicles may therefore not have to recycle via the trans-Golgi reticulum to form high-density chromaffin granules in order to re-enter the regulated secretory pathway.

Adrenal Medulla↗

PubMed1995

Effect of veratridine on miniature endplate current amplitudes at the rat neuromuscular junction and acetylcholine uptake by Torpedo synaptic vesicles.

Veratridine produces a marked elevation in spontaneous quantal release from nerve endings through its ability to enhance sodium-channel activity, leading to sustained membrane depolarization. In the course of an electrophysiological investigation into the effects of vesamicol, an inhibitor of the synaptic vesicle acetylcholine transporter, on veratridine-induced acetylcholine release from rat motor nerve terminals we observed that veratridine itself has an effect on miniature endplate current amplitude distributions suggestive of an effect of the compound on the filling of cholinergic synaptic vesicles with acetylcholine. This effect of veratridine is release-dependent, being inhibited by either removal of extracellular calcium ions or by the addition of the sodium channel blocking toxin, tetrodotoxin. Biochemical studies using synaptic vesicles isolated from Torpedo electroplaque confirmed the ability of veratridine to directly inhibit the vesicular transport of acetylcholine. This appears to be a consequence of its ability to dissipate the trans-vesicular membrane proton gradient, which normally drives the active transport of acetylcholine into synaptic vesicles. We discuss how such an action of veratridine could lead to the observed release-dependent effects of the compound on electrophysiologically monitored spontaneous quantal acetylcholine release. The action of veratridine on cholinergic synaptic vesicles could be of considerable import when using this agent to elicit neurotransmitter release from either peripheral or central nerve endings.

Acetylcholine↗

PubMed1991

Link between cell apical morphology and H+ secretion in salamander small intestine.

To determine the morphological basis for the cellular secretion of H+ by in vitro salamander jejunum the transepithelial proton flux (JH) was measured under different conditions and compared with the ultrastructure of the villus absorptive cells using transmission electron microscopy in conjunction with morphometric analysis. Norepinephrine (NE) stimulated JH and significantly increased the density of small cytoplasmic vesicular and tubular profiles in the region immediately below the apical plasma membrane of the absorptive cells. Cytoplasmic tubular elements were often observed in direct contact with the plasma membrane at the base of the microvilli, suggesting that expansion of the plasma membrane had occurred. The alpha 2-antagonist, yohimbine, blocked NE-stimulated H+ secretion and reduced the number of tubulovesicles. Theophylline reduced the rate of spontaneous acid secretion and the density of apical tubules and vesicles. Exposure to horseradish peroxidase revealed a physical connection between the extracellular space and the apical tubulovesicular compartment. The direct relationship between the number of apical vesicular and tubular profiles and JH suggests that the luminal membrane plays a fundamental role in the cellular events in H+ secretion. The membranes of the tubules and vesicles may be elements of a dynamic pool of transporting membranes under the control of exogenous effectors such as catecholamines.

Animals↗

PubMed1987

Coordinated release of ATP and ACh from cholinergic synaptosomes and its inhibition by calmodulin antagonists.

Isolated cholinergic synaptosomes from elasmobranch electric organ release both ACh and ATP when depolarized in the presence of calcium. The conditions that trigger ATP release are the same as those known to stimulate neurosecretion. The ratio of ACh to ATP released is the same as that found in purified elasmobranch synaptic vesicles. Both ACh and ATP release are inhibited either by the removal of extracellular calcium or the addition of the "calmodulin antagonist" trifluoperazine (TFP). Taken together, these data suggest that both ACh and ATP are released by exocytosis from synaptic vesicles within nerve terminals. A number of drugs, collectively known as calmodulin antagonists, were examined for their effect on neurosecretion. TFP completely inhibited secretion in a rapid and reversible manner. Other related drugs had similar effects, though with relative efficacies different from those reported for inhibiting calmodulin. It therefore seems likely that a calmodulin-like component of the nerve terminal, distinct from calmodulin itself, is essential for the process of neurosecretion.

Acetylcholine↗

PubMed1997

Specific binding of [3H]GppNHp to extracellular membrane receptors in chick cerebellum: possible involvement of kainic acid receptors.

Guanine nucleotides (GNs), including GMP, displace [3H]kainic acid binding to chick cerebellar lysed and vesiculated membranes. Saturation studies of [3H]GppNHp binding, under conditions that prevent the occupation of the nucleotide binding sites in G-proteins, demonstrate the existence of extracellular membrane receptors specific for guanine nucleotides. Affinity-labeling of a vesicle preparation with [alpha-32P]GTP gives one single labeled band, upon electrophoresis, with an apparent molecular mass of 50 kDa. Additional experiments with partially purified kainate receptors suggest that the GN extracellular sites may overlap, at least partially, the kainic acid binding sites, being then responsible for the displacement of [3H]kainic acid by GNs. The physiological significance of these findings remains unclear.

Animals↗

PubMed1993

Extracellular calcium modulates insulin's action on enzymes controlling cyclic AMP metabolism in intact hepatocytes.

Absence of physiological concentrations of extracellular Ca2+ in the Krebs-Henseleit incubation buffer did not affect the ability of 10 nM glucagon (< 5%) to increase hepatocyte intracellular cyclic AMP concentrations, but severely ablated (by approximately 70%) the ability of 10 nM insulin to decrease these elevated concentrations. Cyclic AMP metabolism is determined by production by adenylate cyclase and degradation by cyclic AMP phosphodiesterase (PDE). In the absence of added extracellular Ca2+ (2.5 mM), insulin's ability to activate PDE activity was selectively compromised, showing a failure of insulin to activate two of the three insulin-stimulated activities, namely the 'dense-vesicle' and peripheral plasma-membrane (PPM) PDEs. In the absence of added Ca2+, insulin's ability to inhibit adenylate cyclase activity in intact hepatocytes was decreased dramatically. Vasopressin and adrenaline (+ propranolol) failed to elicit the activation of either the 'dense-vesicle' or the PPM-PDEs. The presence of physiological concentrations of extracellular Ca2+ in the incubation medium is shown to be important for the appropriate generation of insulin's actions on cyclic AMP metabolism.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1978

Ultrastructural localization of calcium in calcifying tendinitis.

To determine the site of mineralization in calcifying tendinitis, we studied the ultrastructure of tendon fragments that were obtained during surgical interventions on ten patients with that condition. We found that calcification was occurring extracellularly and essentially in areas where the tendon had undergone cartilaginous transformation. Calcific crystals were present in matrix-vesicle-like structures that were seen either singly among collagen fibers or in aggregates. When they were in aggregates, the calcifying vesicles tended to fuse with each other. Thus, the process of mineralization in calcifying tendinitis resembled those calcifying conditions that have already been described in man and animals, both normal and pathological, in which the apatite crystals are initially localized in matrix vesicles.

Calcinosis↗