Noradrenaline content correlated to matrix density in small noradrenergic vesicles from rat seminal ducts

The relation between matrix density and noradrenaline content has been investigated in a fraction of small noradrenergic storage vesicles obtained from seminal ducts of castrated rats by density gradient centrifugation. This preparation contained numerous dense-cored small vesicles with an increased diameter but with a similar appearance to those in nerve terminals in the intact vas deferens.After incubation of the vesicle fraction for 20 min at 30°C in the presence of noradrenaline, Mg²⁺ and adenosine 5'-triphosphate, about 50–60% of the vesicles with a diameter of 40–70 nm contained dense cores, in some regions of the sediment even up to 80%. This correlated well with a high level of sedimentable noradrenaline, 150% of control.Incubation of vesicles without exogenous noradrenaline in the presence of Mg²⁺ and adenosine 5'-triphosphate yielded mostly electron-lucent vesicles, and biochemically only 40% of the control level of noradrenaline was retained in the particulate fraction after centrifugation. A similar depletion of electron dense-core material was observed after incubation with tyramine, Mg²⁺ and adenosine 5'-triphosphate, where only 55% of the sedimentable noradrenaline as compared to control was retained. In this respect the small dense-cored vesicles differed markedly from the large dense-cored vesicles purified from bovine splenic nerve, where the dense cores were retained in spite of a 70–80% depletion of noradrenaline after incubation in the presence of tyramine.Incubation with 5-hydroxydopamine did not significantly increase the electron density of the small dense-cored vesicles, in contrast to the marked effect of this agent when administered in vivo. Incubation with reserpine and atractyloside did not affect either noradrenaline release or vesicle morphology significantly, whereas treatment with N-ethylmaleimide gave a 60% decrease in particulate noradrenaline content but only a moderate reduction in electron density.The results indicate that both noradrenaline and, to a lesser extent, other matrix material in the presence of Mg²⁺ and adenosine 5′-triphosphate are capable of forming electron-dense cores, and that noradrenaline is more important for dense-core formation in small noradrenergic vesicles than it is in the large vesicles.     

Neuronal and subcellular localization of acetylcholine in the posterior pituitary of the rabbit

1. Acetylcholine and neurohypophysial hormones were measured in subcellular fractions of rabbit neurohypophysis.2. Differential and density gradient centrifugation, followed by bio-assay and electron microscopy were used to separate and characterize the subcellular particles, or isolated nerve endings.3. Acetylcholine in the rabbit neurohypophysis was found to occur in small vesicles of about 40 nm diameter which had similar characteristics to the synaptic vesicles from central nervous tissue.4. Examination of intact nerve endings isolated from the rabbit neurohypophysis indicated that at least some acetylcholine and neurohypophysial hormones occurred in separate nerve endings.     

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.     

Latency of dopamine β-hydroxylase in purified noradrenergic vesicles

Purified large dense core noradrenergic vesicles isolated from bovine splenic nerve were investigated to determine the percentage of total dopamine β-hydroxylase which existed in a latent or masked form. The effects of the nonionic detergent Triton X-100 in concentrations between 0.0025 and 0.1% were studied. At 0.01%, Triton produced a maximum three-fold increase in accessible dopamine β-hydroxylase activity to reach 0.64μmol octopamine per mg protein per min at 37°C, but released only a third of the enzyme into the soluble phase. About 90% of the noradrenaline, 42% of the phospholipids and 17% of the vesicle protein were released. At 0.05%, Triton produced the same maximum enzyme activity with complete solubilization of the enzyme. All the noradrenaline, 90% of the phospholipids, and 48% of the vesicle protein were released into the soluble phase. Ultrastructural examination of the treated vesicles revealed only partial lysis with 0.01% Triton, while 0.05% produced essentially complete disruption of vesicles into membrane fragments and fine granular matrix contents. Equilibrium centrifugation of the control and treated vesicles on a second sucrose-D₂O density gradient showed that emptied and partially depleted vesicles and released matrix granules redistributed from the heavy vesicle peak to less dense regions of the gradient, including the zone of the light vesicle peak.It is proposed that the latency of dopamine β-hydroxylase activity in this noradrenergic vesicle fraction is best explained by the occurrence of the enzyme as an inaccessible complex in the vesicle matrix which may include phospholipid.     

Subcellular distribution of adenylate cyclase and cyclic adenosine monophosphate phosphodiesterase in rat and guinea-pig vas deferens

Cyclic adenosine 3′:5′-monophosphate (cyclic AMP) is assumed to play a role in catecholamine synthesis and release. In order to determine if cyclic AMP metabolism at the level of the storage granule is important in this respect the subcellular distribution of adenylate cyclase and cyclic AMP phosphodiesterase was determined in vas deferens from normal rats, castrated rats and castrated guinea-pigs. The phosphodiesterase activity was mainly found in the soluble fractions, while the adenylate cyclase was associated with sedimentable material. When vas deferens homogenates from both intact and castrated rats were subjected to sucrose density gradient centrifugation the main part of the adenylate cyclase activity was found associated with membrane fragments at 0.5–0.6 m sucrose. The distribution of adenylate cyclase activity in the density gradient parallelled that of 5′-nucleotidase, but was different from that of noradrenaline. Also in the guinea-pig, adenylate cyclase tended to have a different distribution from that of noradrenaline.The adenylate cyclase activity in, all fractions was stimulated by fluoride and guanosine triphosphate. Noradrenaline, prostaglandin E₂, 2-chloroadenosine and phenylisopropyladenosine stimulated adenyl cyclase activity in nuclear and mitochondrial fractions, but only to a small extent, if at all, in the fractions collected from the density gradient.The results do not indicate that adenylate cyclase activity in vas deferens homogenates is associated with catecholamine storage vesicles. Hence, cyclic AMP metabolism at the level of the storage granule is probably not involved in transmitter turnover.     

Sympathetic axons and nerve terminals: the protein composition of small and large dense-core and of a third type of vesicles

Homogenates of bovine splenic nerve and of vas deferens were subjected to differential and density gradient centrifugation to investigate their noradrenaline-storing organelles. The subcellular fractions obtained were analysed by immunoblotting in order to define the presence of various antigens in small dense-core and large dense-core vesicles. In both large granule and microsomal fractions from splenic nerve only one type of noradrenaline-storing vesicle was found, which represents the large dense-core vesicles. These organelles contained chromogranin A, chromogranin B, cytochrome b-561, carboxypeptidase H, glycoprotein II, glycoprotein III, dopamine beta-hydroxylase and the monoamine carrier which are also present in adrenal chromaffin granules. The subcellular distribution of synaptin/synatophysin was more complex since this protein was apparently present in two organelles: in a light vesicle which did not contain significant amounts of antigens found in large dense-core vesicles (dopamine beta-hydroxylase, cytochrome b-561 and the monoamine carrier) and in the dense fractions of the gradient, possibly within large dense-core vesicles. In the microsomal gradient from vas deferens several markers (catecholamines, synaptin/synaptophysin and dopamine beta-hydroxylase) were found in a bimodal distribution, which is consistent with their presence in small and large dense-core vesicles. When the larger granules were removed with higher centrifugation speed a microsomal fraction containing only light vesicles was obtained. After gradient centrifugation of this fraction several components (catecholamines, dopamine beta-hydroxylase, cytochrome b-561, the monoamine carrier and synaptin/synaptophysin) were concentrated in a peak at low density; apparently only small dense-core vesicles were now present.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biochemical and functional properties of large and small dense-core vesicles in sympathetic nerves of rat and ox vas deferens

A comparative study of the noradrenaline storing vesicles in vas deferons from ox and rat was performed. Microsomal fractions were subjected to density gradient centrifugation. In rat, noradrenaline and dopamine β-hydroxylase were mainly present in the upper fractions of the gradient, which is consistent with the predominance of light (small dense-core) vesicles in this species. In ox, noradrenaline, dopamine and dopamine β-hydroxylase were found in the gradient in a bimodal distribution. This is consistent with the presence of about equal numbers of small and large dense-core vesicles in this species. On the other hand, chromogranin A, immunologically related proteins and enkephalin-like immunoreactivity were only present in the dense (large dense-core) vesicle population. In order to study the capability of light and dense vesicles to synthesize noradrenaline we “pulse-labelled” ox vasa deferentia with [³H]tyrosine. Already 3.5 min after the pulse both types of vesicles contained [³H]noradrenaline and [³H]dopamine. During longer “chase” periods the amount of [³H]dopamine gradually declined.We conclude that dense (large dense-core) vesicles contain chromogranin A, immunologically related proteins and enkephalin-like immunoreactivity whereas light (small dense-core) vesicles are devoid of these components. Both types of vesicles contain dopamine β-hydroxylase and can synthesize noradrenaline from dopamine under in vivo conditions.     

Opioid peptides and noradrenaline co-exist in large dense-cored vesicles from sympathetic nerve

The possibility that opioid peptides and noradrenaline co-exist not only in the desheathed bundle of bovine splenic nerve which contains ~98% sympathetic C-fibers, but also in the population of large dense-cored noradrenergic vesicles from these fibers, has been investigated. The primary fraction of large dense-cored vesicles which can be prepared at about 85% purity has been further subjected to density gradient and fractional centrifugation procedures, including D₂O-loading and unloading on modified second gradients, in an attempt to separate any minor population of particles which potentially could contain opioid peptides and contaminate the large dense-cored vesicle fraction. Measurement of opioid peptides, noradrenaline, dopamine and dopamine β-hydroxylase activity supports the conclusion that opioid peptides are stored in the primary population of large dense-cored vesicles per se, rather than in a minor population of contaminating particles from cells other than sympathetic C-fibers.This conclusion has implications for exocytotic release and the physiological role of the opioid peptides intra- and extra-neuronally. Nerve vesicle opioid peptides have a size less than 5000 daltons, in contrast to the high proportion of large peptides containing enkephalin sequences in the bovine adrenal medulla.     

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.     

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.     

An electron microscopic study of autonomic nerve cells in the cloacal region of the lamprey,Lampetra japonica

Summary Two types of autonomic nerve cell in the cloacal region of lamprey,Lampetra japonica have been studied by electron microscopy. Large ganglion cells (LGC) were unipolar and individually invested with a satellite cell sheath. The LGC-satellite cell complex measured 24 m × 38 m on average. Granular endoplasmic reticulum and cored vesicles (80–140 nm in diameter) were scattered in the perikaryon. Two kinds of peculiar cytoplasmic filament were seen in LGC: one type was about 20 nm in diameter with periodic dense material on the surface and the other had a diameter of about 8 nm and showed an undulating profile. Nerve endings containing abundant small clear vesicles (30–50 nm in diameter) and a few larger cored vesicles (50–100 nm in diameter) were found in synaptic contact with LGC. Small ganglion cells (SGC) were also unipolar and covered incompletely by a satellite cell sheath. The SGC-satellite cell complex measured 6 and 12 m on average. The SGC was packed with organelles and the perikaryon appeared more electron dense than that of LGC. SGC perikaryonal cytoplasm contained dispersed granular endoplasmic reticulum and numerous large cored vesicles (55–220 nm in diameter). Nerve endings containing numerous large cored vesicles (70–170 nm in diameter) and variable numbers of small clear vesicles (30–50 nm in diameter) were seen on the surface of SGC.     

Morphological effects of osmolarity on purified noradrenergic vesicles.

Large dense core vesicles (LDV) were purified from bovine splenic nerve homogenates by the sucrose-D2O density gradient method. Vesicles were subjected to a 50% increase and decrease in osmolarity from the control 330 mosmol 1(-1) by adjusting sucrose or potassium phosphate buffer during pre- and/or postfixation. Control vesicles with a mean diameter of 717 A readily swelled to approximately 1050 A and shrunk to approximately 600 A in the hypotonic and hypertonic media, respectively, with either sucrose or phosphate buffer. The dense core responded similarly but to a lesser degree. Prefixation in glutaraldehyde had little effect on vesicle sensitivity to subsequent tonicity change, not did the fixative per se exert an obvious osmotic effect. Thus, final vesicle size was largely determined by the OsO4 postfixation medium and principally by the vehicle rather than the fixative. In controls there was a mixture of spherical to oblate vesicles mostly filled with an electron-dense matrix. Upon swelling, more vesicles became spherical and nearly all had a prominent translucent halo between core and membrane. Upon shrinking, more vesicles became oblate, the halo was obliterated and the electron-density of the matrix increased. Frequency distributions of vesicle diameters at different tonicity clearly indicated that the diameter of LDV could overlap the 400-500 A range characteristic of small dense core vesicles; however, there was no suggestion of a population of the latter in the purified LDV fraction. Implications are discussed concerning the biochemical and morphological identification of 'light' and 'heavy' density peaks of noradrenaline and dopamine beta-hydroxylase from mixed vesicle populations and the possible relevance of changes in vesicle shape to a functional state in situ.     

The re-distribution of cytochrome oxidase, noradrenaline and adenosine triphosphate in adrenergic nerves constricted at two points

1. The experiments correlate certain changes in the ultrastructure of cat hypogastric nerves constricted at two points with the distribution of a mitochondrial enzyme (cytochrome oxidase), noradrenaline (stored in some of the vesicles with an electron dense core, i.e. granular vesicles) and adenosine triphosphate (ATP) (present in noradrenaline storage granules, mitochondria and the soluble fraction of the axon).2. Noradrenaline (NA) and granular vesicles accumulated proximal but not distal to both constrictions. The total amount of NA and the concentration of granular vesicles above the first constriction was greater than that present in a similar piece of normal nerve, indicating that the cell body was continuing to produce the transmitter despite injury to its axon. The granular vesicles proximal to the first constriction were found in swollen or distorted axons and in new axonal outgrowths. It was concluded that the movement of NA in these constricted nerves was only centrifugal in direction.3. Mitochondria and cytochrome oxidase accumulated on both sides of the two constrictions, indicating a bi-directional movement of mitochondria in the damaged axons. The possibility that some of the increase in the cytochrome oxidase could be related to an increase in the number of mitochondria in cells other than neurones is considered.4. The adenosine triphosphate content increased on both sides of the two constrictions. This increase developed more slowly and was less marked than that of the other two substances.5. It was concluded that (a) there was a close correlation between the behaviour of noradrenaline and granular vesicles and between cytochrome oxidase and mitochondria, (b) the dense cored vesicles and the mitochondria moved independently of one another and at different rates after constriction of non-myelinated axons, (c) while some of the changes may be attributed to an obstruction to the free movement of axoplasm others may be due to an active reaction to axonal injury, and (d) localized intraaxonal synthesis of noradrenaline and cytochrome oxidase did not occur between the two constrictions.     

大颗粒小泡非突触部位的胞吐——论神经肽释放的另一种形式

本文在以前的工作基础上,进一步用电镜及免疫细胞化学方法,研究了大颗粒小泡非突触部位胞吐作用。实验结果表明,切除大鼠刚髭部皮肤1—24小时之后,术侧延髓后角浅层大颗粒小泡胞吐比对照侧明显增多(P<0.01),术后3—9天复又下降(近似对照动物),术后14—15天又急剧上升(P<0.01)。这些胞吐大部分出现于延髓后角浅层四种轴突终末的非突触部位,少最也发生于树突及轴突中。从术后第6天开始,术侧P物质明显减弱,而甲硫-脑腓肽略有增强。研究结果提示;1)后角浅层胞吐增多,P物质下降及脑腓肽增高,反映了中枢内不同神经元对去传入神经的功能调整作用;2)大颗粒小泡在非突触部位释放神经肽,弥散地作用于远距离的受体,可能起着神经调制物的作用。     

Evidence for the coexistence and co-release of [met]enkephalin and noradrenaline from sympathetic nerves of the bovine vas deferens

The localization and neurosecretion of methionine-enkephalin was studied in sympathetic nerves of the bovine vas deferens. Immunostaining showed methionine-enkephalin-like immunoreactivity in a network of varicose nerve fibres in the smooth muscle layers of the vas deferens. When vas deferens homogenates were subjected to differential and sucrose density gradient centrifugation, methionineenkephalin was found to parallel the distribution of noradrenaline in the more dense region of the gradient, where “heavy” or large dense-cored vesicles are present. Electron microscopic immunochemistry confirmed this finding and showed methionine-enkephalin-like immunoreactivity in large dense-cored vesicles. The release of methionine-enkephalin upon electrical stimulation was studied in superfusion experiments. The methionine-enkephalin secretion was shown to be Ca²⁺-dependent and was inhibited by adding the adrenergic neuron blocking drug guanethidine to the superfusion medium.

We conclude that in the bovine vas deferens methionine-enkephalin is only present in large dense-cored vesicles of adrenergic neurons and that the peptide is released from these vesicles together with noradrenaline by a Ca²⁺-dependent mechanism.     

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.     

Reinnervation of Pacinian corpuscles by CNS axons after transplantation to the dorsal column: incidence and ultrastructure

Summary We have investigated the capacity of injured axons in the spinal dorsal columns of young adult rats to reinnervate grafted Pacinian corpuscles. A branch of the hindlimb interosseous nerve with a group of crural Pacinian corpuscles attached to it was autotransplanted to the surface of the spinal cord and the nerve stump was implanted into the dorsal column. Two to three months later 16 grafts were removed for examination by light and electron microscopy. By 3 months after transplantation almost all Schwann cell columns of the grafted nerve branch were occupied by regenerated myelinated and unmyelinated axons. Of 41 corpuscles examined by electron microscopy 24 were reinnervated by 1–3 myelinated fibres which gave rise to multiple terminals in the inner core. The remaining corpuscles appeared to be denervated. Only two of the reinnervated corpuscles contained regenerated endings which reiterated the distinct ultrastructure of normal presynaptic terminals of CNS axons, characterized by clusters of lucent vesicles and paramembranous densities. All other corpuscles were reinnervated by terminals which resembled peripheral mechanosensory endings as they contained mitochondria and very few vesicles. One such corpuscle was coinnervated by small terminals filled with large dense cored vesicles. We assume that the majority of grafted Pacinian corpuscles have been reinnervated by dorsal column axons and that the regenerated terminals with the ultrastructure of peripheral mechanosensory endings derive from central axons of primary sensory neurons, which are apparently capable of constructing mechanosensory-like terminals in response to signals from the Pacinian corpuscles. The vesicle-filled endings are probably formed by second order sensory neurons, corticospinal neurons and small peptidergic neurons unable to adjust their terminals to the new target.     

Bovine splenic nerve: characterization of noradrenaline-containing vesicles and other cell organelles by density gradient centrifugation

1. Homogenates of bovine splenic nerves were subjected to differential and sucrose density gradient centrifugation. From the low-speed supernatant a high-speed sediment (mitochondria, lysosomes, microsomes and noradrenaline (NA) vesicles) was obtained. By density gradient centrifugation of this sediment it was shown that NA vesicles are slightly less dense than mitochondria, but denser than microsomes.2. In further experiments a mitochondrial and a microsomal sediment were obtained. The mitochondrial sediment was fractionated with a short centrifugation time over a density gradient ranging from 0.6 to 1.2 M sucrose. Mitochondria (fumarase and succinate-dehydrogenase) and lysosomes (acid ribonuclease and deoxyribonuclease) sedimented to the bottom of the tube. The highest concentration of NA vesicles was found in a medium position. There was only a small amount of microsomes (glucose-6-phosphatase) present.3. The microsomal sediment was centrifuged for 150 min over a density gradient ranging from 0.8 to 1.4 M sucrose. The microsomes remained on the top of the gradient. There were also some mitochondria and lysosomes present. The NA vesicles were found in highest concentration in the middle of the gradient (at about 1.2 M sucrose).4. With the use of these two density gradients, the subcellular distribution of dopamine-beta-hydroxylase, monoamine oxidase and ATPase was studied. Dopamine-beta-hydroxylase was found to be localized in the NA vesicles. Monoamine oxidase was mainly recovered in mitochondria; a small part of the enzyme appeared to be microsomal. ATPase was present in microsomal elements.     

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.     

Purine salvage at the cholinergic nerve endings of the Torpedo electric organ: the central role of adenosine

The uptake and metabolism of adenosine, adenine, inosine and hypoxanthine were studied at the cholinergic nerve endings of the Torpedo electric organ. In isolated synaptosomes there is a linear uptake (measured up to 60 min) for adenosine and adenine at concentrations of 0.3 μM Uptake of adenosine exceeds that of adenine by a factor of 10. Adenosine is transported into synaptosomes via a saturable uptake system (K_m, 2 μM;V_max, ～- 30 pmols/min/mg protein). 2′-Deoxyadenosine is a competitive inhibitor of synaptosomal adenosine uptake. The nerve terminal possesses anabolic pathways for the formation of adenosine 5′-triphosphate from both adenosine and adenine. Adenosine becomes phosphorylated rapidly after entry into synaptosomes to form adenosine 5′-monophosphate; adenosine 5′-diphosphate and adenosine 5′-triphosphate were also major metabolites (70%). Adenine, inosine and hypoxanthine first accumulate in the synaptosomes. However, adenine leads to major formation of nucleotides (41% adenosine 5′-triphosphate after 60 min). Only traces of adenosine-3′:5′ cyclic monophosphate are formed from both adenosine and adenine. If adenosine 5′-triphosphate is added to a suspension of intact synaptosomes it becomes degraded to adenosine.We conclude that cholinergic nerve endings in the Torpedo electric organ possess an effective purine salvage system. Adenosine 5′-triphosphate released from either a pre- or a postsynaptic source would become degraded to adenosine in the extra-cellular medium and be re-used via an uptake system for renewed synthesis of adenosine 5′-triphosphate in nerve terminals.