Purification of small dense vesicles from stimulated Torpedo electric tissue by glass bead column chromatography

Synaptic vesicles were isolated from perfused tissue blocks of Torpedo electric organ using sucrose density gradient centrifugation in swing-out rotors. After application of [³H]acetate and low frequency stimulation (0.1 Hz) a denser peak of [³H]acetylcholine could be separated from the main and coincident peak of the vesicle constitutents acetylcholine and adenosine 5'-triphosphate in accordance with previous findings using zonal centrifugation (Zimmermann & Denston, Neuroscience2, 715–730, 1977). Further separation of subcellular particles sedimenting in the range of synaptic vesicles, by chromatography through columns of porous glass beads, yielded three main fractions which were eluted in the order, large (esterase-containing) membrane particles in the void volume, larger synaptic vesicles containing acetylcholine of low specific radioactivity (peak I) and smaller vesicles containing acetylcholine of higher specific radioactivity (peak II). After stimulating the electric tissue (which causes the appearance of a large proportion of synaptic vesicles about 25% smaller in diameter; Zimmermann & Denston, Neuroscience2, 695–714, 1977), the peak of larger vesicles (peak I) also contains vesicles of smaller diameter. The glass bead column thus separates membrane fragments from synaptic vesicles, but only partially resolves larger and smaller vesicles. This is also reflected by the decrease in the ratio of the specific radioactivity of acetylcholine of peak I to that of peak II, from 8.2 for unstimulated control to 4.0 for stimulated tissue.The results demonstrate that using glass bead chromatography the smaller vesicles, which appear on stimulation-induced transmitter release, contain acetylcholine of high specific radioactivity and can be completely separated from any membrane contaminants which could possibly contain a pool of nonvesicle-bound acetylcholine.     

Separation of synaptic vesicles of different functional states from the cholinergic synapses of the Torpedo electric organ

Synaptic vesicles were isolated on sucrose zonal gradients from perfused tissue blocks of Torpedo electric organ. They give rise to a coincident peak in the concentrations of acetylcholine and adenosine 5′-triphosphate. On low-frequency stimulation (0.1 Hz) of the nerve attached to the tissue block a distinct population of synaptic vesicles is found that sediments further into the density gradient forming a second (denser) vesicle peak. When dextran is added to the perfusate, only these denser vesicles contain electron-dense granules. This second (denser) peak contains about 25% of all vesicular acetylcholine and about 30% of the adenosine 5′-triphosphate and most of the newly synthesized acetylcholine as shown by incorporation of radiolabelled acetate. The specific radioactivity of acetylcholine in the denser vesicles after 1800 impulses is on average 16.5 times higher than that of the vesicles sedimenting at the original density and 9.2 times higher than the average of all vesicles isolated. The specific radioactivity of total tissue acetylcholine is lower than the average for all vesicles.It is concluded that stimulation makes apparent metabolic and morphological heterogeneity of synaptic vesicles. The increase in density of the vesicles containing newly synthesized acetylcholine could be due to endocytotic uptake of sucrose contained in the perfusate after the vesicle has undergone exocytosis. The results suggest that synaptic vesicles can be reloaded with transmitter and re-used even after uptake of extracellular marker.     

Turnover of adenine nucleotides in cholinergic synaptic vesicles of the Torpedo electric organ

Synaptic vesicles were isolated from perfused blocks of electric tissue on sucrose density gradients in a zonal rotor. In vesicles from control tissue the composition was ATP (83%), ADP (15%) and AMP (2%): the corresponding figures for stimulated tissue were 69, 22 and 6% respectively: thus ATP is the predominant vesicular adenine nucleotide in both types of vesicle. Stimulation of the nerves to the tissue at a frequency (0.1 Hz) which does not cause a fall in vesicle numbers induces an approx. 50% loss of total vesicular nucleotides, the same as the degree of loss of acetylcholine in previous experiments.When tissue blocks are perfused with a solution containing [2-³H]adenosine for several hours, 85% of the radiolabel recovered in the isolated vesicle fraction is in the form of ATP. Besides some radiolabelled ADP and a reproducible but small contribution of inosinemonophosphate, traces of radiolabelled AMP, adenosine, adenine, hypoxanthine and inosine were detected. On stimulation of nerves to tissue blocks at 0.1 Hz two populations of synaptic vesicles can be isolated, the denser one of which contains the bulk (70%) of the newly synthesized vesicular ATP as well as acetylcholine. Vesicles sedimenting at the original sucrose density lose both ATP and acetylcholine. The specific radioactivity of ATP in the denser vesicles after a simulation of 1280–1800 impulses was about four times higher than that of vesicles equilibrating at the original sucrose density.The results suggest that adenosine is an effective precursor of vesicular adenine nucleotides. On stimulation nucleotides are lost from synaptic vesicles together with the neurotransmitter. The new population of vesicles appearing on stimulation has a high turnover rate for both ATP and acetylcholine.     

Origin of transmitters released by electrical stimulation from a small metabolically very active vesicular pool of cholinergic synapses in guinea-pig cerebral cortex.

The turnover of true and false transmitters was measured to find out from which subcellular pool in guinea-pig cerebral cortex acetylcholine is released after electrical stimulation. [N-Me-¹⁴C]choline was injected along with [N-Me-³H]pyrrolidinecholine or [N-Me-³H]homocholine into cortical tissue. Both choline analogues were acetylated, although two to five times less favourably than choline. All acetylated forms were incorporated into synaptic vesicles; this vesicular uptake was enhanced by low-frequency electrical stimulation (0.1 Hz) and occurred preferentially into a metabolically very active subpopulation of synaptic vesicles closely attached to the presynaptic membrane which can be isolated at the interphase between 0.8 M and 1.0 M sucrose (fraction H), using the sucrose density gradient separation technique. Acetylhomocholine was preferentially localized within the cytoplasmic compartment and taken up into synaptic vesicles to a smaller extent than acetylpyrrolidinecholine.Locke's solution was placed in Perspex cylinders (cups) applied to the surface of the cortex and acetylcholine as well as acetylpyrrolidinecholine and acetylhomocholine were released into these cups by diffusion. High-frequency stimulation (30 Hz) approximately doubled the amount of transmitters released. The molar ratios of the transmitters ([¹⁴C]acetylcholine/[³H]acetylpyrrolidinecholine or [¹⁴C]acetylcholine/[³H]acetylhomocholine) released during stimulation closely approximated those found in fraction H but differed significantly from the molar ratios determined in the monodisperse synaptic vesicle fraction (fraction D) at the 0.4 M sucrose level and also from that in the cytoplasmic compartment. Moreover, the specific activity of the released transmitter was nearly identical with that in fraction H.It is concluded that electrical stimulation releases acetylcholine from a distinct, metabolically very active vesicular pool in the guinea-pig cortex and that synaptic activation in some way facilitates the vesicular uptake of transmitters.     

The relationship of transmitter release and storage to fine structure in a sympathetic ganglion

Summary Cat sympathetic ganglia were prepared for electron microscopy by perfusion fixation with glutaraldehyde in the presence of Mg⁺⁺. At resting boutons de passage the populations of synaptic vesicles were 6400 per bouton. The vesicle distributions displayed many of the features of spheres in close-packing. Calculations based on a vesicle close-packing hypothesis gave a figure of 8000 vesicles per bouton. In ganglia stimulated for 20 min at 20/s the vesicle populations were reduced to 25%, and to 28.5% in ganglia in which acetylcholine (ACh) synthesis was inhibited by hemicholinium (HC-3). The reduction was to 34% when stimulation was for 1 min. In ganglia stimulated for 20 min at 1/s and 4/s the vesicle populations were reduced to 44% and 46% respectively. Even following 1 min stimulation at 4/s over half the boutons showed significant loss of vesicles. ACh stores in ganglia are known not to be depleted by any of these procedures except stimulation in the presence of HC-3. The fraction of ganglionic ACh stores known to be released by stimulation for 1 min at 20/s or 4/s and by 20 min stimulation at 1/s is substantially less than the fraction of vesicles lost. The observations therefore were not readily accounted for by the vesicle theory of transmitter storage and release. They were consistent with the idea the ACh is stored in vesicles at rest, but that during maintained activity over half the bouton ACh is free in the cytoplasm. The concentration of cytoplasmic ACh was calculated to be approximately 50–150 mm 1^–1. Examination of the hypothesis that ACh may be released from this cytoplasmic pool during synaptic activation indicated an efflux of approximately 1.5–3.0 × 10^–12 M Ach cm^–2 synapsing membrane/impulse.Medical Research Associate of the Medical Research Council of Canada.     

Vesicular storage and release of a false cholinergic transmitted (acetylpyrrolcholine) in the Torpedo electric organ.

The choline analogue N-[Me-³H]N-hydroxyethyl-pyrrolidinium (pyrrolcholine) was studied in the Torpedo electric organ. Pyrrolcholine is transported into isolated nerve terminal sacs by the choline high affinity uptake system (K_T = 5.7 μm). If blocks of electric tissue are perfused in the presence of both [³H]pyrrolcholine and [¹⁴C]choline both compounds become acetylated and are taken up into synaptic vesicles. This process is enhanced by low frequency stimulation (~0.1 Hz). Upon subsequent stimulation of the nerve at 5 Hz both acetylpyrrolcholine and acetylcholine are released into the perfusate by a calcium-sensitive mechanism; their molar ratio in the perfusate is the same as that for their loss from the vesicle fraction. Acetylpyrrolcholine is a potent agonist on the frog rectus abdominis muscle although 12.7-fold less active than acetylcholine.We conclude that pyrrolcholine is a precursor of a cholinergic false transmitter in the Torpedo electric organ. Acetylpyrrolcholine has been shown to fulfil all the criteria for the definition of a false transmitter, including storage in synaptic vesicles.     

Recycling of synaptic vesicles in the cholinergic synapses of the Torpedo electric organ during induced transmitter release.

Blocks of innervated Torpedo electric tissue can be perfused in vitro and remain functioning for more than 24 h. Low frequency stimulation (0.1 Hz) of the attached nerve leads to a decay in electrical response and tissue content of acetylcholine although the number of vesicles counted in terminals does not fall. Stimulation leads to the appearance of a population of vesicles about 25% smaller in diameter than normal. If dextran (Molecular weight 10,000–40,000) is added to the perfusate an increasing number of vesicles becomes labelled during stimulation. After 720–1800 impulses 74% of all vesicles are found to contain dextran in their lumen. The label is contained mainly in the new small vesicle population. Perfusion with dextran per se does not lead to significant fine structural changes or uptake of dextran. After stimulation, dextran-containing vesicles are also found in the unmyelinated part of the terminal axon.It is concluded that stimulation-induced transmitter release is accompanied by a recycling of synaptic vesicles which leads to uptake of extracellular marker into the lumen of the vesicles. Thus synaptic vesicles become heterogeneous as a result of their previous exo- and endocytotic activity.     

Spontaneous and evoked release of acetylcholine and a cholinergic false transmitter from brain slices: comparison to true and false transmitter in subcellular stores

Rat cerebral cortex slices were incubated with [¹⁴Ccholine and [³Hhomocholine to allow synthesis of [¹⁴Cacetylcholine and [³Hacetylhomocholine; release of the acetylated compounds was tested in the presence of eserine and hemicholinium-3. Both [¹⁴Cacetylcholine and [³Hacetylhomocholine were released spontaneously by a Ca²⁺-independent mechanism; exposure of the tissue to a high K⁺ medium resulted in a Ca²⁺-dependent increase in [¹⁴Cacetylcholine release (30-fold) and in [³Hacetylhomocholine release (5 to 7-fold). Thus, spontaneous and K⁺-evoked transmitter release could be distinguished on the basis of their molar ratios of true to false transmitters and these ratios were compared to the molar ratios of the transmitters in subcellular fractions prepared from the incubated tissue. The molar ratio of acetylcholine to acetylhomocholine released from the tissue spontaneously differed from the ratio in subcellular fractions containing occluded transmitters. The molar ratio of acetylcholine to acetylhomocholine released by K⁺ differed from the ratio in a fraction (H) containing occluded transmitters but was similar to the ratio in the fraction (D) containing monodisperse synaptic vesicles and the fraction (0) containing the majority of a soluble cytoplasmic marker. Comparison of the transmitter contents of subcellular fractions from unstimulated and K⁺-stimulated tissue showed that the three fractions (D, H and O) lost equal proportions of both transmitters as a result of K⁺ stimulation.It is concluded that acetylhomocholine may be released from brain slices both spontaneously and in response to stimulation via mechanism similar to those that release acetylcholine, but that there must be some differences in specificity of the acetylcholine storage and/or release processes. The results also indicate that spontaneous transmitter release originates from extravesicular stores; the results are consistent with a vesicular site of origin of evoked transmitter release but do not distinguish between nerve terminal vesicles and cytoplasm as the immediate source of evoked transmitter release.     

The effects of nerve stimulation and hemicholinium on synaptic vesicles at the mammalian neuromuscular junction

1. Electron micrographs of nerve terminals in rat phrenic nerve—diaphragm preparations have been studied. This has been done before and after prolonged nerve stimulation. The effectiveness of nerve stimulation has been monitored by intracellular micro-electrode recordings from the muscle cells.

2. Characteristic changes in the form and distribution of the nerve terminal mitochondria were noted after nerve stimulation.

3. Synaptic vesicle numbers in the region of nerve terminal less than 1800 Å from the synaptic cleft were significantly greater in tissue taken 2 and 3 min after nerve stimulation, than in unstimulated preparations.

4. The long and short diameters of the synaptic vesicle profiles less than 1800 Å from the synaptic cleft were measured. Analysis of the distribution of the diameters indicated synaptic vesicles to be basically spherical structures. Estimates of synaptic vesicle volume were made from the measurements. Synaptic vesicle volume was significantly reduced in tissue taken 2 and 4 min following nerve stimulation.

5. If hemicholinium, a compound which inhibits acetylcholine synthesis, was present during the period of nerve stimulation, much greater reductions in synaptic vesicle volume occurred. Synaptic vesicle numbers in the region of nerve terminal less than 1800 Å from the synaptic cleft were also reduced, compared with unstimulated control preparations.

6. These results are regarded as support for the hypothesis that the synaptic vesicles in nerve terminals at the mammalian neuromuscular junction represent stores of the transmitter substance, acetylcholine.

     

The water spaces in cholinergic synaptic vesicles from torpedo measured by changes in density induced by permeating substances

The water spaces in cholinergic synaptic vesicles isolated from the electromotor terminals of Torpedo marmorata electric organ have been determined as a fraction of the total vesicle volume by measuring the density changes induced in the vesicles by the addition of permeating substances to iso-osmotic density gradients. Three permeating substances were selected for study: deuterium oxide, dimethylsulphoxide and glycerol. The water spaces measured by these three substances were not equal, being 83%, 72% and 65% of the vesicle volume, respectively. When vesicles were lysed in dilute (10 mM) Hepes buffer (pH 7.0), the deuterium oxide space was not detectably changed, but the dimethylsulphoxide and glycerol spaces assumed the same value of 74–75%. This was interpreted to mean that lysis resulted in the loss of highly hydrated core constituents (presumably mainly acetylcholine and adenosine 5'-triphosphate) whose bound water can be replaced by dimethylsulphoxide and deuterium oxide but not by glycerol. When intact or lysed vesicles were exposed to highly hyperosmotic CsCl gradients, the changes in the density and in the deuterium oxide and glycerol spaces showed that the vesicles had undergone collapse due to osmotic dehydration; 91–96% of the glycerol space is osmotically active water. The density of the membrane was estimated to be 1.11 to 1.135 depending on its protein content.These results confirm, by an independent method, conclusions already reached in this laboratory from the protein and lipid analysis of vesicles (Ohsawa, Dowe, Morris & Whittaker, Brain Res.161, 447–457, 1979) and from density measurements at varying osmotic pressures (Breer, Morris & Whit-taker, Eur. J. Biochem.87, 453–458, 1978), namely, that the vesicle is a highly hydrated structure with a diameter of 80–100 nm and a lipid-rich membrane 4–5 nm in thickness.The implications of the results for measurements of vesicular membrane potential and intravesicular pH are discussed.     

Synthesis,storage and release of [14C]acetylcholine in isolated rat diaphragm muscles

1. Segments of rat diaphragms were kept in choline-free media for 4 hr and were then exposed to a physiological concentration of [¹⁴C]-choline (30 μM) at 37° C. The synthesis, storage and subsequent release of [¹⁴C]acetylcholine by the muscles was assessed by isotopic- and bio-assays after isolation of the transmitter by paper electrophoresis.2. Replacement of endogenous acetylcholine (0·92 μ-mole/kg) with labelled acetylcholine proceeded slowly at rest, but rapidly during nerve stimulation. [¹⁴C]Acetylcholine accumulated most rapidly when hydrolysis of the released transmitter, and thus the re-use of endogenous choline, was prevented by an esterase inhibitor. Fully replaced stores were maintained during nerve stimulation by synthesis rates sufficient to replenish at least 35% of the store size in 5 min.3 In the presence of hemicholinium-3, which inhibits choline uptake, acetylcholine stores declined rapidly during stimulation, and residual synthesis was slight, indicating little intraneural choline. Net choline uptake into nerve terminals was estimated from the highest observed synthesis rate and from previous measurements of the number and size of terminals, as 3-6 p-mole/cm² sec.4. Transmitter synthesis was localized in the region of end-plates, and was reduced to a few per cent of normal 6 weeks after phrenic nerve section. Release experiments suggested that at least half of the acetylcholine in phrenic nerves is in their terminals; from this content and the morphology of the terminals, the average concentration of transmitter in the whole endings would appear to be about 50 m-mole/l. Homogenization of the muscles freed choline acetyltransferase into solution, but left some [¹⁴C]acetylcholine associated with small particles, presumably synaptic vesicles.5. Resting transmitter release was about 0·013% of stores/sec. With 360 nerve impulses at 1-20/sec, release increased up to 0·43% of stores/sec, and amounted to 3·5-7 × 10^-18 moles per end-plate per impulse. The release rate was unaffected by the doubling of store size which occurred with eserine, but the extra transmitter did help to maintain releasable stores during prolonged stimulation. Experiments with fractional store labelling indicated that newly synthesized acetylcholine was preferentially released.6. Preformed [³H]acetylcholine was not taken up and retained by muscle or nerve cells in the absence of an esterase inhibitor. With eserine present, labelled acetylcholine was taken up uniformly by muscle segments; when eserine was then removed, radioactive acetylcholine remained only near neuromuscular junctions.     

Amyotrophic lateral sclerosis immunoglobulins G enhance the mobility of Lysotracker-labelled vesicles in cultured rat astrocytes

Aim: We examined the effect of purified immunoglobulins G (IgG) from patients with amyotrophic lateral sclerosis (ALS) on the mobility and exocytotic release from Lysotracker‐stained vesicles in cultured rat astrocytes. Methods: Time‐lapse confocal images were acquired, and vesicle mobility was analysed before and after the application of ALS IgG. The vesicle counts were obtained to assess cargo exocytosis from stained organelles. Results: At rest, when mobility was monitored for 2 min in bath with Ca²⁺, two vesicle populations were discovered: (1) non‐mobile vesicles (6.1%) with total track length (TL) < 1 μm, averaging at 0.33 ± 0.01 μm (n = 1305) and (2) mobile vesicles (93.9%) with TL > 1 μm, averaging at 3.03 ± 0.01 μm (n = 20 200). ALS IgG (0.1 mg mL^?1) from 12 of 13 patients increased the TL of mobile vesicles by approx. 24% and maximal displacement (MD) by approx. 26% within 4 min, while the IgG from control group did not alter the vesicle mobility. The mobility enhancement by ALS IgG was reduced in extracellular solution devoid of Ca²⁺, indicating that ALS IgG vesicle mobility enhancement involves changes in Ca²⁺ homeostasis. To examine whether enhanced mobility relates to elevated Ca²⁺ activity, cells were stimulated by 1 mm ATP, a cytosolic Ca²⁺ increasing agent, in the presence (2 mm ) and in the absence of extracellular Ca²⁺. ATP stimulation triggered an increase in TL by approx. 7% and 12% and a decrease in MD by approx. 11% and 1%, within 4 min respectively. Interestingly, none of the stimuli triggered the release of vesicle cargo. Conclusion: Amyotrophic lateral sclerosis‐IgG‐enhanced vesicle mobility in astrocytes engages changes in calcium homeostasis.     

Metal ion content of cholinergic synaptic vesicles isolated from the electric organ of Torpedo: Effect of stimulation-induced transmitter release

Cholinergic synaptic vesicles were isolated from the Torpedo electric organ by a combination of differential and density gradient centrifugation. Iso-osmolar solutions of glycine or NaCl were used as homogenization and preparation media. The metal content of intact tissue and subcellular fractions were determined by atomic absorption spectroscopy. In the synaptic vesicle fraction ratios of metals to acetylcholine (g atom/mol) were: Na, 0.30; K, 0.10; Mg, 0.07; Ca, 0.28. Filtration of isolated vesicles revealed that the strength of metal binding depends on the ionic potential of the metal cation. Thus alkali metal ions are bound to synaptic vesicles less tightly than alkaline earth metals. Incubation of vesicles with elevated levels of NaCl led to a partial exchange of Na with K but external concentrations of CaCl₂ in the physiological range were without effect on vesicle metal ion content. Stimulation of the electric organ in vivo (5000 impulses, 5 Hz) caused a depletion of the acetylcholine and adenosine 5′-triphsophate content of the vesicles whereas the levels of metal ions were increased.It is suggested that the release of acetylcholine from synaptic vesicles exposes free negative charges to which extracellular metal cations can bind in ion exchange.     

Parallel changes in ultrastructure and noradrenaline content of nerve terminals in rat vas deferens following transmitter release

Transmural electrical stimulation and exposure to incubation media where some or all of the Na⁺ had been replaced with K⁺ were used to elicit transmitter release. Changes in noradrenaline content and ultrastructure of the nerve terminal varicosities in rat vas deferens were measured. Electrical stimulation in the presence of 4-aminopyridine had little effect, but high [K⁺] solutions caused a parallel reduction in noradrenaline content and the number of small dense-cored vesicles; large densecored vesicles showed no change, and small clear vesicles increased in number. In spite of a reduction in total vesicle number there was no evidence of expansion of the varicosity membrane.The parallel fall in noradrenaline content and in the number of small dense-cored vesicles suggests that the latter are the source of the released noradrenaline under the conditions of high [K⁺] stimulation we have used.     

The effects of nerve stimulation and hemicholinium on synaptic vesicles at the mammalian euromuscular junction

1. Electron micrographs of nerve terminals in rat phrenic nerve-diaphragm preparations have been studied. This has been done before and after prolonged nerve stimulation. The effectiveness of nerve stimulation has been monitored by intracellular micro-electrode recordings from the muscle cells.2. Characteristic changes in the form and distribution of the nerve terminal mitochondria were noted after nerve stimulation.3. Synaptic vesicle numbers in the region of nerve terminal less than 1800 A from the synaptic cleft were significantly greater in tissue taken 2 and 3 min after nerve stimulation, than in unstimulated preparations.4. The long and short diameters of the synaptic vesicle profiles less than 1800 A from the synaptic cleft were measured. Analysis of the distribution of the diameters indicated synaptic vesicles to be basically spherical structures. Estimates of synaptic vesicle volume were made from the measurements. Synaptic vesicle volume was significantly reduced in tissue taken 2 and 4 min following nerve stimulation.5. If hemicholinium, a compound which inhibits acetylcholine synthesis, was present during the period of nerve stimulation, much greater reductions in synaptic vesicle volume occurred. Synaptic vesicle numbers in the region of nerve terminal less than 1800 A from the synaptic cleft were also reduced, compared with unstimulated control preparations.6. These results are regarded as support for the hypothesis that the synaptic vesicles in nerve terminals at the mammalian neuromuscular junction represent stores of the transmitter substance, acetylcholine.     

Nucleotide uptake by isolated cholinergic synaptic vesicles: Evidence for a carrier of adenosine 5'-triphosphate

The in vitro uptake of [³H]nucleotides was studied using cholinergic syaptic vesicles isolated from Torpedo electric organ, with a resting membrane potential of 50–60 mV. The osmotically sensitive uptake of [³H]adenosine 5'-triphosphate (ATP) was markedly influenced by temperature and external pH, and was maximal after 40–50 min; longer incubation resulted in loss of accumulated radiolabel. Similar characteristics were also observed for adenosine 5'-mono- and diphosphate and guanosine and uridine triphosphates, all of which acted as competitive substrates for the saturable system which transported ATP (K_T 1.15 mM). Breakdown of [³H]nucleotides in the medium was not a significant factor, and adenosine, guanosine and adenine were very poorly incorporated. Under conditions of V_max, vesicle to medium ratios of [³H]ATP of 20–25 were observed; the amount of radiolabel was equivalent to 20–50% of the initial endogenous amount of ATP in the vesicles. Atractyloside specifically inhibited nucleotide transport with no modification of hemicholinium-3 sensitive acetylcholine uptake. Antisera raised (a), to whole Torpedo vesicle extract, and (b), to a single purified vesicle polypeptide, greatly stimulated ATP uptake without effect on simultaneous influx of either acetylcholine or glucose.It is concluded that isolated vesicles contain a nucleotide carrier of wide pharmacological specificity (possibly the 34,000 molecular weight protein of Stadler & tashiro [1979]), which is likely to be of physiological relevance. Implications for vesicular refilling mechanisms are discussed.     

Solitary membrane associated particles in normal and experimentally enlarged synaptic vesicles

Freeze-etch replicas of synaptic vesicles were obtained from the vertebrate central nervous system and from the frog motor endplate. Large solitary particles were found to be associated mainly to the cytoplasmic fracture face of the vesicle membrane. They are especially conspicuous in the initial stages of Wallerian degeneration when the synaptic vesicles are abnormally large. The finding of these particles bears a striking resemblance to the previous observations of cytochemically verified vesicular calcium binding sites. The possible role of these particles for control by Ca²⁺ of transmitter release is discussed.     

Distribution of plasmelemmal vesicles on arterial endothelial surface as determined by freeze cleavage

The freeze-fracture technique was used to study the density and distribution of plasmalemmal vesicles at the endothelial surface of canine carotid arteries. The fractured surface of the endothelium can be divided into areas with vesicles (A_ves) and areas without vesicles (A_nves), the latter being located at the parajunctional zone. With morphometric analysis, A_ves and A_nves were found to be 75% and 25% of the endothelial surface, respectively. The average width of A_nves (distance from the intercellular cleft) is approxmately 0.4 μ. In A_ves, the density of vesicles is 120 μm^?2, and approxmately 16% of A_ves is covered by the vesicle orifices. The tight junctions appear as long and straight strands, 8–9 nm in width. The number of the strands varies from one to five. The gap junctions consist of closely packed particles 9–10 nm in size which form patches or plaques from 80 to 800 nm in size. These findings provide the quantitative information needed for the theoretical modeling of transendeothelial vesicular transport of macromolecules.     

Acetylcholine release from rat cortical slices during postnatal development and aging

Acetylcholine release from cortical slices superfused with choline-enriched Krebs solution containing physostigmine was investigated at birth, at 7, 20 and 30 days, and at 3 and 24 months of age, in order to assess age influence on the functional efficiency of the cortical cholinergic network. The slices were electrically stimulated at frequencies from 1 to 10 Hz for 5 min periods, preceded and followed by rest periods. The superfusate was collected every 5 min and acetylcholine content quantified by bioassay. In the newborn and 7 day-old pups acetylcholine release was approximately 50% lower than that of the 3 month-old rats at all frequencies tested. The highest release was elicited in the 30 day-old rats. Beginning with this age the evoked ACh release underwent a decline which in the 24 month-old rats brought it back to the same level as in the newborn ones. The blockade of the muscarinic autoreceptors by atropine 1.5 × 10^?8 M caused an increase in acetylcholine release at 20 day, 3 and 24 months of age but not in the newborn and 7 day-old pups. Adenosine 3 × 10^?5 M decreased acetylcholine output in newborn and adult but had no effect in the senescent rats.