[125I]MIBG uptake and release in different regions of the rat brain

Radioiodinated m-iodobenzylguanidine ([¹²⁵I]MIBG) and tritiated norepinephrine ([³H]NE]) uptake and release were compared, in different regions of the brain of the rat. The classification of the regions according to uptake was the same for both tracers: striatum > hypothalamus > hippocampus > cortex > brainstem. Tetrabenazine (TBZ), a granular monoamine uptake inhibitor reduced the uptake in the different regions. The inhibition rate was higher for [³H]NE uptake than for [¹²⁵I]MIBG. The spontaneous release was the same for [¹²⁵I]MIBG and [³H]NE and was the lowest in the striatum. The K⁺ stimulated release of [³H]NE was more complete than the release of [¹²⁵I]MIBG and was the most important in the striatum. From these results, it is inferred that MIBG enters the brain tissue via NE uptake mechanisms. It appears that MIBG is stored in the chromaffin granules, as NE, but also in the cytoplasm. A modified molecule derived from MIBG which would cross the blood-brain barrier, would then appear as a potential scintigraphic marker of monoamine uptake, storage and release.     

Release of norepinephrine from the cat ovary: changes after ovulation.

The distribution of intraneuronal constituents involved in norepinephrine (NE) storage, uptake, and release were used to estimate changes in NE secretion from the cat ovary after ovulation induced with eCG plus hCG. The content of NE and ATP, which are principally stored in small noradrenergic vesicles (isolated at a density of 1.041 g/ml in Percoll gradient), decreased after ovulation. However, the activity of dopamine beta-hydroxylase, which is principally associated with large noradrenergic vesicles (isolated at a density of 1.033 g/ml in Percoll gradient), was only slightly decreased. Mg(2+)-dependent ATPase, located in both large and small storage vesicles, decreased only in the small storage vesicles, suggesting that preferential secretion from small noradrenergic vesicles occurred. The hormonal treatment also affected the functional capacity of the vesicles, as evidenced by the decrease in uptake and storage capacity as well as the decrease in the stimulated release of 3H-NE observed after ovulation. The aforementioned changes are characteristically seen after a sympathetic discharge; thus they strongly support the notion that ovarian sympathetic activity increases during the ovulatory process, resulting in the postovulatory decrease in both the size and functional capacity of the intraneuronal compartment where NE is stored.     

KCl stimulation increases norepinephrine transporter function in PC12 cells

The norepinephrine transporter (NET) plays a pivotal role in terminating noradrenergic signaling and conserving norepinephrine (NE) through the process of re-uptake. Recent evidence suggests a close association between NE release and regulation of NET function. The present study evaluated the relationship between release and uptake, and the cellular mechanisms that govern these processes. KCl stimulation of PC12 cells robustly increased [3H]NE uptake via the NET and simultaneously increased [3H]NE release. KCl-stimulated increases in uptake and release were dependent on Ca2+. Treatment of cells with phorbol-12-myristate-13-acetate (PMA) or okadaic acid decreased [3H]NE uptake but did not block KCl-stimulated increases in [3H]NE uptake. In contrast, PMA increased [3H]NE release and augmented KCl-stimulated release, while okadaic acid had no effects on release. Inhibition of Ca2+-activated signaling cascades with KN93 (a Ca2+ calmodulin-dependent kinase inhibitor), or ML7 and ML9 (myosin light chain kinase inhibitors), reduced [3H]NE uptake and blocked KCl-stimulated increases in uptake. In contrast, KN93, ML7 and ML9 had no effect on KCl-stimulated [3H]NE release. KCl-stimulated increases in [3H]NE uptake were independent of transporter trafficking to the plasma membrane. While increases in both NE release and uptake mediated by KCl stimulation require Ca2+, different intracellular mechanisms mediate these two events.     

Correlation of Rates of Calcium Entry and Release of Endogenous Norepinephrine in Rat Brain Region Synaptosomes

Voltage-dependent 45Ca2+ uptake and endogenous norepinephrine (NE) release were measured simultaneously in synaptosomes isolated from rat hypothalamus, brainstem, and cerebellum at 1, 3, 5, 15, and 30 s. In synaptosomes depolarized by 125 mM KCl, 45Ca2+ uptake and NE release exhibited fast and slow components. Rates of NE release and 45Ca2+ uptake were fastest from 0 to 1 s. NE release and 45Ca2+ uptake rates from 1 to 5 s were less than 15% of 0-1 s rates. Both resting (5 mM KCl) and depolarization-induced (125 mM KCl) NE release paralleled 45Ca2+ uptake from 1 to 30 s. Voltage-dependent NE release was approximately 1% and 2% of total synaptosomal NE content at 1- and 30-s measurement intervals, respectively, and did not differ between the three brain regions studied. Calcium and potassium dependence studies showed that NE release was stimulated by increased potassium and that depolarization-induced NE release was dependent on the presence of external calcium. These results show that calcium-dependent NE release from synaptosomes is correlated with calcium entry. Both processes exhibit fast and slow temporal components.     

β-NEUROTOXIN REDUCES NEUROTRANSMITTER STORAGE IN BRAIN SYNAPSES

β-neurotoxin, a component of Bungarus multicinctus venom, is known to cause neuromuscular blockade by first increasing the rate of spontaneous ACh release and then inhibiting the nerve impulse-induced release of ACh. We report that the toxin also affects the storage of several neurotransmitters in rat brain and it is active on synaptosomes and brain minces. Synaptosomes prepared from brain tissue that had been treated with β-toxin have a reduced ability to accumulate radioactive NE, GABA, serotonin and the ACh precursor, choline. The toxin also causes a release of previously accumulated NE and GABA from synaptosomes, suggesting that the storage process rather than the uptake transport process is affected. The toxin does not contain phospholipase A, phospholipase C, protease or hyaluronidase activity.     

Presynaptic Mechanisms Underlying the γ-Aminobutyric Acid-Evoked Receptor-Independent Release of [3H]Norepinephrine in Rat Hippocampus

The effects of gamma-aminobutyric acid (GABA) on the spontaneous efflux of [3H]norepinephrine ([3H]NE) were studied in synaptosomes prepared from rat hippocampus and prelabelled with [3H]NE. It had been observed previously that, when synaptosomes were exposed in superfusion to GABA, the basal release of the tritiated catecholamine was enhanced, apparently with no involvement of the known GABA receptors. The mechanisms underlying this effect have now been investigated. The potency of GABA as a releaser of [3H]NE was decreased by lowering the Na+ content of the superfusion medium, and its effect disappeared at 23 mM Na+. The GABA-induced [3H]NE release was counteracted by the GABA uptake inhibitor N-(4,4-diphenyl-3-butenyl)nipecotic acid (SKF 89976A), but it was unaffected by the NE uptake blockers desmethylimipramine and nisoxetine. The GABA-induced release of [3H]NE was Ca2+-dependent and tetrodotoxin-sensitive. The data support the hypothesis that GABA provoked [3H]NE release by a novel mechanism which involves penetration into the noradrenergic nerve terminals through a GABA carrier located on the NE terminals themselves. This uptake process might be electrogenic and provoke depolarization of the nerve terminals, causing an exocytotic release of [3H]NE.     

LOCALIZATION OF TRITIATED NOREPINEPHRINE IN VASCULAR SYMPATHETIC AXONS OF THE RAT INTESTINE AND MESENTERY BY ELECTRON MICROSCOPE RADIOAUTOGRAPHY

The distribution of infused tritiated norepinephrine (NE-³H) in small mesenteric arteries and intestinal arterioles in rats was investigated with electron microscopic radioautography. Silver grains, indicating the presence of the tritium label on the sections, were found lying mainly over axon bundles, but some were present over collagen and smooth muscle cells. Axons with the highest concentrations of silver grains had been sectioned at points where they were naked of Schwann cell sheath, were dilated into varicosities, and contained small granular vesicles. This finding was taken as confirmatory circumstantial evidence that the small granular vesicles were the sites of uptake and storage of NE. The short interval between the start of infusion and the fixation of the tissue appeared to rule out any process other than a direct uptake of NE by the peripheral axons. If axonal sites of uptake of NE-³H correspond to sites of release of NE, then the evidence suggests that such sites of release are widespread over the terminal part of the axon and are not confined to those parts of the axon which are in close contact with smooth muscle cells. Since the fixation and embedding procedures will remove NE which is not strongly bound to tissues, the localization of NE-³H in the radioautographs does not necessarily correspond to the distribution of all the NE present in vivo.     

Reserpine sensitivity of catecholamine metabolism in murine neuroblastoma clone N1E-115.

—Neuroblastoma cells of clone NIE-115, originally obtained from the murine tumor C1300, resemble normal noradrenergic neurons in that they have high levels of tyrosine 3-monooxygenase (EC 1.14.16.2; l -tyrosine, tetrahydropteridine: oxygen oxidoreductase (3-hydroxylating)) and dopamine β-monooxygenase (EC 1.14.17.1; 3,4-dihydroxyphenylethylamine, ascorbate: oxygen oxidoreductase (β-hydroxylating)) activities, dense core versicles (100–300 nm in dia), long neurites and excitable membranes. These studies show that reserpine, a blocker of vesicular uptake in noradrenergic neurons, inhibits the accumulation and storage of catecholamines, as well as the conversion of dopamine to NE in neuroblastoma cells. Differentiated monolayer cultures took up [³H]dopamine [10⁻⁴] at a rate of 37 pmol/min per mg protein. Reserpine [5 × 10⁻⁵m ] did not affect the initial rate of uptake, but reduced the extent of uptake at saturation by 60%. Chromatographic examination of cell extracts showed that dopamine was converted to NE in control cultures, but not in reserpine treated cultures. Cells labelled with [³H]dopamine for 60 min and then exposed to release buffer without dopamine for an additional 60 min, retained approximately 40% of the label, 10% as dopamine and 30% as NE. Thirty-five per cent of the radioactivity retained was found, after homogenization and high speed centrifugation, to be associated with a particulate, subcellular fraction. Reserpine, present during release incubations, also reduced the ability of cells to store catecholamines. These results show that N1E-115 cells synthesize and store NE by reactions similar to those in normal noradrenergic neurons.     

The effects of ricin on the sympathetic vascular neuroeffector system of the rabbit

Ricin is a toxic lectin that inhibits protein synthesis. Because ricin decreases arterial pressure and causes cardiovascular collapse, its effects on the vascular neuroeffector system were investigated. Rabbits were given either of two doses of ricin, and then norepinephrine (NE) release from aorta to transmural stimulation, NE uptake into aorta, NE content of aorta, monoamine oxidase activity, and catechol-O-methyl transferase activity in aorta were determined 18 hours, 4 days or 7 days later. Norepinephrine uptake and enzyme activities in the aorta were not altered by ricin administration. Norepinephrine release and content of aorta were increased at most time periods following ricin administration, significantly so for NE content at 4 days and for release at 18 hours following the lower dose of ricin. We conclude that the mechanisms involved in the release of NE from sympathetic nerves in the vasculature are not impaired by ricin administration, but rather show changes that indicate increased compensatory activity.     

Reserpine-induced reduction in norepinephrine transporter function requires catecholamine storage vesicles

Treatment of rats with reserpine, an inhibitor of the vesicular monoamine transporter (VMAT), depletes norepinephrine (NE) and regulates NE transporter (NET) expression. The present study examined the molecular mechanisms involved in regulation of the NET by reserpine using cultured cells. Exposure of rat PC12 cells to reserpine for a period as short as 5 min decreased [³H]NE uptake capacity, an effect characterized by a robust decrease in the V_max of the transport of [³H]NE. As expected, reserpine did not displace the binding of [³H]nisoxetine from the NET in membrane homogenates. The potency of reserpine for reducing [³H]NE uptake was dramatically lower in SK-N-SH cells that have reduced storage capacity for catecholamines. Reserpine had no effect on [³H]NE uptake in HEK-293 cells transfected with the rat NET (293-hNET), cells that lack catecholamine storage vesicles. NET regulation by reserpine was independent of trafficking of the NET from the cell surface. Pre-exposure of cells to inhibitors of several intracellular signaling cascades known to regulate the NET, including Ca²⁺/Ca²⁺–calmodulin dependent kinase and protein kinases A, C and G, did not affect the ability of reserpine to reduce [³H]NE uptake. Treatment of PC12 cells with the catecholamine depleting agent, α-methyl-p-tyrosine, increased [³H]NE uptake and eliminated the inhibitory effects of reserpine on [³H]NE uptake. Reserpine non-competitively inhibits NET activity through a Ca²⁺-independent process that requires catecholamine storage vesicles, revealing a novel pharmacological method to modify NET function. Further characterization of the molecular nature of reserpine's action could lead to the development of alternative therapeutic strategies for treating disorders known to be benefitted by treatment with traditional competitive NET inhibitors.     

Norepinephrine release in the heart atria of diabetic rats

The content, release and uptake of norepinephrine (NE) in the sympathetic nerves of the rat heart atria were studied in the course of diabetes and in age-matched controls. Diabetes was induced by streptozotocin (STZ) and rats were subjected to further experiments 1, 4 or 7 months later (STZ1, STZ4, STZ7). Isolated atria were superfused with oxygenated Krebs-Henseleit (KH) solution. After equilibration, four 10-min fractions were collected: B1, basal release of NE; S1, potassium-evoked release (KER), where NE outflow was stimulated by depolarisation with 50 mmol/l KCl; B2, basal release of NE under the influence of the neuronal uptake blocker desipramine (DES); S2, KER under the influence of DES. The content of NE was measured by radioimmunoassay. In STZ4 and STZ7 rats, NE concentrations were significantly lower in both atria compared to controls. B1 and S1 were significantly higher in STZ4 than in control atria. DES increased KER of NE in controls only. In contrast, DES caused a significant decrease in B2 and S2 in STZ4 atria, suggesting that a substantial portion of NE release was due to a calcium-independent carrier-mediated process. In experiments with calcium-free KH solution in fractions B2 and S2, KER ill controls was nearly abolished. However, in STZ4 and STZ7 atria, S2 was still significantly higher than B2. In conclusion, the NE-releasing mechanism may be different in the chronically diabetic animals than in healthy subjects and may contribute to the decreased NE concentration in the STZ atria.     

Rapid changes in synaptic vesicle cytochemistry after depolarization of cultured cholinergic sympathetic neurons

Sympathetic neurons taken from rat superior cervical ganglia and grown in culture acquire cholinergic function under certain conditions. These cholinergic sympathetic neurons, however, retain a number of adrenergic properties, including the enzymes involved in the synthesis of norepinephrine (NE) and the storage of measurable amounts of NE. These neurons also retain a high affinity uptake system for NE; despite this, the majority of the synaptic vesicles remain clear even after incubation in catecholamines. The present study shows, however, that if these neurons are depolarized before incubation in catecholamine, the synaptic vesicles acquire dense cores indicative of amine storage. These manipulations are successful when cholinergic function is induced with either a medium that contains human placental serum and embryo extract or with heart-conditioned medium, and when the catecholamine is either NE or 5-hydroxydopamine. In some experiments, neurons are grown at low densities and shown to have cholinergic function by electrophysiological criteria. After incubation in NE, only 6% of the synaptic vesicles have dense cores. In contrast, similar neurons depolarized (80 mM K+) before incubation in catecholamine contain 82% dense-cored vesicles. These results are confirmed in network cultures where the percentage of dense-cored vesicles is increased 2.5 to 6.5 times by depolarizing the neurons before incubation with catecholamine. In both single neurons and in network cultures, the vesicle reloading is inhibited by reducing vesicle release during depolarization with an increased Mg++/Ca++ ratio or by blocking NE uptake either at the plasma membrane (desipramine) or at the vesicle membrane (reserpine). In addition, choline appears to play a competitive role because its presence during incubation in NE or after reloading results in decreased numbers of dense-cored vesicles. We conclude that the depolarization step preceding catecholamine incubation acts to empty the vesicles of acetylcholine, thus allowing them to reload with catecholamine. These data also suggest that the same vesicles may contain both neurotransmitters simultaneously.     

Catecholamines in human saliva.

Catecholamines are readily detectable in human saliva but their origin is unclear. Norepinephrine (NE) was stable in saliva stored at 4 degrees for 2 hours but 11 +/- 3% degraded after storage at 25 degrees for 1 hour. We intravenously infused 3H-NE into humans and measured levels of 3H-NE and its metabolites in both saliva and forearm venous plasma (a site whose plasma NE levels reflect both local uptake and release of NE). 3H-NE levels in saliva continued to rise for 1 hour even though forearm plasma levels had plateaued by 5 min. By 65 min into the infusion the ratio of 3H-NE:non-radioactive NE was similar in saliva and forearm venous plasma. The ratio of NE:epinephrine (E) was similar in saliva and forearm venous plasma at all time points. Chewing induced salivation, and at least tripled the amount of NE, E and 3H-NE released into saliva per minute, but decreased their concentration in saliva by as much as one half. Saliva NE level was unaltered after 15 min of standing but was increased by 31% after 1 hour of upright posture. Our data imply that the NE present in human saliva comes from both the bloodstream and from salivary sympathetic nerves. The finding that diffusion of blood NE into saliva takes roughly 1 hour to complete suggests that NE in saliva is a poor index of acute changes in sympathetic activity.     

Effect of noradrenaline on its own internal liberation from cortex synaptosomes in the rat]

Norepinephrine (NE) uptake velocity in rat cerebral cortex synaptosomes does not depend on internal NE contents: continuous exchange occurs. NE enhances spontaneous release and inhibits release elicited by KCl. Phenylephrine an a agonist, produces the same effect. Phentolamine, an a antagonist, did not modified the spontaneous release but enhanced release elicited by KCl 25 mM.     

Changes in noradrenergic vesicle markers of rabbit oviducts during progesterone treatment

The effect of progesterone (P) on norepinephrine (NE), [3H] norepinephrine ([3H]NE) and dopamine beta-hydroxylase (DBH) in noradrenergic vesicles from rabbit oviducts was studied after daily injections of the hormone during different periods (4, 7 and 15 days). Progesterone induced a concomitant increase in NE and DBH activity and [3H]NE uptake. To study the mechanism involved in such effects, 4 tissue fractions were obtained by differential centrifugation of the oviducts of which the vesicular fraction was applied over continuous sucrose gradients (0.3-2 M). The changes induced by P in markers of tissue and gradient fractions showed an increase of the NE storage capacity which could be ascribed to an increase in the number of storage vesicles, and/or to a higher extravesicular storage capacity. The occurrence of these mechanisms during pregnancy or after P treatment could account for the (long-lasting) high levels of NE observed in such instances.     

B and C Types Natriuretic Peptides Modify Norepinephrine Uptake and Release in the Rat Adrenal Medulla

Vatta, M. S., M. F. Presas, L. G. Bianciotti, M. Rodriguez–fermepin, R. Ambros and B. E. Fernandez. B and C types natriuretic peptides modify norepinephrine uptake and release in the rat adrenal medulla. Peptides 18(10) 1483–1489, 1997.—We have previously reported that atrial natriuretic factor (ANF) modulates adrenomedullar norepinephrine (NE) metabolism. On this basis, the aim of the present work was to study the effects of B and C types natriuretic peptides (BNP and CNP) on the uptake, intracellular distribution and release of ³H-NE. Experiments were carried out in rat adrenal medulla slices incubated “in vitro.” Results showed that 100 nM of both, CNP and BNP, enhanced total and neuronal NE uptake. Both peptides (100 nM) caused a rapid increase in NE uptake during the first minute, which was sustained for 60 min. NE intracellular distribution was only modified by CNP (100 nM), which increased the granular fraction and decreased the cytosolic pool. On the other hand, spontaneous as well as evoked (KCl) NE release, was decreased by BNP and CNP (50 and 100 nM for spontaneous release and 1, 10, 50 and 100 nM for evoked output). The present results suggest that BNP and CNP may regulate catecholamine secretion and modulate adrenomedullary biological actions mediated by catecholamines, such as blood arterial pressure, smooth muscle tone, and metabolic activities.     

Levonorgestrel and norethindrone alter insulin action on skeletal muscle of the female rat

Prospective studies of women receiving oral contraceptives suggest that the progestin component may induce insulin resistance and variable deterioration of glucose tolerance. Because the tissue sites and nature of this insulin antagonism are not well-defined, we studied the effects of two parenterally administered progestins, levonorgestrel (NG) and norethindrone (NE), on insulin-regulated glucose uptake and phenylalanine release by the perfused rat hindquarter. Female rats were injected sc for 14 days with NG or NE (10 or 30 micrograms/kg/day). Low-dose NG and high-dose NE approximate the per kg dose received by women taking a high-dose progestin oral contraceptive. Phenylalanine release and glucose uptake (nmole/min/g) by the perfused hindquarters were calculated from the A-V difference for each. Progestin treatment (30 micrograms/kg/d) significantly reduced phenylalanine release from hindquarters perfused without exogenous insulin. Hindquarters from the high dose NG and low and high dose NE rats perfused with insulin (100 microU/ml) released 22% less phenylalanine than control rats perfused with the same insulin concentration (P less than 0.01) but the net suppression below baseline was similar in the control and steroid-treated groups. High-dose progestin treatment did not alter glucose uptake by hindquarters perfused without exogenous insulin. Insulin (100 microU/ml) increased glucose uptake by hindquarters of control and progestin-treated rats as compared to animals in the same treatment group perfused without exogenous insulin (P less than 0.01). High dose NE impaired insulin-stimulated glucose uptake 24% below values of the control group (P less than 0.01). The other NE and NG doses had no effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

Potassium evoked catecholamine release from the nucleus tractus solitarius invitro

The release of endogenous catecholamines (CA) from rat brain slices containing the nucleus tractus solitarius (NTS) was measured using a sensitive radioenzymatic assay. KCl (35 to 75 mM) induced a dose-related increase in norepinephrine (NE) release. Dopamine (DA) release was maximal with 50 mM KCl. An increase in epinephrine (E) release was only observed with 75 mM KCl. NE and E release was totally calcium-dependent whereas DA release was only partially calcium-dependent. Subsequent administrations of KCl released less CA. The calcium dependency of the KCl induced release of E, NE, and DA suggests a neurotransmitter function in the NTS for these CA. A difference in storage sites and/or mechanisms may be responsible for the observed differences in sensitivity to KCl and to extracellular calcium.     

Compartmental analysis of the efflux of l-[3H]norepinephrine from isolated perfused rat lungs

Isolated rat lungs, pretreated with 100 microM pargyline and 100 microM U-0521 (3',4'-dihydroxy-2-methylpropriophenone) to block metabolism of norepinephrine (NE), were perfused with 0.3 microM 3H-labeled l-norepinephrine (1-[3H]-NE) for 30 min. Efflux samples were then collected for 30 min during washout of the tissue with amine-free Krebs solution. Compartmental analysis (nonlinear least-squares regression) of the efflux of tissue l-[3H]NE content vs. time indicates that NE is accumulated in a large slowly equilibrating compartment (t 1/2 = 58.15 +/- 6.84 min) in addition to distribution in the vascular (blue dextran tracer) and extracellular ([3H]sorbitol tracer) fluid compartments of the lung. Pretreatment of the lungs with 100 microM cocaine hydrochloride reduces the total l-[3H]NE space from 7.44 +/- 1.91 to 2.48 +/- 0.23 ml/g (P less than 0.05) by selectively decreasing the size of the slow NE compartment from 6.99 +/- 1.97 to 1.67 +/- 0.14 ml/g (P less than 0.05). The large size, cocaine sensitivity, and long efflux half time of this compartment suggest that neuronal uptake contributes to the pulmonary vascular inactivation of l-[3H]NE.