Rapid refilling of neurosecretory terminals with secretory granules after an acute stimulus to the isolated neural lobe of the rat

Summary Quantitative ultrastructural techniques were used to study changes in the distribution of intracellular organelles in neurosecretory terminals of the rat neurohypophysis during recovery from a depolarising stimulus in vitro. The volumetric density of neurosecretory granules which, in profiles of nerve terminals sectioned through the area of contact with the basal lamina, was decreased as a result of stimulation, returned to control values within 2 h after cessation of stimulation. We conclude that, even in the absence of the cell body and action potentials propagated from it, granules can migrate quickly within the terminal region of the neurosecretory axons to refill a depleted compartment.     

HPLC Analysis of Neurophysin Proteins from Neurosecretory Granules

Abstract: Neurosecretory granules were obtained from neurolobes of porcine pituitary glands. From the granules, highly purified neurophysins were prepared by HPLC. According to polyacrylamide gel electrophoresis, isoelectric focusing, N- and C-terminal amino acid residue determination, and amino acid composition, the neurophysins I₁ I₂, and II were identical to the neurophysins obtained from whole posterior lobes. Since degradation could not have occurred, we conclude that neurophysin I₁ and I₂ originated in the neurosecretory granules.     

Pseudopodia formation by neurosecretory granules

Summary Ultrastructural studies of the mouse neurohypophysis, under various experimental conditions, revealed a number of neurosecretory granules (NSG) bearing single pseudopodia-like protrusions. Some NSG adhered to the axolemma via pseudopodia; other NSG, distant from the axolemma, budded electron lucent microvesicles from the tip of the pseudopod.Pseudopodia counts were made on electron micrographs, and calculated as a percentage of the NSG population. In neural lobes from intact mice, small numbers of pseudopodia were observed (0.3%); the count increased significantly after injections of large doses of horseradish peroxidase (HRP) (9.4–14.5%); hypertonic saline augmented the count, as did histamine.In vitro incubation experiments with isolated neural lobes in Krebs Ringer revealed concomitant pseudopodia formation and elevated vasopressin release (measured by antidiuretic bioassay) in the presence of HRP and di-butyryl cyclic AMP respectively. Histamine and excess potassium also increased hormone secretion, but did not induce pseudopodia formation in vitro; pseudopodia were observed neither in controls, nor in the presence of ineffective secretagogues.It is suggested that the pseudopod may represent the active site on the granule membrane. Different ultrastructural images of granule release suggest that several modes of hormone release may be operative in the neurohypophysis. The role of HRP in pseudopodia formation and vasopressin release is enigmatic.This study is dedicated to Prof. Berta Scharrer, esteemed mentor and beloved friendProf. Zwi Selinger, Department of Biological Chemistry of the Hebrew University, kindly collaborated in the in vitro experiments. Thanks are due to Mrs. Ilana Sabnai and Mrs. Sara Eimerl for excellent technical assistance. Research supported by the Binational Israel-United States Science Foundation (BNSF), grant 200     

Morphology and distribution of the synapses to the spinal motoneuron of the frog

Summary Neurosecretory axons and their dilatations in the pars nervosa of the human neurohypophysis were studied electron microscopically. The axons are of two different types based on their content of neurosecretory granules (NSGs): (i) NSGs of Type A are 100–300 nm, and (ii) NSGs of type B are 50–100 nm in diameter.While fibers (or axons) of type B were scarce, showing simple swellings and terminal formations, fibers of type A were ubiquitous in the human pars nervosa, exhibiting numerous dilatations with a diversity of internal structure, apparently representing the ultrastructural manifestation of intraaxonal turnover of neurohypophysial hormones. Based on the predominating aspect of their internal structure, dilatations of type A-fibers were classified into six different types, with various transitional forms: Type I is characterized by abundant NSGs; type II by prominent mitochondria; type III by abundant lysosomal bodies; type IV by an electron-lucent matrix with few organelles; type V by prominent tubuloreticular profiles; and type VI by numerous microvesicles. The functional significance of each type is discussed and a scheme of possible interrelationships between these dilatations is proposed.     

Isolation and characterization of neurosecretory granules of the rat posterior pituitary gland

Summary Neurosecretory granules (NSG) of rat posterior pituitary glands were prepared by differential centrifugation techniques mainly according to the procedure as described by Barer, Heller and Lederis (1963). As revealed by electron microscopy, the recovery of neurophysin and the contents of enzymes, purified NSG were obtained in a pellet at 30 000 g/60 min (0.44 M sucrose). Eighteen h after injection of (³⁵S) cysteine into the supraoptic nucleus 60% of the recovered radioactivity in the neural lobe was found in the NSG, whereas 20% was found in the final supernatant (100 000 g/120 min). Sixteen days after injection the NSG and the final supernatant fraction contained fairly equal amount of (³⁵S) cysteine (approximately 40%). It is suggested that after a period of intragranular maturation neurophysin is extruded into an extragranular pool of neurosecretory material.With the use of conventional polyacrylamide-gel electrophoresis it was shown that the predominating proportion of radioactivity in the NSG after a hypothalamic injection of (³⁵S) cysteine was located within the neurophysin fraction A and in fraction B. Fraction B is suggested to be partly bound to the NSG membranes. When the NSG soluble and NSG insoluble proteins, obtained after lysis of NSG, were separated on polyacrylamide gels in the presence of sodium dodecylsulphate, the highly radioactive soluble protein was shown to consist of two components with average molecular weights of 12 300 and 14 600. Most of the proteins in the lysate were found in the NSG membranes, though less radioactive. A component with a mol.wt. of 37 000 was enriched in the membrane fraction. At longer times after isotope injection the high mol.wt. proteins, particularly those of the NSG membranes, contained increased amounts of radioactivity.Abbreviations NSG neurosecretory granule - NSM neurosecretory material - SON supraoptic nucleus The present investigation was supported by grants from Svenska Livförsäkringsbolags nämnd för medicinsk forskning from Svenska Sällskapet för Medicinsk forskning, from the Medical Faculty, University of Göteborg, and from the Swedish Medical Research Council (B 72-12X-2543-04A).We are indebted to Mrs. Marie-Louise Eskilsson, Mrs. Wally Holmberg and Mrs. Ulla Svedin for technical assistance, and to Miss Gull Grönstedt for careful secreterial work.     

Effect of Tetanus Toxin on Oxytocin and Vasopressin Release from Nerve Endings of the Neurohypophysis

The effect of tetanus toxin on neuropeptide hormone release from isolated nerve endings of the neural lobe of rat pituitaries (neurosecretosomes) was measured in a perfusion system. Tetanus toxin inhibited depolarization-evoked release of oxytocin and vasopressin in a time- and dose-dependent manner. At 1 microgram/ml, tetanus toxin blocked stimulated release by 85%. Tetanus toxin that was preincubated with a neutralizing monoclonal antibody or heated to 100 degrees C had no effect on hormone release. The ionophores A23187 and ionomycin were potent stimulators of hormone release in control nerve endings, but were not able to overcome the effect of tetanus toxin in intoxicated nerve endings. 8-Bromo-cyclic GMP, which has been reported to reverse the action of tetanus toxin in PC12 cells, had no effect on the action of tetanus toxin in neurosecretosomes. Neurosecretosomes are the first system in which tetanus toxin has been shown to block release from peptidergic nerve terminals. They appear to be a valuable in vitro system for studying the biochemical mechanism of tetanus toxin action.     

Morphometric classification of neurosecretory granules in the neurohypophysis of the hagfish,Eptatretus burgeri

Summary Neurosecretory axons in the neurohypophysis of the hagfish, Eptatretus burgeri, were statistically classified into six types according to the size of secretory granules. These types are comparable with those in higher vertebrates. The concentration of each axon type is different in three regions: anterior dorsal wall, posterior dorsal wall, and ventral wall. The regional differences of the hagfish neurohypophysis are discussed in relation to the regional differentiation of the tetrapod neurohypophysis into the median eminence and the pars nervosa.We wish to express our appreciation to Professor Hideshi Kobayashi for the guidance of this study. It was aided by grants from the Ford Foundation and the Ministry of Education to Professor H. Kobayashi, and also by grants from the Population Council, New York and the Ministry of Education to Professor S. Ishii.     

Cytochemical duality of neurosecretory material in the hypothalamo-posthypophysial system of the rat as related to hormonal content

Summary Cytochemical methods using silver proteinate, silver methenamine and potassium ferrocyanide + OsO₄ for ultrastructural detection of glycoproteins allow, in the posthypophysis and the magnocellular nuclei of the rat, differentiation of two types of fibres and neurons: one type containing negative granules with a homogeneous content of low electron density, the second type containing granules which demonstrate a ring-shaped deposit either of silver or of potassium ferrocyanide-osmium complex, likely to be related to a glycoproteic component. The difference between these two types is increased by prestaining en bloc with uranyl acetate before the silver proteinate reaction. A similar investigation was carried out on the vasopressin deficient Brattleboro rat; the neurosecretory material, present in some endings and neurones only, is of the nonreactive type, so that it appears justified to correlate the reactivity of granules with vasopressin, and consequently to distinguish neurones and fibres containing vasopressin from those in which oxytocin is quantitatively the main hormonal peptide. This conclusion is strongly supported by the fact that percentages of reactive and negative endings, as determined on this basis in the posthypophysis of normal rats from two different strains, are in good agreement with biochemical data reported in the literature.     

Ultrastructural organization of actin filaments in neurosecretory axons of the rat

Summary The ultrastructural organization of actin filaments was studied in the neurohypophysial system of the rat after heavy meromyosin (HMM) labeling. This structural pattern is characterized by (1) a straight arrangement of the filaments parallel to the axonal axis in the proximal nondilated parts of axons, (2) a central location within axonal dilatations, and (3) a higher concentration within axonal endings where the filaments form a complex three-dimensional network. The relationships of the filaments to other axonal structures and organelles was further studied by use of electron microscopic stereoscopy. The actin filaments frequently appear anchored to the axolemma with either polar arrangements of the arrowhead decoration (i) at structurally undifferentiated sites, and (ii) more particularly within perivascular endings, at sites with electron-dense thickenings. In all axonal divisions actin filaments are also found to bind to filamentous material surrounding the microtubules and to various organelles. Within the terminal portions of the axons actin filaments exhibit close relationships to neurosecretory granules and to the numerous smooth microvesicles found in this region. Such preferential relationships are particularly observed both in axon terminals and in pituicytes, with coated vesicles frequently binding actin filaments. In water-deprived rats, the concentration of actin filaments is conspicuously increased along the axons and more clearly in the axonal swellings and endings, where they form a more complex and interconnected network. These data are discussed in the light of a possible involvement of contractile proteins in the mechanisms of axonal transport and terminal release of neurosecretory products.     

Ultrastructural changes in neurohaemal tissue of the stick insect,Carausius morosus,induced by the ionophores Br-X-537 A and A-23187

Summary The antibiotic ionophores Br-X-537A and A-23187 alter the ultrastructure of neurohaemal tissue on the transverse nerve of the stick insect, Carausius morosus. Br-X-537A induces dramatic changes in the ultrastructural appearance of all three types of neurosecretory fibres present in the neurohaemal tissue. The neurosecretory granules become more electron-lucent and the mitochondria become more electron-opaque. The bounding membrane of the granules is frequently ruptured. A-23187, on the other hand, has no effect on two of the three types of fibres, but does produce an increase in the number of exocytotic profiles in the third.The two ionophores therefore have different effects on the same tissue. The results are discussed in the light of previous work with the use of these ionophores.We wish to thank Mrs. J. Birch for assistance with the electron micrographs, and Roche Products Ltd. and Lilly Research Centre Ltd. for gifts of the ionophores Br-X-537A and A-23187. The work was supported by the Science Research Council     

Isolation and Immunological Characterization of a Glycoprotein from Adrenal Chromaffin Granules

A glycoprotein (s-GP III) was isolated from the soluble lysate of chromaffin granules by chromatography with immunoaffinity and lectin columns. An identical protein (m-GP III) was shown to be present in the granule membranes. The apparent molecular weight of these glycoproteins as determined by the electrophoresis system of Laemmli (1970) was 43,000 under reducing conditions. In the absence of mercaptoethanol they aggregated to dimers. Antisera were raised against both the soluble and the membrane-bound forms of this glycoprotein. With these antisera GP III was further characterized: Immunoreplicas were obtained after two-dimensional electrophoresis of soluble and membrane-bound proteins of chromaffin granules. GP III was identified as a protein with a rather broad pI (4.6-5.3), indicating microheterogeneity. As shown by subcellular fractionation, m-GP III is specifically confined to chromaffin granules. GP III can therefore be used as a marker for the membranes of these organelles. The soluble form is secreted from adrenal medulla during stimulation with carbamylcholine chloride. An immunologically identical antigen was detected in adeno- and neurohypophysis. The physiological function of GP III is still unknown. It does not demonstrate any of the enzymatic activities so far known to occur in chromaffin granules.     

The ultrastructure of the developing neural lobe

Summary The ultrastructure of the developing neural lobe of rats was studied. The results revealed three periods in its development. The first period lasts till the 17th day of fetal life. At its beginning the anlage of the neural lobe is formed as a mass of cells very similar in appearance and in connection with the subependymal cells of the future median eminence. During the first period the cells of the anlage differentiate into pituicytes, and the penetration of the first nerve fibres and blood vessels among them is seen. The second period is from the 18th day of fetal life till one month after birth. At its beginning the first signs of neurosecretory activity were detected. During the period increasing numbers of neurosecretory fibres penetrate into the neural lobe, and the pituicytes show morphological signs characteristic of active cells. An increase in the functional activity of the neural lobe is also detected. The third period is from the end of the 1st till the end of the third month. During this period the development of the neural lobe proceeds and at the end it has the appearance of the adult gland. During this period the pituicytes gradually lose the signs of activity and at the end of the period they look like those observed in adult animals. Considering the results from the study, together with some data from previous investigations it is suggested that the pituicytes exert some stimulating and regulative influences on the process of neurosecretion in the neural lobe.Research fellow of the Alexander von Humboldt Foundation.     

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     

Localization of neurophysin in the rat supraoptic nucleus

Summary Localization of neurophysin in neurons of the supraoptic nucleus was accomplished using an unlabeled-antibody, post-embedding, immunoperoxidase technique. Neurophysin was exclusively associated with neurosecretory granules within cell bodies of supraoptic neurons and their processes.Supported by U.S.P.H.S. Grant HD-08867     

Newly Synthesized Cholecystokinin in Subcellular Fractions of the Rat Brain

Abstract: The subcellular localization of in vivo synthesized cholecystokinin (CCK) in different parts of the rat brain was studied after intracisternal pulse injections of [³⁵S]methionine. The rats were decapitated 1 h after the injection, and the brain was divided into cortex, hippocampus and remainder. Subcellular fractions were obtained according to Whittaker's method. De novo synthesized CCK in the crude mitochondrial-synaptosomal fraction, P₂, and in the purified synaptosomal fraction was demonstrated by affinity chromatography, using antibodies specific for the COOH-terminal sequence of CCK. By subsequent gel chromatography two molecular forms of labelled CCK occurred, with elution constants, K_av , of 1.1 (corresponding to the COOH-terminal octapeptide) and of 1.40 (a component which may correspond to the COOH-terminal tet-rapeptide amide, CCK-4). The findings support the idea that the small molecular forms are the transmitter forms of CCK.     

Calcium and sulphur in neurosecretory granules and calcium in mitochondria as determined by electron microscope X-ray microanalysis

Summary Sections of neurosecretory cells fixed in glutaraldehyde and osmium tetroxide were studied by means of an EMMA-4 analytical microscope. Secretory granules in neurosecretory cells of the corpus cardiacum and of the brain, both in the desert locust Schistocerca and in the blowfly Calliphora, as well as neurosecretory granules in posterior pituitaries of the frog Rana and of the albino rat all contain a high concentration of calcium. A distinct sulphur peak was also a constant feature.In neurosecretory cells of the corpus cardiacum of Schistocerca the chromatin contained a high concentration of calcium. The mitochondria also contained much calcium, but part of this disappeared during preparation except when fixative and wash contained calcium chloride. By block staining with uranyl acetate most calcium is displaced from the mitochondria, whereas most of the calcium remains in the neurosecretory granules. Since the calcium peaks in spectra from neurosecretory granules appear of similar size, regardless of variations in the preparative procedure, this calcium must be firmly bound. The possible role of the calcium bound to the neurosecretory substance is discussed.The presence of sulphur in insect neurosecretory granules indicates the presence of a protein besides the hormone, i.e., an insect neurophysin.We wish to thank Dennis Greer for the operation of the analytical microscope. This work was supported by a Wellcome-Carlsberg Travelling Research Fellowship awarded to T.N. The EMMA-4 facility is supported by a grant from the British Science Research Council     

Immunocytochemical localization of angiotensin-(1–7) in the rat forebrain

Recent findings by this group have led us to reconsider the view that amino (N−) terminal fragments of angiotensin (Ang) II are inactive degradation products of renin-angiotensin system. To further examine this possibility, an antibody to Ang-(1–7), the N-terminal heptapeptide, was produced to demonstrate the neuroanatomical distribution of the rat brain. Ang-(1–7)-immunoreactivity was found in paraventricular, supraoptic, and suprachiasmatic nuclei, bed nucleus of the stria terminalis, substantia innominata, median eminence, and neurohypophysis. This distribution of Ang-(1–7) in the rat forebrain, together with our previous demonstrations of vasopressin secretion in response to this peptide, suggest that Ang-(1–7) functions as a neuromodulator.     

Isolation and Characterization of Chromogranins A, B, and C from Bovine Chromaffin Granules and a Rat Pheochromocytoma

Bovine chromaffin granules from adrenal medulla contain three acidic secretory proteins: chromogranins A, B, and C. For isolation of these proteins, methods based mainly on high performance liquid chromatography were developed. After removal of contaminating glycoproteins by lectin affinity chromatography, chromogranins were separated by high performance anion-exchange, gel-filtration, and reverse phase liquid chromatography. As a final purification step sodium dodecyl sulfate-gel electrophoresis was performed. Amino acid analysis of isolated bovine chromogranins revealed a similar composition of all three proteins, with glutamic acid being the most prominent amino acid. The methods developed for bovine proteins also proved suitable for isolating rat chromogranins A and B from a transplantable pheochromocytoma. Chromogranin C was not present in sufficient amounts to be isolated from this tissue. The chromogranins purified by these methods were used to raise specific antibodies in rabbits. The use of purified chromogranins together with specific antisera may be valuable in understanding the still undiscovered function of these proteins.     

RNA解旋酶和应激颗粒

应激颗粒（stress granules, SGs）是细胞在环境压力刺激下停止蛋白质翻译后,mRNA与多种细胞蛋白组装而成的胞质颗粒结构.RNA 解旋酶家族作为生物体内普遍存在的一类高度保守的蛋白质酶类,参与了RNA代谢各个环节,近年来其家族成员被陆续发现是一类新的SG重要组分.本文综述了RNA解旋酶参与应激颗粒形成过程,RNA解旋酶家族蛋白的结构和其参与应激颗粒形成的研究进展.     

The distribution of pancreatic polypeptide in the nervous system and gut of the blowfly,Calliphora vomitoria (Diptera)

Summary The distribution of a neuropeptide, previously shown to have the same or a very similar amino acid composition as vertebrate pancreatic polypeptide (PP), has been studied in the nervous system and gut of the blowfly, Calliphora vomitoria. Neurones immunoreactive to a bovine PP antiserum occur in the thoracic and abdominal ganglionic components of the central nervous system, in addition to the brain and suboesophageal ganglion. Pancreatic polypeptide appears to be relayed from its cells of origin to a neurohaemal organ in the dorsal sheath of the thoracic ganglion. PP immunoreactivity is also found in cells of the hypocerebral ganglion of the stomatogastric nervous system and in associated nerve fibres. The mid-gut contains PP-positive material in flask-shaped cells of its epithelial lining.