Axonal agranular reticulum and synaptic vesicles in cultured embryonic chick sympathetic neurons

Cultured chick embryonic sympathetic neurons contain an extensive axonal network of sacs and tubules of agranular reticulum. The reticulum is also seen branching into networks in axon terminals and varicosities. The axonal reticulum and perikaryal endoplasmic reticulum resemble one another in their content of cytochemically demonstrable enzyme activities (G6Pase and IDPase) and in their characteristic membrane thicknesses (narrower than plasma membrane or some Golgi membranes). From the reticulum, both along the axon and at terminals, there appear to form dense-cored vesicles ranging in size from 400 to 1,000 Å in diameter. These vesicles behave pharmacologically and cytochemically like the classes of large and small catecholamine storage vesicles found in several adrenergic systems; for example, they can accumulate exogenous 5-hydroxydopamine. In addition, dense-cored vesicles at the larger (1,000 Å) end of the size spectrum appear to arise within perikaryal membrane systems associated with the Golgi apparatus; this is true also of very large (800–3,500 Å) dense-cored vesicles found in some perikarya.     

The axonal reticulum in the neurons of the superior cervical ganglion of the rat as a direct extension of the Golgi apparatus

Summary Adrenergic neurons from the superior cervical ganglion of the rat have been studied with the chromaffin reaction and the zinc iodide-osmium tetroxide method. Phosphotungstic acid staining at low pH and a combined acid phosphatase reaction and phosphotungstic acid staining have also been performed on glycolmethacrylate-embedded tissue. The results indicate that phosphotungstic acid-positive elements lacking acid phosphatase activity are present at the inner side of the Golgi apparatus. These elements give rise directly to reticular differentiations, carrying catecholamines, or to tubular extensions, representing the origin of the axonal reticulum. On these tubules, reticular differentiations can again be formed at any level. In the cell body, the differentiations are mainly found close to the neurolemma. In the axons, they are especially abundant at the axon terminals. Large granules may be associated with the reticular differentiations and small and large granules may detach from them.It is concluded that the whole catecholamine-producing and/or-storing system in sympathetic neurons can be considered as a direct extension of the Golgi apparatus, set up for local catecholamine synthesis. The relative importance of small and large granules along this system may reflect the functional status of the nerve cell.     

Neurosecretory granule formation in ligated axons: additional arguments for a local differentiation from a Golgi apparatus extension

The sorting domain for the different types of granules and small synaptic vesicles in neurosecretion is still largely a matter of debate. Some authors state that an exocytotic process has to precede granule formation. In previous studies, we favoured the idea that neurosecretory packages in terminals are assembled from axonal reticulum membranes simply by differentiation at the axon ending, the axonal reticulum being an extension of the Golgi apparatus. By ligating bovine splenic nerve, a de novo differentiation can be induced. After ligation, granules and granulo-tubular complexes appear. They were immunoreactive for SV2, VMAT2 and synaptobrevin II, which are all known to be highly enriched in large dense granules. Previously the granulo-tubular structures have already been recognized as precursor stadia of neurosecretory granules.It is concluded that at a de novo differentiation, a sorting out and aggregation is taking place of molecules typical for large dense granules. The small dense granules and tubules can be considered unripe, precursor forms of the large dense granules. All this occurs in the absence of signs of exocytosis. The present findings corroborate the view that granule formation occurs via local differentiation at an axon ending.     

内质网及其标志酶在离体培养脊髓神经元中的发育变化

In an attempt to elucidate the relationship between synapse formation and cell development, the morphology and cytochemistry of the endoplasmic reticulum and its enzymic marker, glucose-6-phosphatase (G-6-Pase), in cultured mouse spinal neurons were investigated ultrastructurally. It was found that in the early period of the development, neurons were characterized by scarceness of organelles; only a few of granular or agranular endoplasmic reticulum and mitochondria were seen. The endoplasmic reticulum and nuclear envelope were packed specifically with G-6-Pase resection product but the product was weak. After a period of culture, most of the neurons had well-developed endoplasmic reticulum, Golgi apparatus, mitochondria and microtubules, etc. The Golgi apparatus was relatively large, having some cisternae associated with vesicles. Either concave of convex face of the saccules was labeled by thiamine pyrophosphatase (TPPase) specifically. GERL, labeled by cytidine monophosphatase (CMPase), was also seen close to the inner or outer face of some Golgi apparatus. The endoplasmic reticulum at this stage was distributed throughout the cytoplasm, including that in dendrites; its enzyme marker (G-6-Pase) localized consistently within the lumen of all endoplasmic reticulum, nuclear space and subsurface cisternae, and frequently in the concave saccules of the Golgi apparatus. After a long-term culture, some neurons became "aged". The endoplasmic reticulum cisternae enlarged and G-6-Pase reaction reduced. Along with the neuronal development, especially maturation of the endoplasmic reticulum and its enzymic marker, synapse formation was begun at the neuropile area. The axo-dendritic synapses always occurred between the axonal terminals and dendrites where the endoplasmic reticulum had showed positive G-6-Pase reactions. Considering the fact, it suggests that the appearance and change of these specific enzymes may be related to the maturation of the neurons in vitro, and also related to the synapse formation between neurons.     

Colchicine effects on neurosecretory neurons and other hypothalamic and hypophysial cells,with special reference to changes in the cytoplasmic membranes

Summary The morphological effects of colchicine on the entire neurosecretory (NS) tract and on various hypothalamic nuclei have been studied. The perturbation in axonal flow, indicated by the accumulation of NS material, coincide with fragmentation of the cytoplasmic membranes, i. e. the Golgi apparatus and the endoplasmic reticulum, whereas the neurotubules remain relatively well preserved. Autophagic destruction of NS material is observed along the entire length of the NS fibres. The rapid and systematic changes in the axoplasmic reticulum, known to store calcium, lead us to envisage a role for this system — similar to that of the sarcoplasmic reticulum — in controlling the transport of NS vesicles. The junctional zone between the stalk and the neural lobe seems to play a particular rôle in the transport of NS material to the posthypophysial terminals of the NS axons. Colchicine provokes an increase in dense-cored vesicles in most of the neurons of the other hypothalamic nuclei studied: arcuate, suprachiasmatic, periventricular and ventromedial. Membranous alterations are also observed in these sites. Colchicine administered to animals which were hypothyroid, castrated or adrenalectomized, reveals stimulated neurons, identified by their excessive content of dense-cored vesicles. These neurons display no specific localization, for they occur in all hypothalamic nuclei, irrespective of the stimulation. The frequency of stimulation of neurons of the periventricular nucleus is striking.     

AChE synthesis in cholinergic neurons: electron histochemistry of enzyme translocation

Summary Endoplasmic reticulum of cholinergic nerve cells exhibits acetylcholinesterase activity. In central neurons that exert an ephemeric acetylcholinesterase activity only during some ontogenetical states, enzyme reaction product is present also in the Golgi system. Neurotubular and neurofilamentar structures exert an acetylcholinesterase in terminal axons of young animals. From these electron histochemical studies it is concluded that enzyme protein molecules, synthesized in the endoplasmic reticulum, are translocated to the active sites (surface membranes) via axonal filaments (or tubules), or will be extruded from the neuron via the Golgi system.Our thanks are due to Dr. P. Röhlich, head of the Budapest Electron Microscope Laboratory, for his generous help in various aspects of the electron microscopic work.     

Fine structure of nerve cells in a planarian

The fine structure of the nerve cell types in the white planarian Procotyla fluviatilis were described. Ganglion cells comprise the major portion of the brain. These cells are irregular in shape with several cytoplasmic processes and contain ribosomes, a sparse endoplasmic reticulum, microtubules, lysosomes, and a Golgi apparatus with numerous small vesicles. Granule-containing cells are situated in the peripheral regions of the brain and along the nerve cords. These cells contain ribosomes, rough-surfaced endoplasmic reticulum and a Golgi apparatus with associated dense granules. The granules occupy most of the cytoplasm and are ～ 750A in diameter with moderately dense contents, ～ 750A with opaque contents, and ～ 1000A with contents of medium density. These granules are similar to those in the nervous systems of higher animals that contain epinephrine, norepinephrine, and neurosecretory substance, respectively. Each cell contains predominantly one type of granule although there is some intermixing of granules and intermediate types between the three most abundant granules. Small clear vesicles, resembling cholinergic synaptic vesicles, and all types of dense granules occur in the neuropil and within nerve endings.     

Detection of sialic acid residues in the axonal reticulum of rat superior cervical ganglion cells by lectin-gold cytochemistry

Highly glycosylated compounds have been demonstrated in the axonal reticulum elements of the superior cervical ganglion cells of the rat, and this is considered to suggest a connection of the reticulum with the trans Golgi side. In the present study, the axonal reticulum and the Golgi elements were further characterized by post-embedding methods of lectin-gold cytochemistry to determine their carbohydrate residues and to see, more specifically, if sialic acid residues could be detected in the axonal reticulum elements. Therefore, the affinity of neuronal cell structures for Limax flavus agglutinin (LFA), wheat germ agglutinin (WGA), and Ricinus communis agglutinin I (RCA-I) was tested in ultra-thin sections of glycolmethacrylate-embedded material, counterstained with phosphotungstic acid (PTA) at low pH. The trans Golgi network, the Golgi-associated axonal reticulum, the reticulum within axons, the large dense-cored vesicles, and the plasma membranes were reactive for all three lectins used. We conclude that the axonal reticulum elements carry sialic acid residues, relating them to the trans Golgi network. The present results support the concept that the axonal reticulum is an extension of the trans network of the Golgi apparatus specialized for neurosecretion.     

Annulate lamellae and whorl bodies in the diffuse supraoptic nucleus of the hamster

The ultrastructure of neurons of the diffuse supraoptic nucleus of the hamster has been studied. These neurons show two specializations of the endoplasmic reticulum: annulate lamellae and whorl bodies. From one to three whorl bodies are found in the same neuron. The annulate lamellae and the whorl body cisterns are continuous with the cisterns of the rough endoplasmic reticulum. These neurons present an extraordinarily developed rough endoplasmic reticulum, small mitochondria, neurosecretory vesicles and a Golgi complex filled with electron-dense material. Astrocytic processes of different thickness surround the neurosecretory cells.     

Thiamine pyrophosphatase activity in the axonal smooth endoplasmic reticulum of neurosecretory neurons

Summary Neurosecretory cells of the supraoptic-neurohypophysial system of normal mice were investigated with the use of the cytochemical reaction for thiamine pyrophosphatase (TPPase) at the ultrastructural level. In the hypothalamic perikarya dense lead precipitates occur within the cisterns of the mature face of the Golgi apparatus, these being the cisterns that give rise to neurosecretory granules (NSG). Smooth endoplasmic reticulum is occasionally confluent with TPPase-positive Golgi cisterns. Along axons, within swellings, and within terminals distinct profiles of TPPase-positive tubules and cisterns are revealed, apparently part of a network of axonal smooth endoplasmic reticulum (AER). Some NSG appear to be confluent with AER. NSG with TPPase-positive tubular protrusions (likely vestiges of AER) are seen. Apart from reaction product (lead precipitate), the AER often contains an electron dense substance optically similar to that of NSG. TPPase-containing AER is often associated with mitochondria. Profiles of electron-lucent, precipitate-free tubules and cisterns are occasionally seen alongside reactive AER. Optimal TPPase activity in the AER occurs at pH 7.0–7.4, whereas in the Golgi complex intense marking is in the range of pH 6.0–8.5. A faint peppering of precipitate occasionally appears in the AER in controls (incubation medium without substrate), but neither in density nor in extent is this comparable to the reaction product seen after incubation in the presence of TPP. Preliminary comparison has been made between the AER revealed by the TPPase reaction, and that visualized after heavy metal impregnation according to the method of Alonso and Assenmacher (1978a). The nature of the close association between NSG and AER, and the possible roles of this membrane system in neurosecretory cells is discussed.Abbreviations AER axonal smooth endoplasmic reticulum - NSG neurosecretory granules - TPPase thiamine pyrophosphatase - SON supraoptic nucleus Research supported in part by a grant from the Israel Academy of Sciences to M.C.We thank Mrs. Ilana Sabnay for excellent technical assistance     

内质网及其标志酶在离体培养脊髓神经元中的发育变化

本文采用形态学与细胞化学相结合的方法,在超微结构水平观察了与突触酶、受体和结构蛋白的合成有关的内质网和高尔基复合体、GERL以及它们的标志酶的发育变化。结果表明神经元本身有一发育过程,发育早期的细胞器较少,成熟时逐渐增多,以内质网和高尔基复合体最为明显。用G一6一Pase、TPPase和CMPase可分别标记内质网及同源结构、高尔基复合体的成熟而膜囊和GERL。这些酶的出现及阳性水平与神经元的发育呈同步关系。可作为判断细胞分化程度和功能状态的指标。G-6-Pase还分布在突触后树突的内质网中,突触形成大都从含G-6-Pase阳性内质网的树突开始。本文对内质网及G-6-Pase在神经元中的发育变化及功能进行了讨论。     

Cellular organization of the brain renin-angiotensin system

A model of intracellular Ang II formation (Figure 1) implies that angiotensinogen neurons exist and that CNS Ang II acts both as a neurotransmitter as well as a neurohormone. Such a mechanism is consistent with the immunocytochemical localization of a fraction of brain Ang II in neurosecretory vesicles. To date, several dozen peptide neurotransmitters and neurohormones have been studied. Those assigned to peptidergic systems follow the generalized pathway of biosynthesis shown in Figure 1. In peptidergic systems, a prohormone and all of its processing enzymes are synthesized in the rough endoplasmic reticulum of a cell and move into the Golgi apparatus (Figure 1: #1-3). In the Golgi the prohormone and processing enzymes are packaged into the same vesicle (#3). These secretory vesicles then migrate toward the plasma membrane, frequently via axonal or dendritic projections to terminals. Within these cytoplasmic vesicles and prior to release, the processing enzymes are activated (#4) and the prohormone enzymatically processed, yielding the active peptide (#5-6). Only then do the vesicles fuse with the plasma membrane (in a calcium-dependent process), releasing their contents (#7-8). Once released, the active peptide migrates across the extracellular space and interacts with specific cell surface receptors to initiate a response (#9). Finally, receptor-bound peptide degradation is initiated by receptor-mediated endocytosis (#10-11). For angiotensin peptides to be produced intracellularly, the cell must present only one secretory pathway for Golgi packaging of renin and angiotensinogen; otherwise current theories of protein sorting would predict that these two proteins would be segregated even if synthesized within the same cell. Small quantities of co-packaged renin and angiotensinogen occurring via "spill-over" between compartments seems an unsatisfactory process for a regulated hormone system. Figure 2, depicting an extracellular mechanism for producing Ang II in the brain, has also been proposed. The mechanism of extracellular angiotensin formation is consistent with the molecular information encoded within the component proteins, known mechanisms of protein secretio, well-defined systemic renin-angiotensin enzymatic cascades, and demonstration of all the components of the renin-angiotensin system in the extracellular compartments of the brain. This model (Figure 2) allows independently coordinated gene expression and synthesis of renin (#1R), angiotensinogen (#1A), and angiotensin-converting enzyme (# 1C) in the same or different cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Virus-neuron interactions in the mouse brain infected with Japanese encephalitis virus

The virus-host interactions between Japanese encephalitis (JE) virus and mouse brain neurons were analyzed by electron microscopy. JE virus replicated exclusively in the rough endoplasmic reticulum (RER) of neurons. In the early phase of infection, the perikaryon of infected neurons had relatively normal-looking lamellar RER whose cisternae showed focal dilations containing progeny virions and characteristic endoplasmic reticulum (ER) vesicles. The reticular RER, consisted of rows of ribosomes surrounding irregular-shaped, membrane-unbounded cisternae and resembled that observed in JE-virus-infected PC12 cells, were also seen adjacent to the lamellar RER. The appearance of the reticular RER indicated that RER morphogenesis occurred in infected neurons in association with the viral replication. The fine network of Golgi apparatus was extensively obliterated by fragmentation and dissolution of the Golgi membranes and their replacement by the electron-lucent material. As the infection progressed, the lamellar RER was increasingly replaced by the hypertrophic RER which had diffusely dilated cisternae containing multiple progeny virions and ER vesicles. The Golgi apparatus, at this stage, was seen as coarse, localized Golgi complexes near the hypertrophic RER. In the later phase of infection, RER of infected neurons showed a degenerative change, with the cystically dilated cisternae being filled with ER vesicles and virions. Small, localized Golgi complexes frequently showed vesiculation, vacuolation, and dispersion. The present study, therefore, indicated that during the viral replication the normal lamellar RER which synthesized neuronal secretory and membrane proteins was replaced by the hypertrophic RER which synthesized the viral proteins. The hypertrophic RER eventually degenerated into cystic RER whose cisternae were filled with viral products. The constant degenerative change which occurred in the Golgi apparatus during the viral replication suggested that some of the viral proteins transported from RER to the Golgi apparatus were harmful to the Golgi apparatus and that increasing damage to the Golgi apparatus during the viral replication played the principal role in the pathogenesis of JE-virus-infected neurons in the central nervous system.     

Identification of the 16 degrees C compartment of the endoplasmic reticulum in rat liver and cultured hamster kidney cells

In many systems transfer between the endoplasmic reticulum and the Golgi apparatus is blocked at temperatures below 16 degrees C. In virus-infected cells in culture, a special membrane compartment is seen to accumulate. Our studies with rat liver show a similar response to temperature both in situ with slices and in vitro with isolated transitional endoplasmic reticulum fractions. With isolated transitional endoplasmic reticulum fractions, when incubated in the presence of nucleoside triphosphate and a cytosol fraction, temperature dependent formation of vesicles occurred with a Q10 of approximately 2 but was apparent only at temperatures greater than 12 degrees C. A similar response was seen in situ at 12 degrees C and 16 degrees C where fusion of transition vesicles with cis Golgi apparatus, but not their formation, was blocked and transition vesicles accumulated in large numbers. At 18 degrees C and below and especially at 8 degrees C and 12 degrees C, the cells responded by accumulating smooth tubular transitional membranes near the cis Golgi apparatus face. With cells and tissue slices at 20 degrees C neither transition vesicles nor the smooth tubular elements accumulated. Those transition vesicles which formed at 37 degrees C were of a greater diameter than those formed at 4 degrees C both in situ and in vitro. The findings show parallel responses between the temperature dependency of transition vesicle formation in vitro and in situ and suggest that a subpopulation of the transitional endoplasmic reticulum may be morphologically and functionally homologous to the 16 degrees C compartment observed in virally-infected cell lines grown at low temperatures.     

Intracellular transport of albumin through the secretory apparatus of rat liver parenchymal cells. An immunocytochemical study

The fine structural localization of albumin in rat liver parenchymal cells was determined by an improved immunocytochemical method and serial sectioning. Albumin in the secretory apparatus of the parenchymal cells was present in segments of the rough endoplasmic reticulum, interrupted with negative segments, in transport vesicles, Golgi saccules, finely anastomosed tubules and vesicles on the trans side of the Golgi complex, and in secretion granules. Horizontally sectioned Golgi saccules contained lipoprotein particles on one side and albumin on the other side. After transport, the vesicles that contained albumin fused with the so-called rigid lamellae on the trans-side of the Golgi complex. Ultrathin serial sections revealed no true structural continuity between the endoplasmic reticulum and the cis-aspect of the Golgi complex. We concluded that secretory proteins are transported from the endoplasmic reticulum to the Golgi complex by transport vesicles that bud from the endoplasmic reticulum and fuse with the Golgi saccules. These vesicles fuse regularly with the Golgi saccules on the cis-side and occasionally with tubular elements on the trans-aspect that may belong to the so-called GERL.     

The fine structure of the collar cells in the optic tentacles of Helix aspersa

Summary At the base of the optic tentacular ganglion there is a group of large monopolar cells containing numerous secretory inclusions. These are the collar cells. Secretory material can be seen accumulating in swollen portions of the granular endoplasmic reticulum. It is postulated that this material is transported to the Golgi bodies and thus the limiting membrane of the inclusions is derived from the Golgi membranes. The Golgi bodies appear to be polarized and small vesicles resembling secretory inclusions are associated with one face of these organelles. The secretory inclusions fuse together to form large membrane-bound secretory pools in the perikaryon. The collar-cell processes are packed with secretory inclusions. These processes traverse the digital extensions of the tentacular ganglion and pass into the epithelium covering the tip of the tentacle. The secretory inclusions do not resemble neurosecretory inclusions in other situations. The collar cell processes receive a nerve supply from single axons containing granular and agranular vesicles. The evidence that these cells may be modified neurons is only minimal.This work was supported by the Australian Research Grants Committee.     

Signalling organelle for retrograde axonal transport of internalized neurotrophins from the nerve terminal

The retrograde axonal transport of neurotrophins occurs after receptor-mediated endocytosis into vesicles at the nerve terminal. We have been investigating the process of targeting these vesicles for retrograde transport, by examining the transport of [125I]-labelled neurotrophins from the eye to sympathetic and sensory ganglia. With the aid of confocal microscopy, we examined the phenomena further in cultures of dissociated sympathetic ganglia to which rhodamine-labelled nerve growth factor (NGF) was added. We found the label in large vesicles in the growth cone and axons. Light microscopic examination of the sympathetic nerve trunk in vivo also showed the retrogradely transported material to be sporadically located in large structures in the axons. Ultrastructural examination of the sympathetic nerve trunk after the transport of NGF bound to gold particles showed the label to be concentrated in relatively few large organelles that consisted of accumulations of multivesicular bodies. These results suggest that in vivo NGF is transported in specialized organelles that require assembly in the nerve terminal.     

Isolation of a vesicular intermediate in the cell-free transfer of membrane from transitional elements of the endoplasmic reticulum to Golgi apparatus cisternae of rat liver

Preparations enriched in part-smooth (lacking ribosomes), part-rough (with ribosomes) transitional elements of the endoplasmic reticulum when incubated with ATP plus a cytosol fraction responded by the formation of blebbing profiles and approximately 60-nm vesicles. The 60-nm vesicles formed resembled closely transition vesicles in situ considered to function in the transfer of membrane materials between the endoplasmic reticulum and the Golgi apparatus. The transition elements following incubation with ATP and cytosol were resolved by preparative free-flow electrophoresis into fractions of differing electronegativity. The main fraction contained the larger vesicles of the transitional membrane elements, while a less electronegative minor shoulder fraction was enriched in the 60-nm vesicles. If the vesicles concentrated by preparative free-flow electrophoresis were from material previously radiolabeled with [3H]leucine and then added to Golgi apparatus immobilized to nitrocellulose, radioactivity was transferred to the Golgi apparatus membranes. The transfer was rapid (T1/2 of about 5 min), efficient (10-30% of the total radioactivity of the transition vesicle preparations was transferred to Golgi apparatus), and independent of added ATP but facilitated by cytosol. Transfer was specific and apparently unidirectional in that Golgi apparatus membranes were ineffective as donor membranes and endoplasmic reticulum vesicles were ineffective as recipient membranes. Using a heterologous system with transition vesicles from rat liver and Golgi apparatus isolated from guinea pig liver, coalescence of the small endoplasmic reticulum-derived vesicles with Golgi apparatus membranes was demonstrated using immunocytochemistry. Employed were polyclonal antibodies directed against the isolated rat transition vesicle preparations. When localized by immunogold procedures at the electron microscope level, regions of rat-derived vesicles were found fused with cisternae of guinea pig Golgi apparatus immobilized to nitrocellulose strips. Membrane transfer was demonstrated from experiments where transition vesicle membrane proteins were radioiodinated by the Bolton-Hunter procedure. Additionally, radiolabeled peptide bands not present initially in endoplasmic reticulum appeared following coalescence of the derived vesicles with Golgi apparatus. These bands, indicative of processing, required that both Golgi apparatus and transition vesicles be present and did not occur in incubated endoplasmic reticulum preparations or on nitrocellulose strips to which no Golgi apparatus were added.(ABSTRACT TRUNCATED AT 400 WORDS)     

The smooth endoplasmic reticulum in neurohypophysial axons of the rat: Possible involvement in transport,storage and release of neurosecretory material

Summary The intra-axonal organization of the smooth endoplasmic reticulum was studied in the neurohypophysis of rats during and after water deprivation. Parallel to conventional electron microscopy, the material was treated with a double impregnation staining technique specifically designed to contrast the intracellular membranous system. In conventionally stained ultrathin sections from severely dehydrated rats most axons appeared to be free of membranous organelles, whereas corresponding axons treated with the double-impregnation technique generally exhibited a highly developed system of smooth endoplasmic reticulum. In axonal endings, both techniques revealed a profusion of microvesicles in intimate relationship with tubular elements of the smooth endoplasmic reticulum. In short-term (12 h) rehydrated rats, a similarly developed system of smooth endoplasmic reticulum was still observed at all axonal levels with both procedures. After 24 to 48 h of rehydration the tubules of the smooth endoplasmic reticulum exhibited, in double impregnated material, numerous dilatations which resembled the adjacent neurosecretory granules. In conventionally stained ultrathin sections, an accumulation of electron dense material occurred within tubules of the smooth endoplasmic reticulum in the more proximal axonal segments, while in the more terminal segments, which contained numerous elongated granules, membrane continuity was frequently observed between newly formed granules and the smooth endoplasmic reticulum. After 7 days of rehydration the general pattern of the axonal smooth endoplasmic reticulum was comparable to that in untreated rats. These results are discussed in the light of a suggested involvement of the axonal smooth endoplasmic reticulum in the non-granular transport of neurosecretory material in connection with (1) storage in distally formed granules, and (2) release via microvesicles. Acknowledgements: The authors wish to express their gratitude to Mrs. M. Balmefrézol for her skillful technical assistance     

Monodehydroascorbate as an electron acceptor for NADH reduction by coated vesicle and Golgi apparatus fractions of rat liver

Coated vesicles were isolated from rat liver in about 80% fraction purity as determined from electron microscopy and analyses of marker enzymes and compared with Golgi apparatus and other membrane fractions isolated in parallel. The fractions were enriched in NADH-monodehydroascorbate reductase, ascorbate oxidase and ascorbic acid. The NADH-monodehydroascorbate reductase and ascorbate oxidase of the Golgi apparatus and coated vesicles differed from that of the endoplasmic reticulum in being inhibited by the sodium selective ionophore, monensin, at physiological concentrations while these activities were stimulated by ethylenediaminetetraacetic acid in coated vesicles but not in Golgi apparatus. Activities of both coated vesicles and Golgi apparatus fractions depleted in the coat protein, clathrin, were activated by the addition of clathrin-rich supernatant fractions. The results are discussed in the context of monodehydroascorbate as an acceptor for electron transport-mediated transfer of electrons from NADH by coated vesicles as part of a possible mechanism to drive membrane translocations or to acidify the interiors of vesicles.