Noradrenaline Storage Vesicles Research Articles

Changes in sympathetic nerve terminals in the heart of cold-exposed rats.

Changes in sympathetic nerve terminals of the heart after varying periods of exposure of rats to 4 degrees C were investigated. Two indices were used for changes in the number of noradrenaline storage vesicles, i.e., vesicular dopamine beta-hydroxylase (DBH) activity and noradrenaline storage capacity. The latter was obtained after uptake of [3H]noradrenaline; endogenous content, uptake of exogenous noradrenaline, and degree of saturation of the vesicles were calculated using the specific activity of the [3H]noradrenaline. As a measure of tyrosine hydroxylase activity, whole ventricular noradrenaline, dopamine, and dihydroxyphenylacetic acid content were used. After 4 h of cold exposure there was an increase in vesicular endogenous noradrenaline content, uptake, storage capacity, and DBH activity as well as a large increase in whole ventricular dopamine. After 6 h in the cold, vesicular endogenous noradrenaline content, storage capacity, and DBH activity were decreased. The results suggest that during cold exposure there is an initial increase followed by a decrease in the number of functional vesicles in the nerve terminal, which could explain the fluctuations in the rate of noradrenaline release.

Uptake of 5-hydroxytryptamine in rat isolated atria

1. 1. The mode of 5-hydroxytryptamine (5-HT) uptake by the rat isolated atria was studied and compared to noradrenaline (NA) uptake. 2. 2. Rat isolated atria were incubated with 14C-5-HT (46 μM) or 3H-NA (0.4 μM). After washing, the radioactivity fixed in atria was counted and the total NA, 5-HT and 5-hydroxyindol-3-acetic acid (5-HIAA) atria contents were measured by HPLC. 3. 3. 14C-5-HT uptake was reduced in atria from 6-hydroxydopamine (100 mg/kg, i.p., 48 hr before experiments) or reserpine (2.5 mg/kg, i.p., 24 and 48 hr before experiments) pretreated rats. 4. 4. The incubation of atria with 5-HT (50 μM) at the same time as 3H-NA reduced the 3H-NA value fixed. 5. 5. Addition of desipramine (1 μM) or hydrocortisone (150 μM) before the incubation of atria with 14C-5-HT was without any effect on 14C-5-HT uptake. In contrast, fluvoxamine (1 μM) or indalpine (5 μM) caused a slight inhibition. 6. 6. These data indicate that 5-HT is taken into the NA storage vesicles within the atria sympathetic nerves. This uptake does not use the NA carrier and involves partly the 5-HT carrier. An extraneuronal accumulation was noticed and a part of it is intracellular.

An Improved Method for the Purification of “Light” Noradrenaline Vesicles from Rat Vas Deferens: Some Biochemical Characteristics

"Light" noradrenaline storage vesicles from nerve endings have been isolated by differential centrifugation and differential gradient centrifugation. They have been further purified by isopycnic sucrose/D2O centrifugation. By using these centrifugation techniques, we obtained an isopycnic gradient fraction in which noradrenaline was enriched about 41 times versus a total homogenate. This factor could be raised to 61 by using seminal ducts of castrated rats. Comparison of the distribution patterns in sucrose/D2O isopycnic gradients indicated that light noradrenaline vesicles of nerve endings contain Mg2+-stimulated ATPase and ATP, but that only a minor part of the dopamine beta-hydroxylase can be associated with these vesicles.

Vesicular noradrenaline stores in peripheral nerves of the rat and their modification by tranylcypromine.

1 Vesicular noradrenaline stores were compared in the heart, salivary gland and vas deferens of the rat. 2 Noradrenaline storage vesicles in nerve terminals of different organs differed with respect to the amount of noradrenaline they contain in the endogenous store (content), the amount of exogenous noradrenaline they can take up from the circulation (uptake) and the amount of noradrenaline they contain when they are saturated (total storage capacity). 3 The data suggest that the vesicles in the salivary gland and vas deferens are almost completely filled with transmitter while, in the heart, the vesicular store is filled to only 55% of its total capacity. 4 The monoamine oxidase inhibitor, tranylcypromine, was found to increase not only the size of the endogenous store but also the size of the unfilled store.

Recycling of noradrenergic storage vesicles of isolated rat vas deferens

Del Castillo and Katz established the theory of quantal release of acetylcholine from the neuromuscular junction1. The concept was expanded by showing that quanta of transmitter was packaged in, and released from, storage vesicles of the nerve endings. Noradrenaline (NA) is also believed to be stored in, and released from, storage vesicles of the sympathetic nerves. The requirement of Ca for NA release2 and concomitant secretion of the vesicular enzyme, dopamine-β-hydroxylase3, on stimulation lends strong evidence to the theory of exocytosis. However, it remains unclear whether storage vesicles after fusing with neuronal membrane to release NA into cleft, are once again reused for further storage and release of NA, or are discarded by the nerve endings. I have investigated this question by making use of the fact that tetraethylammonium (TEA) causes profound facilitation of the overflow of 3H-NA when the sympathetic nerves of the isolated vas def erens are electrically excited4, and that this excess release is accompanied by a significant reduction in tissue NA content5. The principal advantage of the method is that the reduction in tissue NA content is effected exclusively by the exocytotic release of NA from the nerve terminals and easy washout of TEA allows one to study uptake of exogenous NA by the tissue. The results presented here support the hypothesis that NA storage vesicles are recycled.

Improved isolation of small noradrenergic vesicles from rat seminal ducts following castration. A density gradient centrifugation and morphological study

Noradrenaline storage vesicles from nerve endings have been prepared by differential and density gradient centrifugation. Seminal ducts from castrated rats have been used as a source since fluorescence micrographs and electron microscopy demonstrated an increased concentration of sympathetic nerve endings in this tissue; probably as a result of muscle atrophy. The fractions obtained were analyzed biochemically and partly by electron microscopy. By centrifugation techniques, a density gradient fraction (at the density of 0.4–0.45 m sucrose) was obtained in which noradrenaline was enriched about 10-fold from the homogenate. By using organs from castrated rats a further 4-fold increase was obtained in the noradrenaline/protein ratio of the density gradient fraction. This gradient fraction was studied by electron microscopy, after incubation with 5-hydroxydopamine as an exogenous marker, and was found to contain a high proportion of granular vesicle profiles. More than ⅓ of all vesicles with a diameter below about 1000A˚had an electron dense core. The vesicle preparation contained low activities of mitochondrial (cytochrome oxidase) and lysosomal (acid phosphatase) marker enzymes, while microsomes and plasma membrane fragments as indicated by NADH-cytochrome c reductase and adenosine triphosphatase had only been partially removed and probably constituted the main contaminants. The distribution of dopamine β-hydroxylase activity suggested that the small, light dense cored vesicles are deficient of this enzyme compared with the large, heavy dense cored vesicles, assumed to occur at the bottom of the gradient. The noradrenaline/adenosine 5'-triphosphate molar ratio was found to be 5.9 in the main noradrenaline fraction, but this fraction was not sufficiently pure to draw any definite conclusions about whether or not noradrenaline is stored together with adenosine 5'-triphosphate in small noradrenergic vesicles.

Changes in noradrenaline and its synthesizing enzymes in the nucleus locus ceruleus after intraventricular administration of colchicine and vinblastin

Concentrations of noradrenaline and its synthesizing enzymes were measured in the locus ceruleus of rats after intraventricular injection of colchicine and vinblastin to determine whether or not neurotubules are involved in the axonal transport of noradrenaline storage vesicles in the central nervous system. Significant increases in the noradrenaline concentration and dopamine-β-hydroxylase activity were evident after 1 day. This together with other available evidence suggested a close relationship between the neurotubules and axonal transport. An increase in tyrosine hydroxylase activity was observed 2 to 4 days after colchicine or, in some cases, vinblastin administration, but a concomitant increase in adrenal tyrosine hydroxylase activity suggests that this increase is due not to the blockage of axonal flow, but to the increased synthesis of enzyme protein in response to stress. On the other hand, the dopamine-β-hydroxylase concentration was later reduced after initial accumulation and marked morphologic changes (chromatolysis) were concomitantly observed. Two possibilities for these changes are discussed.

Effect of chronic denervation on the activities of monoamine oxidase and catechol-O-methyl transferase and on the contents of noradrenaline and adenosine triphosphate in the rabbit ear artery.

When assayed on homogenates of the rabbit ear artery up to 192 h after sympathetic denervation, the activity of monoamine oxidase (MAO) showed a small but significant decrease (maximum 9%). The activity of catechol-O-methyl transferase (COMT) appeared unchanged, although it is possible that the small and variable nature of this enzyme activity compared with that of MAO may have masked a comparable effect of denervation. The maximum decreases in noradrenaline (NA) and adenosine triphosphate (ATP) contents were greater than 90% at 48 h, and 32% at 96 h after denervation. These results confirm the presence of neuronal as well as extra-neuronal MAO, and also the presence of extraneuronal comt in this artery. The decline in ATP contents after denervation suggest that part of the ATP is neuronal. However, the estimated ratio of NA to neuronal ATP is far in excess of that reported for isolated noradrenaline storage vesicles, suggesting that the major portion of the neuronal ATP was not present in these vesicles.

INFLUENCE OF COLD EXPOSURE ON THE ACTIVITIES OF SOLUBLE AND MEMBRANE‐BOUND DOPAMINE β‐HYDROXYLASE IN RAT HEART VESICLES

Abstract— A fraction containing noradrenaline storage vesicles of the sympathetic nerve terminals in the rat heart was obtained by differential centrifugation. In this preparation, 17% of the dopamine β‐hydroxylase was present in a soluble form. Cold exposure (3°C) for periods from 5 to 30 min led to an increase in the activity of soluble dopamine β‐hydroxylase by about 50%, while the activity of membrane‐bound dopamine β‐hydroxylase was simultaneously decreased by approx 30%. The nor‐adrenaline content of the vesicles rose concomitantly with the increase in the activity of soluble dopamine β‐hydroxylase. This rise in noradrenaline content was caused by an enhanced synthesis and not by an alteration in the subcellular distribution. The results are discussed with respect to the fate of dopamine β‐hydroxylase during enhanced sympathetic nerve activity.

Fate of noradrenaline storage vesicles after release

Using [ 3H]noradrenaline (NA), changes in vesicular NA content and storage capacity of rat heart ventricles resulting from 24 h cold stress have been measured. NA content was decreased to 63% of control value; uptake of [ 3H]NA was the same in control and cold treated animals, while NA storage capacity was decreased to 78% of control value. This suggests that vesicles are not immediately available for re-use after NA release. After cold stress the degree of saturation of the vesicular storage capacity was reduced to 78% of control value, suggesting a change in the balance of NA synthesis and storage.

Pharmacological and ultrastructural studies on the electron dense cores of the vesicles that accumulate in noradrenergic axons constricted in vitro

The noradrenaline storage vesicles that accumulate in axons proximal to ligatures applied to cat hypogastric nerves incubated in vitro have been examined in the electron microscope with different fixation and staining procedures after exposure of the nerves to a variety of drugs which alter their noradrenaline content. After glutaraldehyde/osmium tetroxide fixation and lead citrate staining the granules possess electron-dense cores irrespective of their noradrenaline content. After fixation with Palade's osmium tetroxide or glutaraldehyde/dichromate followed by lead citrate staining, dense cores are not seen after treatment with noradrenaline-depleting agents but reappear after staining additionally with uranyl acetate. It is concluded that the matrix of axonal (large) noradrenaline storage vesicles contains some material in addition to catecholamines and this possibility is discussed in connection with the relationship between large and small dense-cored vesicles seen in sympathetic nerve endings.

A correlation between the effects of anti-mitotic drugs on microtubule assembly in vitro and the inhibition of axonal transport in noradrenergic neurones.

1. Podophyllotoxin, colchicine and griseofulvin inhibit the intra-axonal movement of noradrenaline storage vesicles in cat hypogastric nerve/inferior mesenteric ganglion preparations maintained in vitro, cause the disappearance of axonal microtubules and inhibit the assembly of microtubules from tubulin in vitro. The order of potency of the three effects is podophyllotoxin greater than colchicine greater than griseofulvin. 2. Lumicolchicine is without effect on the three parameters and does not interfere with the binding of tritiated colchicine to tubulin. 3. Podophyllotoxin causes a more rapid loss of microtubules from axons than the same concentration of colchicine. 4. The experiments provide strong evidence that microtubules are components of the system responsible for the intra-axonal migration of noradrenaline storage vesicles.

On the mechanism of the accumulation of 3H-bretylium in peripheral sympathetic nerves.

The uptake of bretylium-(N-3H-methyl) iodide in the rat heart atrium in vitro was examined. The uptake was linear for at least 1 hr and was strongly inhibited by (+)-amphetamine, (-)-noradrenaline, desipramine, cocaine, DSP 4 [N-(2-bromobenzyl)-N-(2-chloroethyl)ethylamine hydrochloride], guanethidine and ouabain. The amphetamine sensitive part of the uptake was almost completely abolished by pre-treatment of the rats with 6-hydroxydopamine and was dependent on the presence of sodium ions. Reserpine had no effect. (+)-Amphetamine but not desipramine caused an increase of the efflux of bretylium from the tissue. The apparent Km value of the active bretylium uptake was 3 x 10(-6) M, which was 10 times higher than that of the uptake of (-)-noradrenaline in the rat heart atrium (Km = 3 x 10(-7) M). The inhibition constants (Ki) for bretylium in inhibiting the noradrenaline uptake and for (-)-noradrenaline in inhibiting the bretylium uptake were 7 x 10(-6) M and 4 x 10(-7) M, respectively. The results obtained support the hypothesis that bretylium is taken up by the same mechanism as that carrying noradrenaline into the nerve terminals but is not bound in the noradrenaline storage vesicles.

Effects of low temperatures on microtubules in the non-myelinated axons of post-ganglionic sympathetic nerves

The effect of temperature changes on the number of microtubules in non-myelinated axons has been studied in cat inferior mesenteric ganglion/hypogastric nerve preparations incubated at various temperatures in Eagles minimal essential tissue culture medium in vitro. At 37°C the non-myelinated axons contained 28.4 ± 0.8S.E.M. (54) microtubules per axon. After incubation at 0°C for 4 h this number fell to 2.3 ± 0.1S.E.M. (41) but returned to normal levels when the nerves were rewarmed. This loss of microtubules on cooling the nerves and their reappearance on rewarming was a rapid process; it was independent of the influence of the neuronal cell body and of protein synthesis within the axon. The preservation of the microtubules was improved when D 2O was present in the incubation medium. Reformed microtubules appeared to function normally with respect to their possible role in the transport of noradrenaline storage vesicles along the axons.

Metabolic and ionic requirements for the axoplasmic transport of dopamine beta-hydroxylase.

1. Metabolic and ionic requirements for the intra-axonal transport of dopamine beta-hydroxylase (DBH) were investigated in the cat hypogastric nerve-inferior mesenteric ganglion preparation in vitro by monitoring the enzyme accumulation above a crush, 2-2.5 cm distal to the ganglion.2. DBH accumulation in the proximal segment immediately above the crush increased linearly up to 6 h, during incubation in normal Krebs solution at 37 degrees C. The rate of transport of the enzyme was about 4 mm/h.3. Removal of the ganglion, electrical stimulation or reserpine pretreatment (1-6 days before the experiment) did not modify the rate of DBH accumulation.4. Anoxia and glucose deprivation, singly, did not affect accumulation of DBH; however, the combined treatment of anoxia plus glucose deprivation, or dinitrophenol plus glucose deprivation, very markedly interfered with accumulation.5. Removal of sodium or potassium from Krebs solution markedly inhibited the transport of DBH. Preincubation of the nerve in a high-calcium Krebs solution at 4 degrees C, and then reincubation at 37 degrees C, prevented the enzyme accumulation.6. N-ethylmaleimide, ouabain and oligomycin markedly inhibited the transport of DBH.7. The results suggest that transport of DBH, and therefore of noradrenaline storage vesicles, within the hypogastic nerve is dependent on metabolic energy derived from either glycolysis or oxidative phosphorylation. It is also suggested that the sodium-potassium-activated ATPase may play an important role in the intra-axonal transport of storage vesicles.

Metabolic aspects of the synthesis and intra-axonal transport of noradrenaline storage vesicles.

1. Constricted cat hypogastric nerve/inferior mesenteric ganglion preparations have been maintained for 24 hr in vitro, in either test-tubes or a two compartment box, and used to study the roles of energy metabolism, protein synthesis and calcium ions in the synthesis and transport of noradrenaline storage vesicles in sympathetic neurones.2. When preparations were incubated so that the nerve trunks but not the ganglia were made anoxic, dense-cored vesicles lost their store of noradrenaline and did not accumulate above the ligature. This was accompanied by profound ultrastructural damage to the nerves.3. When in contact with the ligated nerve trunks, sodium fluoride inhibited the intra-axonal movement of dense-cord vesicles but did not deplete them of their stored noradrenaline. When sodium fluoride and pyruvate were present in the medium bathing the nerve trunks the movement of noradrenaline storage vesicles was unaffected.4. Whilst a reduction of the calcium ion concentration in the incubation medium and the inhibition of protein synthesis by cycloheximide prevented the synthesis of dense-cored vesicles in the neuronal perikaryon they had no effect on the movement of preformed dense-cored vesicles along non-myelinated axons.

Evidence for the presence of dopamine- -hydroxylase in both populations of noradrenaline storage vesicles in sympathetic nerve terminals of the rat vas deferens.

Journal of NeurochemistryVolume 20, Issue 1 p. 245-248 Evidence for the presence of dopamine-β-hydroxylase in both populations of noradrenaline storage vesicles in sympathetic nerve terminals of the rat vas deferens M. A. Bisby, M. A. Bisby University Laboratory of Physiology and Department of Pharmacology, OxfordSearch for more papers by this authorM. Fillenz, M. Fillenz University Laboratory of Physiology and Department of Pharmacology, OxfordSearch for more papers by this authorA. D. Smith, A. D. Smith University Laboratory of Physiology and Department of Pharmacology, OxfordSearch for more papers by this author M. A. Bisby, M. A. Bisby University Laboratory of Physiology and Department of Pharmacology, OxfordSearch for more papers by this authorM. Fillenz, M. Fillenz University Laboratory of Physiology and Department of Pharmacology, OxfordSearch for more papers by this authorA. D. Smith, A. D. Smith University Laboratory of Physiology and Department of Pharmacology, OxfordSearch for more papers by this author First published: January 1973 https://doi.org/10.1111/j.1471-4159.1973.tb12122.xCitations: 51 Department of Neurosciences, McMaster University, Hamilton, Ontario. Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume20, Issue1January 1973Pages 245-248 RelatedInformation

ATP hydrolysis and noradrenaline transport in purified vesicles from splenic nerve trunk.

AbstractA fraction of highly purified noradrenaline (NA) storage vesicles isolated from bovine splenic nerve trunk was tested for ATP hydrolytic activity. At 37.5† C and pH 7.2, the specific activity of Mg++‐ and Ca++‐activated hydrolyses were 0.22 and 0.15 μM P1/mg protein/min, respectively. There was no hydrolysis in the absence of divalent cations. The additions of various combinations of Na+ and K+ salts had little effect. Concentrations of NA and dopamine between 0.1 and 10.0 μg/ml had no effect. A concentration of 5×10‐8 M reserpine, which produced 30–50% inhibition of NA exchangeability and influx rate, had no effect on ATP hydrolysis under a wide variety of conditions. This concentration of drug causes no obvious evidence of vesicle damage at the electron microscopic level. The data do not support the possibility of a link between ATP hydrolysis and NA transport in either direction across the membrane of the isolated nerve trunk vesicle.

Studies on the translocation of noradrenaline-containing vesicles in post-ganglionic sympathetic neurones in vitro. Inhibition of movement by colchicine and vinblastine and evidence for the involvement of axonal microtubules.

1. Methods are presented for studying axonal transport mechanisms in preparations of constricted hypogastric nerve/inferior mesenteric ganglia maintained in vitro for periods up to 48 hr. Under these conditions the ultrastructure of the tissue is excellently preserved.2. The proximo-distal movement of noradrenaline and noradrenaline storage vesicles in the non-myelinated axons of these preparations is inhibited by both colchicine (10 mug/ml.) and vinblastine sulphate (1 mug/ml.) whilst the movement of mitochondria appears to be unaffected.3. Neither colchicine nor vinblastine sulphate depletes accumulated dense-cored vesicles of their stores of noradrenaline.4. These drugs reduce the accumulation of noradrenaline and densecored vesicles against a constriction when they are in contact with the nerve trunks only, and are denied direct access to the nerve cell bodies in the ganglion.5. The only other morphological change that can be attributed to the action of colchicine and vinblastine is a marked reduction in the number of axonal microtubules.6. The experiments provide strong support for the view that the axonal system of microtubules is closely involved in the proximo-distal movement of noradrenaline storage vesicles within noradrenergic neurones. The microtubular system does not appear to be involved in mitochondrial movement.

Electron carriers of the noradrenaline storage vesicles from bovine splenic nerves

The spectral properties of the noradrenaline storage vesicles of bovine splenic nerves are essentially the same as those recently reported for the adrenal chromaffin granules (T. Flatmark, O. Terland and K.B. Helle, Biochim. Biophys. Acta, 226 (1971) 9) indicating the presence of flavoprotein(s) and a high-potential b-type cytochrome termed cytochrome b 561. The specific activities of NADH : (acceptor) oxidoreductase (EC 1.6.99.3) were higher than those previously found in the adrenal granules. Based on the assay of these enzymic activities, the flavoprotein(s) also appear to be more stable in the nerve granules than in the adrenal granules upon prolonged storage of the organelles at 0 c.