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At least 19 recordsLinked to original sources

PubMed1981

Maturation of sympathetic neurotransmission in the rat heart. IV. Effects guanethidine-induced sympathectomy on neonatal development of synaptic vesicles, synaptic terminal function and heart growth.

Sympathetic nerve input has been proposed to regulate cardiac growth and differentiation. In the present study, this hypothesis was tested by giving the neurotoxic adrenergic neuron blocking agent, guanethidine (50 mg/kg, s.c.), daily to rats for 21 consecutive days to produce long-term peripheral sympathectomy in neonatal rats. Ontogeny of the sympathetic nerve terminal was measured by the ablity of synaptic vesicle preparations to take up radiolabeled norepinephrine, and heart growth in the sympathectomized animals was monitored by organ weight as well as by RNA and protein synthesis. Guanethidine treatment produced a massive sympathectomy, as synaptic vesicle development was totally arrested; the functional consequence of this treatment was confirmed by the attenuation of chronotropic responses to tyramine, a drug which acts by displacement of norepinephrine from the noradrenergic terminal. Despite the clear-cut effectiveness of guanethidine to prevent formation of functional sympathetic innervation of the heart, no significant alterations in heart growth or RNA and protein synthetic capabilities were observed in the developing rats. These results suggest that the presence of sympathetic innervaton is not obligatory for normal growth of the heart to occur.

Aging↗

PubMed1993

Synaptic vesicle and synaptic membrane glycoproteins during pre- and postnatal development of mouse cerebral cortex, cerebellum and spinal cord.

Glycoproteins of synaptic vesicles and synaptic membranes play an important role during the process of synaptogenesis. In order to study the temporal expression of specific carbohydrates and the expression of selected neural proteins, we used peroxidase-conjugated lectin overlays on Western blots and immunoblots of synaptic vesicles and synaptic membranes isolated from pre- and postnatal mouse cerebral cortex, cerebellum and spinal cord. Our lectin overlays on Western blots showed that (1) the synaptic vesicle glycoproteins, gp80-100, gp47 and gp44, and (2) the synaptic membrane glycoproteins, gp180, gp72, gp70 and gp34, show temporal regulation of expression of carbohydrate moieties. Quite significantly, gp47 showed a decrease in the vesicles coinciding with an increase in membranes suggesting a shift in localization. Anti neural cell adhesion molecule (N-CAM) antibody cross-reacted with gp180. The developmental expression of synaptotagmin 1, a well characterized glycoprotein of synaptic vesicle, was determined by immunoblots analysis. Anti synaptosomal-associated protein 25 (SNAP-25) antibody immunoblots were performed in order to compare our results with a developmentally regulated synaptosomal protein demonstrating expression coincident with synaptogenesis. Our immunoblot studies showed that (1) N-CAM (gp180) immunoreactivity decrease with development; (2) the expression of synaptotagmin 1 and SNAP-25 increases as development progresses, and (3) synaptotagmin 1 and SNAP-25 show a shift in subcellular localization (from synaptic vesicle to synaptic membrane) during development, thereby indicating that these proteins are first identified in a vesicular fraction. Thus, our data suggest that synaptic vesicle and synaptic membrane glycoproteins show temporal regulation of specific carbohydrates as well as protein expression during development, which may be a key factor to our understanding of the process of synaptogenesis.

Animals↗

PubMed1980

A calcium-stimulated alkaline phosphatase associated with synaptic vesicles.

Synaptic vesicles isolated from bovine cerebral cortex were found to contain alkaline phosphatase activity towards p-nitrophenylphosphate and alpha-naphthyl phosphate, but not towards pyridoxal phosphate. The enzyme had an apparent molecular weight of 125,000 and co-purified with the synaptic vesicles in parallel with the specific neurotransmitter content and with the loss of contaminating components, whereas the major phosphatase which was present in the brain homogenate, with an apparent molecular weight of 140,000 purified away. The optimal pH for the enzyme activity on p-nitrophenylphosphate was 9.8. At this pH the activity was 33.4 nmol/mg protein/min, and the apparent Km value was 0.31 +/- 0.05 mM. The pH dependency of the synaptic vesicle phosphatase activity towards p-nitrophenylphosphate differed from that of the Ca2+/Mg2+-dependentt ATP hydrolysis by the synaptic vesicles. Upon mild digestion of lyzed vesicles with trypsin, phosphatase activity was reduced whereas the ATPase activity was retained suggesting that the phosphatase and the ATPase are two different enzymes. The phosphatase was reversibly inhibited by ethyleneglycol bis (aminoethyl ether) N,N'-tetracetic acid (EGTA) and activity was restored by the addition of an equimolar amount of CA2+. Magnesium ions could restore only 30% of the activity. The activity of the synaptic vesicle phosphatase was not affected by o-phenanthroline, zinc ion or by cAMP. Tetranitromethane inactivated the enzyme irreversibly, whereas phenylmethanesulfonylfluoride diisopropylfluorophosphate and p-hydroxymercurybenzoate inhibited the activity partially. The enzyme did not have a diesterase activity. Adenosine mono-, di- and triphosphate inhibited the p-nitrophenylphosphatase activity and were also hydrolyzed by the vesicle preparation. However, the different kinetic parameters obtained with the nucleotide as inhibitors or as substrates suggest that additional enzymes are involved in the hydrolysis of the adenine nucleotides in vesicle preparation.

Alkaline Phosphatase↗

PubMed1979

Stimulation of Ca2+-dependent neurotransmitter release and presynaptic nerve terminal protein phosphorylation by calmodulin and a calmodulin-like protein isolated from synaptic vesicles.

Synaptic vesicles have a Ca(2+)-dependent protein kinase system that may play a role in mediating Ca(2+)-stimulated neurotransmitter release and vesicle function. Calcium's ability to initiate norepinephrine release and protein phosphorylation in synaptic vesicle preparations was shown to be stimulated by the presence of an endogenous heat-stable vesicle protein fraction. The heat stability and characteristics of this endogenous vesicle fraction were similar to those of calmodulin (Ca(2+)-dependent regular protein) isolated from rat and bovine brain. Calmodulin, like endogenous heat-stable vesicle factor, restored calcium's ability to stimulate vesicle neurotransmitter release and protein kinase activity. Calmodulin-like vesicle protein and purified calmodulin were also equally effective in stimulating cyclic nucleotide-dependent phosphodiesterase, further indicating that these two proteins are functionally equivalent. Depolarization-dependent Ca(2+) uptake in intact synaptosomes simultaneously stimulated release of neurotransmitter and phosphorylation of particular synaptic vesicle proteins that were shown in the isolated vesicle preparation to be dependent on Ca(2+) and calmodulin. The results suggest that calcium's effects on neurotransmitter release and presynaptic nerve terminal protein phosphorylation may be mediated by endogenous calmodulin-like proteins.

Animals↗

PubMed2002

Roles of ATP in depletion and replenishment of the releasable pool of synaptic vesicles.

Synaptic terminals of retinal bipolar neurons contain a pool of readily releasable synaptic vesicles that undergo rapid calcium-dependent release. ATP hydrolysis is required for the functional refilling of this vesicle pool. However, it was unclear which steps required ATP hydrolysis: delivery of vesicles to their anatomical release sites or preparation of synaptic vesicles and/or the secretory apparatus for fusion. To address this, we dialyzed single synaptic terminals with ATP or the poorly hydrolyzable analogue ATP-gammaS and examined the size of the releasable pool, refilling of the releasable pool, and the number of vesicles at anatomical active zones. After minutes of dialysis with ATP-gammaS, vesicles already in the releasable pool could still be discharged. This pool was not functionally refilled despite the fact that its anatomical correlate, the number of synaptic vesicles tethered to active zone synaptic ribbons, was completely normal. We conclude 1) because the existing releasable pool is stable during prolonged inhibition of ATP hydrolysis, whereas entry into the functional pool is blocked, a vesicle on entering the pool will tend to remain there until it fuses; 2) because the anatomical pool is unaffected by inhibition of ATP hydrolysis, failure to refill the functional pool is not caused by failure of vesicle movement; 3) local vesicle movements important for pool refilling and fusion are independent of conventional ATP-dependent motor proteins; and 4) ATP hydrolysis is required for the biochemical transition of vesicles and/or release sites to fusion-competent status.

Adenosine Triphosphate↗

PubMed1973

Electrokinetic properties of isolated cerebral-cortex synaptic vesicles.

Synaptic vesicles isolated from guinea-pig cerebral cortex had an electrophoretic mobility of -3.55mum.s(-1).V(-1).cm in saline-sorbitol, pH7.2, at 25 degrees C (ionic strength 0.015g-ions/1). The mobility was pH-dependent, varied with ionic strength and indicated that the vesicular surface contained weak acidic functions with a pK(a) in the range 3.0-3.8. Although the vesicular surface was determined to be highly negatively charged, treatment with neuraminidase had no effect on mobility and indicated that the relatively strong carboxyl groups of sialic acid do not contribute significantly to vesicular electrokinetic properties. Treatment of synaptic vesicles with trypsin or trypsinized concanavalin A resulted in increases in mobility, but treatment with ribonuclease, deoxyribonuclease, chrondroitinase ABC or hyaluronidase had no significant effect on mobility. Mn(2+) or Ca(2+) was more effective in decreasing vesicle mobility than was Mg(2+), Sr(2+) or Ba(2+). The electrokinetic properties of the synaptic vesicle surface are discussed and contrasted with the properties of the synaptosomal membrane.

Animals↗

PubMed1994

Proline clustering in proteins from synaptic vesicles.

Synaptic vesicle proteins share a specialized and common fate throughout the life cycle of the vesicle, and thus may need to respond to some common signals. It is therefore expected that these proteins will share some common motifs. However, sequence comparison among many of these proteins has not revealed any obvious motifs. Such a motif may be formed by the relative abundance of proline residues, which are not randomly distributed along the sequence but rather are clustered at the cytoplasmatic face of vesicular proteins. We propose that proline clusters serve as structural spacers between functional domains as well as potential target sites for protein-protein interactions. In view of the proline-rich nature of SH3 binding proteins, some of the proline-rich synaptic vesicle proteins may also participate in SH3 binding. Such binding may modulate certain signalling pathways in nerve terminals. Surprisingly, the consensus sequence between the proline clusters of synaptic vesicle proteins is found in a large family of abundant proline-rich proteins of the secretory organelles of the parotid exocrine gland.

Amino Acid Sequence↗

PubMed2006

ATP dependence of the non-specific ion channel in Torpedo synaptic vesicles.

Synaptic vesicles of Torpedo electromotor neurons contain a high amount of ATP. The concentration of total ATP is around 120 mM, whereas the free [ATP] is about 5-6 mM. We examined the effect of intravesicular ATP on the non-specific ion channel in Torpedo-fused synaptic vesicles. It was found that this channel is closed when the ATP concentration is above 2 mM, but it is very frequently open at lower ATP concentrations. Unmasking this ion channel at a low ATP concentration may be significant for post-fusion control of transmitter release by the 'kiss and run' mechanism in normal conditions, while during metabolic stress it may underlie dissipation of important gradients across the vesicle membrane.

Adenosine Triphosphate↗

PubMed2000

Levels of mRNAs encoding synaptic vesicle and synaptic plasma membrane proteins in the temporal cortex of elderly schizophrenic patients.

BACKGROUND: Electron microscopy and biochemical studies indicate that developmental abnormalities in synaptic organization may be present in brains of schizophrenic patients. This study determined whether these synaptic abnormalities are reflected in differential or uniform alterations in the expression of various synaptic protein genes in the left superior temporal gyrus of schizophrenic patients. METHODS: Levels of mRNAs encoding four synaptic vesicle proteins (synaptotagmin I [p65], rab3a, synaptobrevin 1, and synaptobrevin 2) and two synaptic plasma membrane proteins (syntaxin 1A and SNAP-25) were measured postmortem in the left superior temporal gyrus from elderly (58-95 years) schizophrenic patients (n = 14) and age-matched control subjects (n = 9). RESULTS: There were significant negative correlations between age and levels of synaptotagmin I (p65), rab3a, synaptobrevin 1, SNAP-25, and syntaxin 1A mRNAs in schizophrenic patients (-.692 < r < -.517,.003 < p <.030) but not in control subjects. Levels of all six synaptic mRNAs studied were increased in the younger (58-79 years) subgroup of schizophrenic patients compared to control subjects and older (80-95 years) subgroup of schizophrenic patients. CONCLUSIONS: That similar abnormalities were found for mRNAs encoding different synaptic vesicle and synaptic plasma membrane proteins suggests that they reflect overall neurodevelopmental abnormalities in synaptic connectivity in the temporal cortex of schizophrenic patients rather than changes in the number of synaptic vesicles per synapse or abnormalities in a specific synaptic function.

Aged↗

PubMed2000

Efferent function of vestibular afferent endings? Similar localization of N-type calcium channels, synaptic vesicle and synaptic membrane-associated proteins.

We investigated the distribution of N-type voltage-dependent calcium channels that mediate Ca(2+) entry initiating transmitter release in the rat vestibular sensory epithelium. We used confocal microscopy to assess the in vitro labeling by fluorescent specific ligand binding, omega-conotoxin-GVIA and also the immunolabeling of presynaptic soluble N-ethylmaleimide-sensitive fusion factor attachment protein receptor (SNARE) proteins, syntaxin, 25,000 mol. wt synaptosome-associated protein and synaptotagmin: components of the neurotransmitter exocytosis machinery. We found that there was a close anatomical association between the voltage-gated calcium channels, the synaptic vesicle and synaptic membrane-associated proteins on the afferent nerve calyces and probably afferent boutons, which are postsynaptic compartments. Our data suggest that these peripheral afferent endings possess the presynaptic Ca(2+) channels and the components of the presynaptic SNARE proteins involved in synaptic vesicle docking and calcium-dependent exocytosis. They provide additional evidence for a secretory function and efferent role of these endings in hair cell neurotransmission.

Afferent Pathways↗

PubMed1994

Formation of synaptic vesicles.

Synaptic vesicles (SVs) are specialized secretory organelles used for the fast and focal signaling between nerve cells. They are small and homogeneous in size (50 nm), and contain non-peptide neurotransmitters such as glutamate, gamma-aminobutyric acid (GABA) and acetylcholine. The exocytosis of SVs occurs at low rates in resting nerve terminals and is greatly stimulated by depolarization-induced Ca2+ influx. Following exocytosis, SV membranes are rapidly retrieved, refilled locally with neurotransmitters and reused for the assembly of new SVs. Over the past few years, significant progress has been made in characterizing the molecular composition of SVs. From these studies, we know that SVs share a conserved set of membrane proteins with transport vesicles involved in other pathways. Furthermore, these findings have provided us with a new understanding about the evolutionary origin of SVs from recycling vesicles present in all cells.

Animals↗

PubMed1979

Kinetics of cation-induced aggregation of Torpedo electric organ synaptic vesicles.

Synaptic vesicles from the Torpedo ray can be induced to aggregate in the presence of Ca2+ and K+ in the 4 mM and 50 mM range, respectively. The reactions are strikingly similar to those of chromaffin granule membranes reported previously (Morris, S.J., Chiu, V.C.K. and Haynes, D.H. (1979) Membrane Biochem. 2, 163-202). The Ca2+-induced reaction includes dimerization and higher order aggregation, and is shown to be due to electrostatic screening interactions and bindng to negatively-charged groups on the membrane surface. The K+-induced reaction includes only dimerization and is shown to be due to screening interactions alone. The kinetics of the dimerization reactions were studied using the stopped-flow rapid mixing technique. The Ca2+-induced reaction has a 'bimolecular' rate constant of 4.77 . 10(8) M-1 . s-1. These values are close to the limit of diffusion control (8.03 . 10(9) M-1 . s-1), indicating that no large energy barriers or structural barriers to aggregation exist. Arrhenius plots for the Ca2+-induced aggregation showed a break at 5 degrees C. Above this temperature, the activation energy is low (+0.65 kcal/mol), consistent with the above. Below this temperature, the activation energy is high, consistent with a membrane structure change increasing theenergetic and structural barriers. This information, and the observation of a high stability constant of the complex, were taken as evidence for the involvement of 'recognition sites' on the membrane surface. The results were analyzed in terms of an encounter complex model in which vesicles with separations of 26-126 A are considered capable of transformation into a stable complex. The rate constant of the transformation step is 1.4 . 10(3) s-1 for Ca2+ and approx. 1.6 . 10(5) s-1 for K+. The values are compared with previous results for chromaffin granule membranes and for phospholipid vesicles derived from chromaffin granule lipids and from acidic phospholipids. The half-time for Ca2+-induced transformation of the encounter complex into the stable complex is 435 microseconds. It is concluded that the recognition sites are almost as optimally deployed as the vesicle plasma membrane recognition sites involved in exocytotic release.

Animals↗

PubMed1975

The duration of aldehyde fixation as a "flattening factor" of synaptic vesicles.

Synaptic vesicle flattening can be induced in the excitatory mossy fibre endings of the rat cerebellum by prolonged immersion in aldehyde during fixation (with or without perfusion). The flattening is found in a greater percentage of vesicles if perfusion has been omitted before the prolonged immersion. This is discussed in relation to the various other factors that are thought to cause flattening and the important problem of the classification of different types of synapse.

Aldehydes↗

PubMed1981

On the specificity of uptake by isolated Torpedo synaptic vesicles.

Synaptic vesicles were isolated from the electric organ of Torpedo marmorata in highly purified form. Their uptake properties were examined using a large number of small organic molecules as substrates. Following incubation at 26 degree C for 1 h, it was found that concentrative accumulation, indicated by a vesicle:medium concentration ratio greater than unity, was achieved by all the choline analogues used and by four biogenic amines, but not by a variety of purine and pyrimidine bases and nucleosides. Amino acids penetrated poorly, as did sugars, and of organic anions, acetate but not citrate or thiocyanate, was almost excluded. Thus Torpedo vesicles are relatively impermeable to compounds which cannot utilize the ACh or ATP carriers, but show a very high rate of amine uptake, which may be linked to a pH gradient.

Amino Acids↗

PubMed1972

The heterogeneity of bound acetylcholine and synaptic vesicles.

Synaptic vesicles containing radioactive acetylcholine have been isolated from slices of Torpedo electric organ incubated with radioactive choline. The recently synthesized radioactive acetylcholine is preferentially removed from the vesicles by iso-osmotic gel filtration. There is therefore a small compartment of loosely bound recently synthesized acetylcholine within the monodisperse vesicle fraction. The specific radioactivity of this compartment correlates most closely with the ;free' acetylcholine of electric organ that is lost when the tissue is homogenized. Membrane-associated vesicles did not contain any particular enrichment of this compartment. On standing at 6 degrees C the loosely bound compartment stabilizes so that it survives iso-osmotic filtration. A study of this phenomenon revealed that it was proportional to the extent of the loss of tightly bound acetylcholine from the vesicles. Incubation with Ca(2+), at pH5.5, or partial hypo-osmotic shock, caused losses of tightly bound acetylcholine and proportional increases in the stabilization of loosely bound acetylcholine of vesicles. Incubation at 20 degrees C caused less loss of tightly bound, and less stabilization of loosely bound, acetylcholine. A theoretical treatment of these exchanges also shows that the random factors promoting loss of tightly bound acetylcholine are statistically correlated with those which cause stabilization of loosely bound acetylcholine. The reciprocal relationship between the exchanges is inconsistent with there being two distinct populations of vesicles, one containing recently synthesized, loosely bound acetylcholine and the other containing tightly bound acetylcholine. It is proposed that all the vesicles contain a core of tightly bound acetylcholine and a surface layer of loosely bound acetylcholine. The origin of the extravesicular acetylcholine and also of the acetylcholine released on stimulation is discussed in the light of these results.

Acetylcholine↗

PubMed1977

Membrane protein and glycoprotein composition of beef brain synaptic vesicles.

Synaptic vesicles were isolated from adult bovine cortical gray matter by differential centrifugation and membrane filtration of a hypoosmotically lysed crude mitochondrial fraction. Vesicle preparations were analyzed for purity by electron microscopy and enzyme assays. Polyacrylamide gel electrophoresis of SDS-solubilized and 2-mercaptoethanol-reduced vesicle membrane proteins revealed 4 major proteins with molecular weights ranging from 17,000 to 60,000, and about 10 minor proteins with molecular weights up to 170,000. The protein profile of the Triton X-100-extracted vesicle membranes was less complex, with 1 major protein and 5 minor bands. The major protein of the Triton extract was identified as a glycoprotein with a molecular weight of 45,000. Two additional minor PAS-positive bands were seen, with molecular weights of 78,000 and 95,000.

Animals↗

PubMedDate not supplied

Evaluation of the evidence for ion channels in synaptic vesicles.

Synaptic vesicles (SVs) have been the focus of much research for many years, however only recently have ion channels from SV membranes been reported. There is now convincing evidence that SVs contain ion channels. This conclusion is based on direct experimental results from several different laboratories using the patch clamp or planar lipid bilayer technique on SVs and neurosecretory granules (NSG). Some limitations of patch clamping and of fusing synamptic vesicles to a bilayer are described and the advantages of the nystatin/ergosterol fusion method are presented. Six different channels appear to exist in SV (or NSG) membranes. Two large channels (250 and 154 pS) have been observed in SVs isolated from mammalian brain, two channels (180 and 13 pS) from Torpedo electric organ, and two channels (130 and 30-40 pS) from NSG. The three larger channels from each set (250, 180 and 130 pS7) are novel in that they have a subconductance state. The 154 pS channel has been identified as synaptophysin but the identity and function of the other channels is unknown. Although some of the channels are gated by voltage, only the 130 pS channel is modulated by Ca2+. Further knowledge of what regulates these channels is mandatory if we are to determine the physiological significance of these channels.

Animals↗

PubMed2003

Synaptophysin I controls the targeting of VAMP2/synaptobrevin II to synaptic vesicles.

Synaptic vesicle (SV) proteins are synthesized at the level of the cell body and transported down the axon in membrane precursors of SVs. To investigate the mechanisms underlying sorting of proteins to SVs, fluorescent chimeras of vesicle-associated membrane protein (VAMP) 2, its highly homologous isoform VAMP1 and synaptotagmin I (SytI) were expressed in hippocampal neurons in culture. Interestingly, the proteins displayed a diffuse component of distribution along the axon. In addition, VAMP2 was found to travel in vesicles that constitutively fuse with the plasma membrane. Coexpression of VAMP2 with synaptophysin I (SypI), a major resident of SVs, restored the correct sorting of VAMP2 to SVs. The effect of SypI on VAMP2 sorting was dose dependent, being reversed by increasing VAMP2 expression levels, and highly specific, because the sorting of the SV proteins VAMP1 and SytI was not affected by SypI. The cytoplasmic domain of VAMP2 was found to be necessary for both the formation of VAMP2-SypI hetero-dimers and for VAMP2 sorting to SVs. These data support a role for SypI in directing the correct sorting of VAMP2 in neurons and demonstrate that a direct interaction between the two proteins is required for SypI in order to exert its effect.

Animals↗