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PubMed2005

A new quantitative (two-photon extracellular polar-tracer imaging-based quantification (TEPIQ)) analysis for diameters of exocytic vesicles and its application to mouse pancreatic islets.

We have developed an imaging approach to estimate the diameter of exocytic vesicles that are smaller than the resolution of an optical microscope and present within intact tissue. This approach is based on two-photon excitation imaging of polar tracers in the extracellular medium, is designated TEPIQ (two-photon extracellular polar-tracer imaging-based quantification), and has three variants. TEPIQ analysis of DeltaV measures vesicle volume with a fluid-phase tracer, sulforhodamine B (SRB). TEPIQ analysis of DeltaS determines vesicle surface area with a polar membrane tracer, FM1-43. TEPIQ analysis of DeltaV/DeltaS estimates vesicle diameter from the SRB/FM1-43 fluorescence ratio. TEPIQ analysis is insensitive to microscope settings because the same setup is used for calibration and actual experiments. We tested the validity of TEPIQ with glucose-induced exocytosis from beta-cells within pancreatic islets. The three TEPIQ variants yielded estimates for the mean diameter of exocytic vesicles of between 340 and 390 nm, consistent with the size of insulin granules. TEPIQ analysis relies on the combination of two-photon excitation imaging, the narrow intercellular spaces of intact tissue, and the presence of diffusible polar tracers in the extracellular medium. It allows quantitative imaging of exocytosis within secretory organs, yielding estimates of vesicle diameter with nanometer resolution.

Anatomy, Cross-Sectional↗

PubMed2002

Molecular determinants for activation of G-protein-coupled inward rectifier K+ (GIRK) channels by extracellular acidosis.

Synaptic cleft acidification occurs following vesicle release. Such a pH change may affect synaptic transmissions in which G-protein-coupled inward rectifier K(+) (GIRK) channels play a role. To elucidate the effect of extracellular pH (pH(o)) on GIRK channels, we performed experiments on heteromeric GIRK1/GIRK4 channels expressed in Xenopus oocytes. A decrease in pH(o) to 6.2 augmented GIRK1/GIRK4 currents by approximately 30%. The channel activation was reversible and dependent on pH(o) levels. This effect was produced by selective augmentation of single channel conductance without change in the open-state probability. To determine which subunit was involved, we took advantage of homomeric expression of GIRK1 and GIRK4 by introducing a single mutation. We found that homomeric GIRK1-F137S and GIRK4-S143T channels were activated at pH(o) 6.2 by approximately 20 and approximately 70%, respectively. Such activation was eliminated when a histidine residue in the M1-H5 linker was mutated to a non-titratable glutamine, i.e. H116Q in GIRK1 and H120Q in GIRK4. Both of these histidines were required for pH sensing of the heteromeric channels, because the mutation of one of them diminished but not abolished the pH(o) sensitivity. The pH(o) sensitivity of the heteromeric channels was completely lost when both were mutated. Thus, these results suggest that the GIRK-mediated synaptic transmission is determined by both neurotransmitter and protons with the transmitter accounting for only 70% of the effect on postsynaptic cell and protons released together with the transmitter contributing to the other 30%.

Acidosis↗

PubMed1991

Intracellular localization of group II phospholipase A2 in rat vascular smooth muscle cells and its possible relationship to eicosanoid formation.

We investigated the localization of group II phospholipase A2 (PLA2-II) in rat vascular smooth muscle cells (VSMCs) by applying immunofluorescence and immunoelectron microscopy with its polyclonal antibody. In unstimulated cells, no immunolabelling was detected in the cells. On the other hand, in the cells stimulated with tumor necrosis factor (TNF) and/or forskolin (FK), intense fluorescence was detected in the cytoplasm. The immunoperoxidase reactions were detected in the cisternae of rough endoplasmic reticulum (rER), trans-cisternae of Golgi apparatus, and small vesicles beneath the plasma membrane. Western blot analysis showed VSMCs secrete PLA2-II after stimulation. Secreted PLA2-II was associated with the plasma membrane and extracellular matrix. Colchicine inhibited PLA2-II synthesis and its secretion to the extracellular space. These observations indicate that in VSMCs PLA2-II is synthesized at rER. transported to Golgi apparatus, discharged into extracellular space via the small vesicles, and microtubules may concern with its process. Furthermore, in VSMCs treated with TNF or TNF + FK, prostaglandin E2 formation was also increased. Actinomycin D and cycloheximide inhibited the potentiation of the prostaglandin E2 formation induced by TNF or TNF + FK, indicating that both RNA and protein synthesis are required for the potentiation. These results suggest an involvement of PLA2-II in the prostaglandin formation.

Animals↗

PubMed1998

Ultrastructural modifications of the extracellular matrix upon calcification of growth plate cartilage as revealed by quick-freeze deep etching technique.

The ultrastructural changes in cartilage matrix that occur during calcification have been examined in chick epiphyseal growth plate cartilage prepared by quick-frozen, deep-etched, and rotary shadowed replicas. The extracellular cartilage matrix contains a reticular network closely associated with an extensive network of collagen. The components of the reticular network, including thick and thin filaments, are attached directly to the cell membrane, matrix vesicle membrane, and collagen fibrils. This network, which interconnects the matrix vesicles and collagen, fills the extracellular matrix. The dimensions of the reticular network seem to remain almost constant in size from the reserve and proliferative zones to the calcifying zone. The collagen fibrils seem to consist of subfibrillar structures that branch and anastomose. In optimally quick-frozen, deep-etched, prepared collagen, a cross-banding pattern was exposed. Globular structures stud the collagen fibrils, which gradually diminish in number from the reserve zone down to the calcifying zone. The matrix vesicles, when fractured, showed a granular appearance. In most cases, the fracture plane passed through the bilayer of the matrix vesicle membrane. The true surface of the matrix vesicle membrane, therefore, was exposed after deep etching. At the calcifying zone, crystal deposition had occurred in needle-like and/or plate-like form within the membrane-bound matrix vesicles. The reticular network was still intact in the vicinity of the calcified matrix, but in the intercrystalline space, neither the reticular structure nor the globular structure was detectable. Within the calcified matrix, both reticular and granular structures had disappeared from the interfibrillar space of the collagen fibrils.

Animals↗

PubMed2005

Impact on fatty acid metabolism and differential localization of FATP1 and FAT/CD36 proteins delivered in cultured human muscle cells.

We compared the intracellular distribution and regulatory role of fatty acid transporter protein (FATP1) and fatty acid translocase (FAT/CD36) on muscle cell fatty acid metabolism. With the use of adenoviruses, FATP1 and FAT genes were delivered to primary cultured human muscle cells. FATP1 and FAT moderately enhanced palmitate and oleate transport evenly at concentrations of 0.05, 0.5, and 1 mM. Long-term (16 h) consumption of palmitate and oleate from the media, and particularly incorporation into triacylglyceride (TAG), was stimulated equivalently by FATP1 and FAT at all fatty acid concentrations tested. In contrast, long-term CO(2) production was reduced by FATP1 and FAT at all doses of palmitate and at the lower concentrations of oleate. Neither FATP1 nor FAT markedly altered the production of acid-soluble metabolic intermediates from palmitate or oleate. The intracellular localization of fusion constructs of FATP1 and FAT with enhanced green fluorescent protein (EGFP) was examined. Independently of fatty acid treatment, FATPGFP was observed throughout the cytosol in a reticular pattern and concentrated in the perinuclear region, partly overlapping with the Golgi marker GM-130. FATGFP was found in the extracellular membrane and in cytosolic vesicles not coincident with GM-130. Neither FATP1 nor FAT proteins colocalized with lipid droplets in oleate-treated cells. We conclude that whereas FAT is localized on the extracellular membrane, FATP1 is active in the cytosol and imports fatty acids into myotubes. Overall, both FATP1 and FAT stimulated transport and consumption of palmitate and oleate, which they channeled away from complete oxidation and toward TAG synthesis.

Biological Transport, Active↗

PubMed2003

Lipopolysaccharide 3-deoxy-D-manno-octulosonic acid (Kdo) core determines bacterial association of secreted toxins.

In contrast to cholera toxin (CT), which is secreted solubly by Vibrio cholerae across the outer membrane, heat-labile enterotoxin (LT) is retained on the surface of enterotoxigenic Escherichia coli (ETEC) via an interaction with lipopolysaccharide (LPS). We examined the nature of the association between LT and LPS. Soluble LT binds to the surface of LPS deep-rough biosynthesis mutants but not to lipid A, indicating that only the Kdo (3-deoxy-d-manno-octulosonic acid) core is required for binding. Although capable of binding truncated LPS and Kdo, LT has a higher affinity for longer, more complete LPS species. A putative LPS binding pocket is proposed based on the crystal structure of the toxin. The ability to bind LPS and remain associated with the bacterial surface is not unique to LT, as CT also binds to E. coli LPS. However, neither LT nor CT is capable of binding to the surface of Vibrio. The core structures of Vibrio and E. coli LPS differ in that Vibrio contains a phosphorylated single Kdo-lipid A, and E. coli LPS contains unphosphorylated Kdo2-lipid A. We determined that the phosphate group on the Kdo core of Vibrio LPS prevents CT from binding, resulting in the secretion of soluble toxin. Because LT binds E. coli LPS, it remains associated with the extracellular bacterial surface and is released in association with outer membrane vesicles. We propose that difference in the extracellular fates of LT and CT contribute to the differences in disease caused by ETEC and Vibrio cholerae.

Bacterial Toxins↗

PubMed2005

Amphetamine induces dopamine efflux through a dopamine transporter channel.

Drugs of abuse, including cocaine, amphetamine (AMPH), and heroin, elevate extracellular dopamine (DA) levels in the brain, thereby altering the activity/plasticity of reward circuits and precipitating addiction. The physiological release of DA occurs through the calcium-dependent fusion of a synaptic vesicle with the plasma membrane. Extracellular DA is cleared by uptake through the Na+/Cl- -dependent DA transporter (DAT). In contrast, the substrate AMPH induces nonvesicular release of DA mediated by DAT. Extracellular AMPH is generally believed to trigger DA efflux through DAT by facilitating exchange for cytosolic DA. Here, in outside-out patches from heterologous cells stably expressing DAT or from dopaminergic neurons, by using ionic conditions in the patch pipette that mimic those produced by AMPH stimulation, we report that AMPH causes DAT-mediated DA efflux by two independent mechanisms: (i) a slow process consistent with an exchange mechanism and (ii) a process that results in rapid (millisecond) bursts of DA efflux through a channel-like mode of DAT. Because channel-like release of DA induced by AMPH is rapid and contains a large number of DA molecules, with a single burst of DA on par with a quantum of DA from exocytotic release of a vesicle, this burst mode of release may play a role in the synaptic actions and psychostimulant properties of AMPH and related compounds. Unlike AMPH, the endogenous substrate DA, when present on both sides of the plasma membrane, inhibits this channel-like activity, thereby suggesting that the DAT channel-like mode cannot accumulate DA against a concentration gradient.

Amphetamine↗

PubMed1991

Accumulation of phosphocreatine and creatine in the cells and fluid of mouse seminal vesicles is regulated by testosterone.

We have recently established that phosphocreatine (PCr), a high-energy phosphate compound known to serve as an intracellular energy reserve, accumulates in the seminal vesicular fluid (SVF) of the mouse and the rat. To investigate whether the accumulation of PCr in the extracellular fluid of the seminal vesicles of mice is androgen-dependent, young adult mice (Swiss Webster, 6-7 wk old) were orchidectomized. Involution of the seminal vesicles following orchidectomy resulted in the total absence of SVF. Administration of testosterone propionate (TP) 2 wk later at a daily dose of 5 micrograms/g b.w. caused a rapid increase in the weights of seminal vesicle tissue (SVT) and SVF and also increased the concentration of PCr in SVF. The concentration of PCr in SVF increased rapidly from 3.0 mumol/g on Day 2 to a peak value of 11.1 +/- 0.5 mumol/g on Day 6. The increase in PCr concentration in SVF coincided with the enhanced rate of secretion of specific proteins by androgen-responsive epithelial cells. The creatine (Cr) concentration in SVF also increased from 12.6 mumol/g on Day 2 to 41.1 +/- 4.6 mumol/g on Day 8. The concentrations of PCr in SVF of TP-treated mice were about twice those in the age-matched intact controls. The concentrations of PCr and of Cr in SVT also increased from 1.1 +/- 0.5 mumol/g and 1.7 +/- 0.8 mumol/g, respectively, on Day 0 to the steady-state levels of 5.3 +/- 0.7 mumol/g and 14.8 +/- 2.7 mumol/g by Day 8. These results demonstrate that the accumulation of PCr in the fluid of mouse seminal vesicle is regulated by androgen.

Adenosine Triphosphate↗

PubMed2000

The presynaptic calcium channel is part of a transmembrane complex linking a synaptic laminin (alpha4beta2gamma1) with non-erythroid spectrin.

Nerve regeneration studies at the neuromuscular junction (NMJ) suggest that synaptic basal lamina components tell the returning axon where to locate neurotransmitter release machinery, including synaptic vesicle clusters and active zones. Good candidates for these components are the synaptic laminins (LNs) containing alpha4, alpha5, or beta2 chains. Results from a beta2 laminin knockout mouse have suggested a linkage of this extracellular laminin to cytosolic synaptic vesicle clusters. Here we report such a transmembrane link at the electric organ synapse, which is homologous to the NMJ. We immunopurified electric organ synaptosomes and found on their surface two laminins of 740 and 900 kDa. The 740 kDa laminin has a composition of alpha4beta2gamma1 (laminin-9). Immunostaining reveals that as in the NMJ, alpha4 and beta2 chains are concentrated at the electric organ synapse. Using detergent-solubilized synaptosomes, we immunoprecipitated a complex containing alpha4beta2gamma1 laminin, the voltage-gated calcium channel, and the cytoskeletal protein spectrin. Other presynaptic proteins such as 900 kDa laminin are not found in this complex. We hypothesize that alpha4beta2gamma1 laminin in the synaptic basal lamina attaches to calcium channel, which in turn is attached to cytosolic spectrin. Spectrin could then organize synaptic vesicle clusters by binding vesicle-associated proteins.

Animals↗

PubMed1984

Reconstitution of purified halorhodopsin.

Asolectin lipid vesicles containing halorhodopsin show light-induced acidification in the presence of proton ionophores. This effect is abolished by triphenyltin chloride, a chloride/hydroxyl antiporter, and is greatly diminished by valinomycin in the presence of potassium ions, which collapse the membrane potential. This indicates that halorhodopsin orients in the lipid vesicles preferentially inside out, pumping chloride into the extravesicular compartment. The absorption maximum of halorhodopsin in asolectin vesicles in 3 M NaCl is at 567 nm, and the action spectrum for the light-induced pH changes followed closely the absorption spectrum. Replacement of chloride by acetate or sulfate causes a shift in the absorption maximum to approximately equal to 559 nm and renders the pump inactive. The different photocycles of the two forms were used to show that 80% of the molecules have the extracellular side exposed to the vesicle interior and that the halide-binding site(s) associated with the spectral transition is accessible from the extracellular side of the molecule. The data presented demonstrate that the purified chromoprotein is the light-driven chloride pump in Halobacterium halobium.

Bacteriorhodopsins↗

PubMed1984

Ectopic mineralization in fibroblast cultures.

A human gingival fibroblast cell line was cultured in a DM-153 medium supplemented with 10% bovine serum. The cells in culture showed both intracellular and extracellular ectopic mineralization. Ultrastructurally, mineralization began within the round and irregularly shaped vesicles contained in large cytoplasmic vacuoles of fibroblasts. The first identifiable needle-shaped crystal was deposited on a point of the inner leaflet of the vesicle membrane. With the increase in size of the crystals, the membranous envelopes were gradually lost, and lattice planes (8.2 A) occurred within the crystal thickness. Crystals radiating from each mineralization center formed mineralized spherules in cytoplasmic vacuoles. The crystal clusters were extruded from the cells through membrane fusion or cellular degeneration in long-term cultures. These liberated clusters formed extracellular mineralizing matrices around the fibroblasts. In extracellular mineral deposition, the initial crystals arose within extracellular matrix vesicles, but were not associated with collagen, elastic fibers, and any other structures. These findings indicate that mineral accumulation and phase transformation of amorphous mineral to a crystalline form take place within the vesicular structures. This provides additional evidence for the view that the initiation of calcification and mineralization requires a microenvironment limited by a membranous (vesicular) structure derived from cells.

Adult↗

PubMed2004

Characterization of a cytotoxic pilin subunit of Xenorhabdus nematophila.

Xenorhabdus nematophila is an insect pathogenic bacterium, known to produce protein toxins that kill the larval host. We have described a cytotoxic pilin subunit of X. nematophila, which is expressed on the cell surface and also secreted in the extracellular medium associated with outer membrane vesicles. A 17kDa pilin subunit was isolated and purified from X. nematophila cell surface. The protein showed cytotoxicity to larval hemocytes of Helicoverpa armigera in an in vitro assay, causing agglutination of the cells, and releasing cytoplasmic enzyme lactate dehydrogenase in the medium. The pilin protein was able to bind to the surface of larval hemocytes. The binding and cytotoxicity of the purified 17kDa protein to hemocytes was inhibited by antiserum raised against the pilin protein. The study demonstrates for the first time a cytotoxic structural subunit of pilin from an entomopathogenic bacterium X. nematophila that is excreted in the extracellular medium with outer membrane vesicles.

Agglutination Tests↗

PubMedDate not supplied

Effects of oscillatory mechanical disturbance on macromolecular uptake by arterial wall.

Transport of 125I-albumin by isolated segments of canine common carotid arteries was studied in vitro at zero transmural pressure. Sinusoidal oscillatory variations in length (peak change 4%) for 15 minutes at frequencies of 5 and 10 Hz caused 40% increase in 125I-albumin uptake, and also a 30% increase in the apparent luminal surface area. Changes in the duration and frequency of oscillation indicate that the total number of oscillations (= frequency X duration) was the critical parameter in causing these effects. The increase in apparent luminal surface area was correlated with regional flattening of the internal elastic lamina and the overlying endothelial cells, as demonstrated by transmission and scanning electron microscopy. Endothelial vesicles were counted with the aid of ruthenium red as a postfixation extracellular marker. The ratio of unstained free vesicles to total vesicles averaged 0.083 in the control state and decreased slightly to 0.070 after oscillation. Although the decrease in free vesicle population indicated an acceleration of vesicle diffusion, our theoretical computations showed that the resulting increase in vesicle flux was negligible. The increase in 125I-albumin uptake by the artery following mechanical oscillation is mainly attributable to the increase in apparent luminal surface area.

Animals↗

PubMed1997

In vitro formation of mineralized cartilagenous tissue by articular chondrocytes.

Study of the deep articular cartilage and adjacent calcified cartilage has been limited by the lack of an in vitro culture system which mimics this region of the cartilage. In this paper we describe a method to generate mineralized cartilagenous tissue in culture using chondrocytes obtained from the deep zone of bovine articular cartilage. The cells were plated on Millipore CMR filters. The chondrocytes in culture accumulated extracellular matrix and formed cartilagenous tissue which calcified when beta-glycerophosphate was added to the culture medium. The cartilagenous tissue generated in vitro contains both type II and type X collagens, large sulfated proteoglycans, and alkaline phosphatase activity. Ultrastructurally, matrix vesicles were seen in the extracellular matrix. Selected area electron diffraction confirmed that the calcification was composed of hydroxyapatite crystals. The chondrocytes, as characterized thus far, appear to maintain their phenotype under these culture conditions which suggests that these cultures could be used as a model to examine the metabolism of cells from the deep zone of cartilage and mineralization of cartilagenous tissue in culture.

Alkaline Phosphatase↗

PubMed2001

The nucleoside triphosphate pyrophosphohydrolase isozyme PC-1 directly promotes cartilage calcification through chondrocyte apoptosis and increased calcium precipitation by mineralizing vesicles.

OBJECTIVE: Aging associated elevations of cartilage extracellular inorganic pyrophosphate (PPi) and PPi-generating nucleoside triphosphate pyrophosphohydrolase (NTPPPH) are linked with degenerative arthritis in chondrocalcinosis. Increased chondrocyte apoptosis and expression of annexin V occur at sites of matrix calcification in degenerative arthritis, and membrane limited chondrocyte apoptotic bodies containing NTPPPH may promote chondrocalcinosis by acting as mineralizing matrix vesicles (MV). Because chondrocytes express 3 related NTPPPH isozymes [PC-1, autotaxin (ATX), and B10/PDNP3], we evaluated the effects on apoptosis and MV mediated calcium precipitation of direct expression of NTPPPH isozymes. METHODS: To achieve "gain of function" of NTPPPH isozymes, we expressed the isozymes in cultured chondrocytic cells. RESULTS: Plasmid cDNA transfection of PC-1, but not ATX or B10/PDNP3, markedly increased apoptosis of cultured chondrocytic knee meniscal cells and increased calcium precipitation by MV fractions. The capacity of PC-1 to increase chondrocyte and meniscal cell apoptosis, and calcium precipitation by MV, further analyzed using adenoviral gene transfer in cultured meniscal cells and articular chondrocytes, was shown to be dependent on integrity of the PC-I NTPPPH catalytic site. The MV-containing fraction released from meniscal cells and chondrocytes that overexpressed wild-type PC-1 had increased annexin V. Use of antibodies to annexin V and PC-1 revealed that both annexin V and PC-1 directly mediated the elevated calcium-precipitating capacity of MV. The increased ability of MV to precipitate calcium from PC-1-overexpressing cells did not require exogenous ATP. CONCLUSION: Upregulated expression of enzymatically active PC-1 directly promotes apoptosis, increased MV annexin V, and an increased capacity of meniscal cell and articular chondrocyte MV to precipitate calcium. These results suggest a direct link between increased PC-1 expression and the pathogenesis of chondrocalcinosis.

Apoptosis↗

PubMed1984

Stereological analysis of the epiphyseal growth cartilage in the brachymorphic (bm/bm) mouse, with special reference to the distribution of matrix vesicles.

The brachymorphic (bm/bm) mutation in the mouse leads to disproportional dwarfism due to a disturbance of endochondral bone formation. The morphological characteristics of bm/bm epiphyseal growth cartilage are signs of cellular degeneration and disintegration and alteration of the composition of the extracellular matrix, with an abnormal mineralization pattern. The present stereological study of the bm/bm growth plate revealed a clearly altered distribution of matrix vesicles as compared with the controls. It was also demonstrated that the bm/bm matrix vesicles have an abnormal size distribution, with an increased mean caliper diameter. The biological significance of these findings is discussed in relation to the different hypotheses on the origin of matrix vesicles and their possible role in the mineralization process. The results support the opinion that extracellular matrix vesicles, at least partly, constitute cellular debris.

Animals↗

PubMed1995

Ultrastructural localization and translocation of nitric oxide synthase in the endothelium of the human cerebral artery.

An electron microscopic immunocytochemical study was undertaken to clarify ultrastructural localization and translocation of nitric oxide synthase (NOS) in endothelial cells (EC) of the human cerebral and superficial temporal arteries (STA) employing antibody against endothelial NOS (EC-NOS). NOS immunoreactivity was found in all EC examined, in association with the plasma membrane and cytoplasmic organelles such as endoplasmic reticulum, Weibel-Palade body and subplasmalemmal vesicles, and in the cytoplasm devoid of organelles and extracellular regions, irrespective of arteries. The immunoreactivity in subplasmalemmal vesicles was, however, demonstrated only in human cerebral arteries. In the human STA exposed to bradykinin which induces EC-NOS phosphorylation, the gold particles significantly increased in the cytosol and decreased in the areas associated with cytoplasmic organelles; however, the number of particles did not change significantly in the plasma membrane. The results implicate that NOS may be translocated from the area associated with cytoplasmic organelles to cytosol following EC exposure to bradykinin.

Adult↗

PubMed1989

The effect of anions on bound acetylcholine in frog sartorius muscle.

1. Frog sartorius muscles were treated with an irreversible cholinesterase inhibitor and then incubated in isotonic potassium propionate solution (isotonic KPr). Total and bound, presumably vesicular, acetylcholine (ACh) in the tissue and ACh in the medium were assayed by mass fragmentography, miniature end-plate potentials (MEPPs) were recorded and the end-plates were investigated by electron microscopy. 2. Incubation in isotonic KPr for 30 min stimulated ACh release and concomitantly decreased total and bound ACh. Nerve stimulation for 30 min by trains of impulses (0.1 s trains of 100 Hz, 1 train s-1) in normal-potassium propionate-containing solution had the same effects. 3. When the tissue was incubated in normal-K+ Ringer solution for 3 h, following chemical or electric stimulation, bound ACh recovered to about 75% of the initial value, provided that Cl- ions were present in the medium. In the presence of propionate instead of Cl- ions almost no recovery of bound ACh took place. There was also recovery of bound ACh in the presence of either NO3- or gluconate ions. In NO3- it was the same as in Cl-, but in gluconate it was less than found in Cl- -containing medium. 4. Recovery of total ACh, in contrast to bound ACh, took place even in the presence of propionate ions, showing that extracellular Cl- is not required for the synthesis of ACh. 5. In terminals recovered in normal Ringer solution, many synaptic vesicles were found, but terminals 'recovered' in propionate solution were depleted of vesicles. 6. From these and other results it is concluded that the recycling of synaptic vesicles normally requires the presence of extracellular chloride.

Acetylcholine↗