Evidence for calcium inactivation during hormone release in the rat neurohypophysis.

1. A study has been made of the relationship between 45Ca uptake into and hormone release from isolated rat neurohypophyses incubated in vitro. 2. Hormone secretion is triggered by high-K (56 mM) but long exposure to the stimulus does not generate a maintained release of hormone. 3. When hormone release began to wane, addition of Ba of La increased hormone output which suggests that the decline in output did not result from depletion of the neurosecretory granules at the nerve terminals. 4. 45Ca uptake is enhanced in the presence of high-K concentration, but the initial high rate declines during long exposure to the potassium stimulus with a time constant similar to that of the decline in hormone release. 5. After a period of incubation in a K-rich, calcium-free medium, addition of calcium to the medium induced hormone release. The magnitude of this release was dependent on the time of exposure to excess potassium. 6. After inactivation of secretion, mobilization of internal calcium by means of a calcium ionophore increased hormone release.     

Ultrastructural changes in isolated peptidergic nerve terminals induced by digitonin permeabilization and K+ stimulation in the sinus gland of the crab,Cardisoma carnifex

Summary The preparation of isolated peptidergic nerve terminals from the sinus gland (a neurohemal organ) of the crab (Cardisoma carnifex) is described. In this species the nerve endings can have diameters up to 30 m. They release neurosecretory material as judged by the decrease in the volumetric density of granules upon depolarization with potassium. Similar results were obtained after permeabilization of the nerve terminals with digitonin, but only in the presence of micromolar concentrations of calcium. This preparation should prove useful in correlating electrical events with other cellular processes involved in stimulus-secretion coupling.     

锯缘青蟹窦腺显微和超微结构研究

借助光学和电子显微镜观察据缘青蟹（Ｓｃｙｌｌａ ｓｅｒｒａｔａ）窦腺的形态结构。窦腺位于眼柄视神经节内髓背侧近外髓处。窦腺呈羹状;腺体壁山神经分泌细胞的末梢和神经胶质细胞组成。神经末梢内充满了电子致密的神经分泌颗粒。根据颗粒的大小、形态及电子致密度等特征,可以区分出４种类型的神经末梢。一些末梢中的多形性颗粒可能是由Ⅱ型末梢中的颗粒转变而成的。一些现象表明,神经分泌物质可以通过胞吐作用或一种类似“顶浆分泌”的方式释放,从而说明神经激素的释放可能是多途径的．     

中华绒螯蟹窦腺神经末梢及X-器官神经分泌细胞的类型

在电子显微镜下观察了性未成熟的中华绒螯蟹黄蟹的窦腺及X－器官的超微结构。X－器官位于眼柄神经节终髓的腹外侧,与窦腺位置斜相对,窦腺主要由神经分泌细胞的末梢和胶质细胞组成。神经末梢含有大量的膜结构包围的颗粒、线粒体、粗面内质网和许多电子透明的小泡,末梢外周有时可见指状突起。依据颗粒的大小、形状、电子致密度以及胞质特征,可区分出6种类型的窦腺神经末梢及7种X－器官神经分泌细胞。观察了末梢中神经分泌颗粒的胞吐作用方式的释放过程,并且尝试对窦腺不同末梢中的颗粒及X－器官神经分泌细胞中的颗粒作了比较,发现二者之间具有较好的对应性,即电子致密度无大的变化,形态特征相似,只是大小稍有增加。     

Ultrastructure of neurosecretory granule exocytosis by crayfish sinus gland induced with ionic manipulations

Neurosecretory granule exocytosis from axon terminals in the crayfish (Orconectes virilis) sinus gland can be effected by alterations in intracellular ionic concentrations through modification of the bathing medium or by electrical stimulation. This may result in increased numbers of exocytotic profiles involving one granule (single exocytosis) or several granules (compound exocytosis), which we interpret as evidence of stimulated neurosecretion. Our results suggest that increased free intracellular Ca⁺⁺ together with a decrease in intracellular K⁺ are prerequisite for and sufficient to elicit increased exocytosis from sinus gland terminals. Similar release, stimulated by horseradish peroxidase, indicates that local surface permeability changes are involved. A minor response to high-K⁺-Ringer, and secretion via compound exocytosis, are uncharacteristic of cells with a neural ancestry.     

Neurosecretion in the sinus gland of the fiddler crab,Uca pugnax

The ultrastructure of the sinus gland of the fiddler crab, Uca pugnax, was investigated and found to be similar to that in other crustaceans. Five types of neurosecretory axon terminals were tentatively identified on the basis of the size, shape, and electron density of granules within the axons. Release of neuro-secretory material appears to be by exocytosis.     

Relationship between Rous sarcoma virus production in golden hamster cells and the cell cycle]

The axon terminals of neurosecretory cells, containing elementary granules of neurosecretory materials, have been described on the basis of light and electron microscopy. No allochthonous neurosecretory elements were found in the sinus gland. The discharge of the granules from some terminals of the sinus gland occur with the changed salinity of the environment. There is a great difference in the structure of the sinus gland when salinity is changed. The structure of the sinus gland and its modification under experimental conditions indicate a possibility of neurohormonal regulation of the hydromineral balance.     

Exo-endocytosis in isolated peptidergic nerve terminals occurs in the sub-second range

Exo- and endocytotic processes induced by depolarization of isolated neurosecretory nerve terminals show a close temporal correlation, which suggests a short time of integration of the neurosecretory granule membrane with the plasma membrane. In order to determine minimal time requirements for exocytosis-coupled endocytosis to occur, we have analyzed by electron microscopy uptake of horserdish peroxidase (HRP) as a fluid phase marker at the onset of depolarization. We have applied rapid mixing and sampling (quenched flow) to assess events in subsecond time peroids after stimulation. A significant number of labelled endocytotic vacuoles was observed during the first second of depolarization. This number then further increased by a factor of about 2 (within 5 s) and 4 (within 50s). Thus, as for exocytosis, the rate of endocytosis decreased considerably during prolonged stimulation. These data indicate i) that a substantial proportion of secretory granules undergoes exocytosis very shortly after stimulation, and ii) that, following exocytosis, the minimal time required for consecutive membrane retrieval is in the sub-second range.     

Exocytosis of neurosecretory granules from the crustacean sinus gland in freeze-fracture

Exocytosis is clearly shown in freeze-fracture preparations to be the mechanism for neurosecretion granule release from axon endings in the crayfish sinus gland. The cytoplasmic leaflet (A-face) of axon ending membrane is characterized by randomly situated depressions representing invaginations of the axolemma, which are in contact with limiting membranes of neurohormone granules in the subjacent cytoplasm. The extracellular leaflet (B-face) of the axolemma at release sites exhibits complementary volcano-shaped protrusions which are cross-fractures through necks of channels formed by invaginating plasma membrane in contact with underlying neurosecretion granules. Structural variation in B-face protrusions is consistent with a spectrum of exocytotic profiles in various stages of formation, and with granules at different stages of passage out of the endings. Evidence in this study suggests that formation of exocytotic structures may begin by alteration of axon membrane structure at the neurosecretory ending-hemolymph interface prior to contact of the neurohormone granules with the axolemma. Limiting membranes of neurosecretory granules exhibit protrusions which appear to interconnect granules adjacent to release sites and to attach granules to the axolemma. Freeze-fracture is clearly shown to be an invaluable tool for monitoring the degree of exocytosis exhibited by sinus glands under normal conditions and under experimental acceleration of hormone release. This technique is capable therefore, of detecting slight increases in numbers of exocytotic profiles much more quickly and accurately than the examination of random thin sections.     

The ultrastructure of the sinus gland of the crayfish Astacus leptodactylus (Nordmann)

Summary An ultrastructural study of the sinus gland of the crayfish Astacus leptodactylus demonstrates that this gland is mainly composed of glial cells, axons and axon terminals. On the basis of the size, shape and electron density of the neurosecretory granules, we could distinguish five different types of axon terminals.     

Release of brain neurosecretory products from the neurohaemal part of the aorta during egg-laying and egg-care in Labidura riparia (Insecta,dermaptera)

The exocytosis level in the neurosecretory terminals of the aorta wall, was studied by electron-microscopy in female Labidura riparia during different stages of the reproductive period: end of vitellogenesis, beginning, middle and end of egg-laying, and during egg-care.The exocytosis level (L. ex) in the terminals which are filled with big dense elementary granules is low at the end of vitellogenesis (L. ex = 0.28), the level increases 8 times when the first egg is passing under the genital plate, reaching a maximum level (2.46) when approximately ten eggs have been laid. The level then decreases at the end of egg-laying (1.8) and abruptly falls when the female begins her egg-care cycle. Throughout this period, the exocytosis level stays between 0.54 and 0.76.These results show a significant release of brain neurosecretory product at the neurohaemal part of the aorta during egg-laying. This suggests that egg-laying could be regulated by a brain hormone.     

The ultrastructure of the sinus gland of Carcinus maenas (Crustacea: Decapoda)

Summary The sinus gland of Carcinus maenas consists of the swollen axonal endings of the neurosecretory cells of the major ganglia and acts as a storage release centre for the membrane bound neurosecretory material. These neurosecretory granules fall into five different types based on size and electron density. Their contents are released by exocytosis of the primary granules or smaller units budded from the primary granules.I thank Professor E. Naylor for his constant advice and Professor E. W. Knight-Jones, Department of Zoology, University College, Swansea, for the provision of laboratory facilities. I am grateful to the Science Research Council for the financial support. Finally, I thank the Electron Microscope Unit, Southampton General Hospital, where the work was completed.     

REGULATION OF GUANOSINE 3'',5'' CYCLIC MONOPHOSPHATE IN THE RAT PINEAL AND POSTERIOR PITUITARY GLANDS

Potassium and norepinephrine stimulate the accumulation of cyclic AMP and cyclic GMP in rat pineal glands and their efflux into the medium. The efflux of both cyclic nucleotides was blocked by probenecid. The accumulation and efflux of cyclic GMP, but not of cyclic AMP, depends upon the presence of intact nerve endings and extracellular calcium. The calcium-dependent release of norepinephrine caused by veratridine was accompanied by the efflux of both cyclic AMP and cyclic GMP. In contrast, the calcium-independent release of norepinephrine caused by tyramine was accompanied by the efflux of cyclic AMP but not cyclic GMP. Changes in cyclic GMP therefore, may be related to exocytosis from the sympathetic nerve endings in the gland. High concentrations of potassium also increased tissue levels of cyclic GMP in the posterior pituitary gland. Veratridine and potassium, but not norepinephrine, stimulated the efflux of cyclic GMP from this neurosecretory gland. Thus, the relationship between cyclic GMP and exocytosis may extend beyond sympathetic nerve endings. The enhanced accumulation of cyclic GMP in the pineal gland after potassium does not appear to be mediated by extracellular (released) norepinephrine. Desmethylimipramine blocked the norepinephrine-stimulated changes in cyclic GMP, but not those caused by potassium. Investigation of the possible relationship between cyclic GMP and release of neurotransmitters is complicated by the apparent seasonal variation in the response of pineal cyclic GMP to potassium or norepinephrine.     

alpha-Latrotoxin as an instrument for studying neurosecretions

alpha-Latrotoxin, a presynaptic toxin from black widow spider venom Latrodectus mactans tredecimguttatus, triggers exocytosis in a variety of neurosecretory cells both in the presence and absence of calcium in the medium. The toxin interacts with two types of membrane the receptors which belong to different families of neuronal proteins and have different structures. Calcium-dependent receptor of alpha-latrotoxin is identified as neurexin I alpha and belongs to the family of neurexins. This family is selectively expressed in nerve tissue. The calcium-independent receptor of alpha-latrotoxin belongs to the family of G-protein-coupled receptors and proteins which homologous to it are found in heart, lung, kidney and spleen tissues. As a result of alpha-latrotoxin interaction with membrane receptor in the calcium medium the toxin forms the ionic channels in plasmalemma and enhances its calcium permeability. The effects of alpha-latrotoxin on exocytosis in the calcium and calcium-free media and question concerning coupling of channel-forming and secretogenic properties of alpha-latrotoxin are discussed.     

Structural studies of nerve terminals containing melanin-concentrating hormone in the eel,Anguilla anguilla

Summary Eels were adapted to black- or white-coloured backgrounds and the pituitary glands were prepared for light and electron microscopy. Immunocytochemical staining was used to study the distribution of the neurohypophysial melanin-concentrating hormone in the neurointermediate lobe. The hormone was located in small, elliptical, electron-opaque neurosecretory granules, measuring approximately 120×90 nm. The neurones terminated on blood vessels in the centre of the neurohypophysis and on the basement membrane separating neural and intermediate lobe tissues. The results of both light and electron immunocytochemistry and of radioimmunoassay are consistent with a higher rate of hormone release from eels adapted to white backgrounds than from those adapted to black backgrounds. In addition to this, when fish that had been adapted to white tanks were transferred to black tanks, there was an accumulation of irMCH in the gland and an increased numerical density of secretory granules at nerve terminals. These results reinforce the proposal that MCH is released during adaptation to a white background, to cause melanin concentration and to inhibit MSH release, and that its release is halted in black-adapted fish.     

Autoradiographic evidence that transport of newly synthesized neuropeptides is directed to release sites in the X-organsinus gland of Cardisoma carnifex

Summary Sections of isolated X-organ — sinus gland neurosecretory systems of the crab, Cardisoma carnifex, were studied by light-and electron microscopy with conventional and autoradiographic procedures. The somata only were exposed to a pulse of ³H-leucine (5 min-5 h) and the entire system perfused with chase medium for various times (1–72 h) before fixation. Within 1 h, radiolabel is concentrated in Golgi complexes and nascent granules of both large and small somata. Label is undetectable in the terminal region following a 10 h chase. It is found in the nerve tract near terminals at 14 h, while after a 19 h chase, label is concentrated in terminal profiles abutting blood sinuses of the neurohemal organ (sinus gland). Following a 72 h chase, label is distributed throughout the terminal region. Each of the six morphologically distinguishable terminal types shows labelling. These observations show that the vast majority of newly formed granules are initially transported to release sites of the perisinus terminals. They thus provide an explanation for previous analyses indicating that newly synthesized peptides are preferentially secreted.     

Fine structure of the neurohemal sinus gland of the shore crab,Carcinus maenas,and immuno-electron-microscopic identification of neurosecretory endings according to their neuropeptide contents

Summary The sinus gland of the shore crab, Carcinus maenas, is a compact assembly of interdigitating neurosecretory axon endings abutting upon the thin basal lamina of a central hemolymph lacuna. Four types of axon endings are distinguishable by the size distribution, shape, electron density and core structure of their neurosecretory granules. One additional type of axon ending is characterized by electron-lucent vacuoles and vesicles. The axon profiles are surrounded by astrocyte-like glial cells. Various fixations followed by epoxy- or Lowicrylembedding were compared in order to optimize the preservation of the fine structure of the granule types and the antigenicity of their peptide hormone contents. By use of specific rabbit antisera, the crustacean hyperglycemic, molt-inhibiting, pigment-dispersing, and red-pigment-concentrating hormones were assigned to the four distinct granule types which showed no overlap of immunostaining. Epi-polarization microscopy and ultrathin section analysis of immunogold-stained Lowicrylembedded specimens revealed that immunoreactivity to Leu-enkephalin and proctolin is co-localized with moltinhibiting hormone immunoreactivity in the same type of granule. The size and core structure of the immunocytochemically identified granule types vary little with the different pretreatments but, in some cases, to a statistically significant extent. The present results are compared with those from earlier studies of sinus glands in different crustaceans. The methods of granule identification used in this study supplement the classical approach in granule typing; they are easier to perform and more reliable for the analysis of release phenomena in identified secretory neurons supplying the neurohemal sinus gland.     

Peroxidase uptake by photoreceptor terminals of the skate retina

The photoreceptors of dark-adapted skate retinas bathed in a Ringer solution containing horseradish peroxidase (HRP) incorporate the tracer into membrane-bound compartments within the synaptic terminal of the cell; after 1 or 2 h of incubation, approx. 10-38% of the synaptic vesicles were labeled. The receptors appeared to be functioning normally throughout the incubation period, since electrical potentials of normal amplitude could be elicited in response to dimphotic stimuli. However, it was possible to block the uptake of peroxidase by a regimen of light adaptation that effectively suppressed light-induced activity in the electroretinogram. If, during incubation with peroxidase, retinas were exposed at 10-min intervals to an intense 1-ms flash from a xenon discharge tube, the receptor terminals were almost completely devoid of peroxidase; fewer than 2% of the vesicles were labeled. The suppression of HRP uptake could also be achieved in dark-adapted retinas by adding magnesium to the bathing solution, suggesting that calcium is necessary for transmitter release from vesicles in the receptor terminals. These findings are consistent with the view that vertebrate photoreceptors discharge a neurotransmitter in darkness, and that light decreases the release of this substance. It seems likely that the incorporation of peroxidase into vesicles of physiologically active receptor terminals reflects a mechanism for the retrieval of vesicle membrane after exocytosis.     

Ouabain evokes exocytosis dependent on ryanodine and mitochondrial calcium stores that is not followed by compensatory endocytosis at the neuromuscular junction

Ouabain is a cardiotonic glycoside that inhibits the sodium potassium ATPase pump leading to sodium accumulation in nerve terminals. At the frog neuromuscular junction, ouabain induces acetylcholine release and a rapid depletion of synaptic vesicles. In the present work, we used FM1–43 vital labeling to dissect the effect of ouabain on synaptic vesicles recycling. We first examined images of nerve-muscle preparations that were stained with FM1–43 by electrical stimulation of the nerve and destained with ouabain. We observed that ouabain induced exocytosis of synaptic vesicles independently of extracellular calcium, implying a mechanism of exocytosis that can bypass the requirement for extracellular calcium. We therefore tested the hypothesis that ouabain induces exocytosis by mobilizing intracellular calcium and we report that calcium release from endoplasmic reticulum through ryanodine receptors is necessary for ouabain-evoked exocytosis. In addition, the ouabain-evoked exocytosis was dependent on calcium released from mitochondria. We also investigated if exocytosis evoked by ouabain is followed by compensatory endocytosis. We observed that muscles incubated with FM1–43 in the presence of ouabain did not present significant staining. In conclusion, our data demonstrate that exocytosis evoked by ouabain is independent on extracellular calcium but dependent on calcium release from endoplasmic reticulum and mitochondrial stores. In addition, we suggest that ouabain can be used as a pharmacological tool to uncouple synaptic vesicles exocytosis from endocytosis at the neuromuscular junction.     

The neurosecretory system of the eyestalk of Carcinus maenas (Crustacea: Decapoda)

The optic ganglia neurosecretory cells of male and female Carcinus maenas during intermoult are distinguishable into six types based on size, location, appearance and method of secretory material release from the perikaryon. Release occurs via the sinus gland and also, in one case, directly into blood capillaries among the neurosecretory cells themselves. The sinus gland consists of axonal extensions of the neurosecretory cells; no secretory granules are produced there and nuclei observed between the axonal endings are those of ill-defined glial cells.