Newly synthesized norepinephrine accumulation in the readily releasable pool of a purified adrenergic vesicle fraction

A highly purified fraction of large dense core adrenergic vesicles was studied after isolation from bovine splenic nerve chilled within 10 to 12 minutes post mortem. In a standard medium containing 5 mM each of Mg⁺⁺ and ATP and 6 μM norepinephrine (NE), this vehicle fraction contained NE in a readily releasable and a more stable pool. When vesicle dopamine β-hydroxylase was activated with 1.33 mM ascorbic acid using 6 μM ¹⁴C-dopamine as substrate at 30°C, ¹⁴C-NE was synthesized at a linear rate during the 45 minute incubation. Net accumulation of NE (p < 0.01) and a proportional net retention of newly synthesized ¹⁴C-NE occurred only when the readily releasable pool could still be demonstrated. The halftime for the fast release pool was doubled from 3 to 6 minutes (p < 0.01) with no effect on the slower released, ATP-facilitated uptake pool. Thus, both during axoplasmic transport and induced NE synthesis $\text{in vitro}$, there is evidence that newly synthesized NE preferentially accumulates in the readily releasable pool, a property also characteristic of the physiologically active pool $\text{in vivo}$.     

Dopamine and norepinephrine stimulate somatostatin release by median eminence fragments invitro

The effect of catecholamines on somatostatin release by median eminence (ME) fragments was evaluated using an $\text{in}$$\text{vitro}$ incubation system. Adult male rats were used as tissue donors. Somatostatin release was readily detected during short-term incubations (10 and 30 minutes). Dopamine (DA) significantly stimulated somatostatin release during a 30 minute incubation period at the two doses tested (0.6 and 6 μM). Under similar conditions, norepinephrine (NE) stimulated somatostatin release only at the 6 μM dose. Using a shorter incubation period (10 min) and a 6 μM dose, only DA stimulated somatostatin release. The effects of DA and NE were specifically blocked by the $\text{in}$$\text{vitro}$ addition of pimozide or phentolamine, respectively, suggesting that dopaminergic and noradrenergic receptors may be present in the somatostatinergic terminals of the ME. The results indicate that both DA and NE may be involved in the regulation of somatostatin secretion.     

Release of endogenous norepinephrine and dopamine from rat brain regions in vitro

Using radioenzymatic assay procedures, we have measured picomolar amounts of endogenous norepinephrine (NE) and dopamine (DA) released $\text{in vitro}$. The release of NE and DA in response to KCl stimulation was examined in 6 brain regions: cortex, hippocampus, hypothalamus, striatum, combined accumbens-olfactory tubercle, and substantia nigra. NE release was detectable in all regions except striatum. Amounts of NE released by 55mM KCl (expressed as % control) were: cortex (313%), hippocampus (227%), hypothalamus (225%), accumbens-tubercle (278%), s. nigra (155%). KCl stimulated release of DA was detected in 3 regions: striatum (414%), accumbenstubercle (282%), and hypothalamus (312%). DA was measurable in filtrates from the s. nigra but levels in control and KCl stimulated samples were equal. Release of NE and DA was also measured in 12 brain regions after incubation of tissue $\text{in vitro}$ with 10^?4M d-amphetamine sulfate. d-Amphetamine stimulated NE outflow when compared to controls in all regions examined. DA outflow was markedly increased in most regions, especially striatum (287%), hypothalamus (387%) and accumbens-tubercle (670%). d-Amphetamine doubled endogenous DA outflow from the s. nigra.     

Effect of cell size, age and anatomical location on the lipolytic response of adipocytes

Studies were conducted on the norepinephrine (NE) stimulated lipolytic sensitivity of adipocytes from epididymal (Epi), perirenal (PR), subcutaneous (SC) and mesentric (M) depots from young (7–8 wk.) and adult (14–16 wk.) male rats. In the young rats dose response curves to NE were similar for Epi, PR and M depots whereas adipocytes from the SC depot showed a diminished effect over the mid-portion of the curve. This difference could not be ascribed to differences in cell size. In the adult rats glycerol release in the Epi depot in response to NE was identical to the younger rats which was in marked contrast to the other depots in which glycerol release was decreased in comparison to the younger animals. This decreased responsiveness was probably largely a result of age and not changes vn adipocyte size within a given depot. In these older rats, glycerol release was greatest in the Epi cells, least in the SC and M depots, and intermediate in PR. When young rats were subjected to a 72-hour fast, loss of triglyceride per cell was the same in all depots as predicted by the $\text{in vitro}$ data whereas in old rats (610 g), triglyceride loss was proportional to cell size with Epi ≥ PR > SC ≥ M. This was also essentially in agreement with the $\text{in vitro}$ lipolytic data from adult rats. These data demonstrate lipolytic differences between depots that are minimal in young rats and which are accentuated with age.     

Central noradrenergic activity and the formation of glycol sulfate metabolites of brain norepinephrine

The present study utilized intraventricular injection of Na₂³⁵SO₄ to detect drug induced changes in the $\text{in vivo}$ formation of the two major metabolites of rat brain norepinephrine (NE) - the sulfate conjugates of 3-methoxy-4-hydroxyphenylglycol (MOPEG-SO₄) and 3,4-dihyd (DOPEG-SO₄). Assays involved the hypothalamus only. Rats pretreated with clonidine showed a reduced formation of both MOPEG-³⁵SO₄ and DOPEG-³⁵SO₄ after intraventricular Na₂³⁵SO₄ as well as reduced synthesis of ³H-NE from intraventricular ³H-tyrosine. Phenoxybenzamine (POB) produced increases in the synthesis of both ³⁵S-labeled conjugates and ³H-NE. Neither drug altered the loss of exogenous ³H-MOPEG-SO₄ but clonidine increased both the accumulation of labeled sulfate and the sulfation of exogenous MOPEG in pheniprazine treated rats. These results show that the rates of formation of the labeled glycol sulfates are sensitive indicators of changes in brain NE turnover but can also be influenced by factors involved in sulfation that are unrelated to NE turnover. Blockade of NE synthesis with alpha methyltyrosine did not affect resting or POB-elevated levels of the labeled conjugates until stores of NE were reduced by 40%. The latter findings suggest that central noradrenergic neurons can release and metabolize NE at a normal rate despite synthesis blockade so long as adequate stores of NE are available.     

Oxaprotiline: Induction of central noradrenergic subsensitivity by its (+)-enantiomer

The effects of the tetracyclic antidepressant oxaprotiline and its two optically active enantiomers on the norepinephrine (NE) receptor coupled adenylate cyclase system were determined in slices of the rat cerebral cortex. While oxaprotiline does not change the response of the cyclic AMP generating system to NE after a single dose, chronic administration of the drug for 3 to 14 days down-regulates the receptor system. The noradrenergic subsensitivity is linked to a reduction in the B_max value of β-adrenergic receptors as assessed by (³H)-dihydroalprenolol binding without changes in the K_d value. The action of oxaprotiline on the NE receptor coupled adenylate cyclase system resides entirely in the (+)-enantiomer which is a potent inhibitor of the neuronal uptake of NE. The (?)-enantiomer of oxaprotiline which is a weak inhibitor of NE reuptake, failed, even in high doses, to modify the noradrenergic receptor system. Though not excluding co-regulatory factors in addition to NE, the studies support the view that an enhanced and persistent NE receptor interaction is one of the prerequisites for the $\text{in}$$\text{vivo}$ down-regulation of central noradrenergic receptor function. The results also suggest that the therapeutic activity of oxaprotiline may reside in its (+)-enantiomer.     

Actions of a nicotinic agonist,DMPP, on intestinal ion transport invitro

High-dose carbachol (10^?3 M) has previously been shown to cause NaCl absorption in short-circuited rabbit ileum. The mechanism of this effect may be norepinephrine release induced by carbachol activation of presynaptic nicotinic receptors on adrenergic neurons. Norepinephrine then interacts with postsynaptic α-adrenergic receptors on intestinal mucosal cells to stimulate neutral NaCl absorption and inhibit electrogenic bicarbonate secretion. The present paper examines the $\text{in vitro}$ intestinal ion transport effects of DMPP an agent which is more specific than carbachol on nicotinic cholinergic receptors. DMPP (10^?5 M) caused a transient increase followed by prolonged depression of the short-circuit current, increased NaCl absorption and increased tissue conductance. This effect was antagonized by hexamethonium and phentolamine. It is concluded that nicotinic cholinergic agents stimulate norepinephrine release from adrenergic nerves and effect intestinal ion transport just as norepinephrine does.     

Alterations in spinal cord norepinephrine levels following experimentally produced trauma in normal and adrenalectomized cats

Spinal cords of normal and adrenalectomized cats were subjected to experimentally produced blunt trauma and then analyzed for changes in concentration of norepinephrine (NE). In normal cats the NE concentrations at the lesion site rose 63% over control after one hour, and by $\text{1}\text{1}\text{2}$ to 2 hours had returned to control level. Little change in concentration was observed in cord segments 2 cm from injury site, but segments adjacent to the injury exhibited increased levels of NE. NE concentrations one hour after trauma in adrenalectomized cats were lower than those measured in normal controls. It is postulated that the mechanism of NE accumulation involves a breakdown of the blood-spinal cord barrier thereby allowing peripherally produced NE to enter the cord parenchyma from the systemic circulation.     

The uptake of (3H)dopamine by homogenates of rat corpus striatum: effects of cations

The uptake of [³H]dopamine was studied with a synaptosomal preparation of the corpus striatum. The accumulation of dopamine was found to be temperature-dependent and very rapid, but linear over time for at least 5 min. at 37°C with characteristics of saturable kinetics. The optimum concentrations for Na⁺ and K⁺ were 150–160 m$\text{M}$ and 2.5–4.8 m$\text{M}$, respectively, while uptake was progressively inhibited at concentrations of K⁺ greater than 5 m$\text{M}$. Rubidium was capable of substituting for potassium whereas cesium was a much less effective replacement. The uptake of DA was blocked by the antibiotics, valinomycin and gramicidin-D which bind K⁺ or both Na⁺ and K⁺, respectively, and thereby might interfere with the transport of cations across neuronal membranes. Similarly, ouabain which blocks the active transport of Na⁺ markedly antagonized the accumulation of DA into striatal homogenates. In contrast, tetrodotoxin which does not prevent the active transport of Na⁺, had no effect. Uptake appeared not to require Ca⁺⁺ and it was not inhibited by increasing total osmolarity to 400 mos$\text{M}$. In general, the cationic requirements for DA-uptake in striatal tissue and its responses to several inhibition of ionic transport, do not appear to be greatly different from those reported for NE with synaptosomes prepared from whole brain.     

Release of the chloride-dependent arginine aminopeptidase from PMN leukocytes and macrophaces during phagocytosis

The zymosan particles induced a time-dependent release of the chloride-dependent arginine aminopeptidase from rat peritoneal macrophages during $\text{in}$$\text{vitro}$ incubations. Intraperitoneal injections of zymosan, a streptococcal cell preparation and a $\text{Micrococcu}$-suspension caused the release of the chloride-activated arginine aminopeptidase into the peritoneal fluid. The arginine aminopeptidases obtained both from the cell cultivation media and the peritoneal washes were partly purified. The enzymes were similar with regard to the following properties: chloride activation with an optimum at physiological concentrations; strong inhibition by 10^?6M $\text{p}$-chloromercuribenzoate; similar elution properties and preferential hydrolysis of mainly the $\text{N}$-L-aminoacyl-2-naphthylamines of arginine and lysine. The chloride-activated arginine aminopeptidase released into the media in $\text{in}$$\text{vitro}$ conditions was inactivated in contrast to the enzyme released into the peritoneal fluid as a result of the intraperitoneal injections. The timing of the release of the chloride-activated arginine aminopeptidase both $\text{in}$ and $\text{in}$$\text{vitro}$ suggests that the enzyme plays a role in the initial phases of inflammation.     

Catecholamine-induced changes in transmembrane potential and temperature in normal and dystrophic hamster brown adipose tissue

Previous studies have shown that norepinephrine (NE) elicits trans-membrane potential changes in skeletal muscle cells from normal and dystrophic (BIO 14.6) hamsters, with the magnitude of these changes being significantly less in dystrophic cells. To determine if the decreased response of the dystrophic muscle cells reflects a more generalized phenomenon, the present study was designed to evaluate the effects of NE on membrane properties of brown adipocytes. $\text{In vivo}$ techniques using glass microelectrodes were similar to those used in the muscle studies. NE injection (2 to 5 μg/kg body wt, i.v.) into anesthetized hamsters was followed by membrane depolarization, the magnitude of which did not significantly differ in the dystrophic and normal adipocytes. For example, upon administration of 5 μg NE/kg body wt, the average depolarization was $\text{14.5 ± 1.3}\text{mV}\text{(}\text{X}\text{±}\text{S.E.}\text{)}$ for 20 dystrophic cells and 14.1 ± 1.8 mV for 18 normal cells. The depolarizations following i.v. infusion of isoproterenol and phenylephrine also had similar amplitudes in both normal and dystrophic cells. Despite this lack of difference in plasma membrane responses, NE induced a significantly smaller rise in interscapular brown fat temperature in the dystrophic (0.09°C) than in the normal hamsters (0.26°C) following administration of 5 μg NE/kg body wt. Thus, the decreased responsiveness to NE of dystrophic sarcolemma did not occur with the plasma membrane of brown adipocytes, although brown fat temperature changes in the dystrophic hamsters were decreased in amplitude.     

Effects of Δ9-tetrahydrocannabinol on neurotransmitter accumulation and release mechanisms in rat forebrain synaptosomes

The effects of (?)?Δ⁹-THC were studied on the release and accumulation of ³H5HT and ³HNE in a rat forebrain synaptosomal preparation. These studies were designed to evaluate the possible sites of action of Δ⁹-THC on these two processes. Δ⁹-THC inhibited the accumulation of ³H-leucine, ³HNE, and ³H5HT, as well as facilitated the release of the latter two amines (to a lesser degree), but had no effect on the release of ³H-leucine. Eighteen-hour pre-treatment with reserpine diminished the ability of Δ⁹-THC to induce release of ³H5HT, but had no effect on the $\text{in vitro}$ inhibition of synaptosomal uptake of this amine. Concentrations of Δ⁹-THC which blocked the uptake of ³H5HT also reduced the conversion of ³H5HT to ³H-5-hydroxy-3-indoleacetic acid. However, Δ⁹-THC, at concentrations which facilitated release of ³H5HT from preloaded synaptosomes, increased the amount of ³H5HIAA found in the medium. Taken together, these data suggest that Δ⁹-THC facilitates release from the synaptic vesicle and retards accumulation at the neuronal membrane.     

Effects of the ionophores,X-537A and A-23187 on catecholamine release from the in vitro frog adrenal

1. The ionophore X-537A increases the rate of catecholamine release from the $\text{in vitro}$ frog adrenal.2. The ratio of epinephrine/norepinephrine measured during X-537A stimulation was the same as that during spontaneous release.3. Even when Ca⁺⁺ was removed from the Ringer, X-537A stimulated catecholamine release, but depolarization by elevated extra-cellular K⁺ was no longer effective.4. X-537A also increases the release of dopamine β-hydroxylase, suggesting that the ionophore acts, at least in part, by stimulating the exocytosis of the chrommaffin granule contents.5. Therefore, it is questionable whether the release of catecholamines by X-537A is owing to its action as a Ca⁺⁺- ionophore.6. The divalent cation ionophore, A-23187 (50μM), did not affect the rate of catecholamine release.     

Conformational preferences of dopamine analogues for inhibition of norepinephrine N-methyltransferase. Conformationally defined adrenergic agents. 7

A series of analogues of dopamine (DA) with varying degrees of conformational flexibility have been examined as potential substrates or competitive inhibitors of the enzyme norepinephrine N-methyltransferase (NMT). A conformationally defined (rigid) analogue of the fully extended conformation of DA, 2-amino-6, 7-dihydroxybenzonorbornene hydrobromide ($\text{3}$; 6, 7-D2HX) proved to be a better substrate than the non-catechol parent 2-aminobenzonorbornene ($\text{4}$; 2HX). However, analogues $\text{3}$ and $\text{4}$ displayed equivalent competitive inhibitory activity toward phenylethanolamine (PEA). Neither 6, 7-ADTN (5), a DA analogue in the 2-aminotetralin (2AT) system, nor 6, 7-DTHIQ ($\text{7}$), a DA analogue in the tetrahydroisoquinoline (THIQ) system, showed substrate activity; 6, 7-ADTN was a poorer competitive inhibitor than the parent 2AT but 6, 7-DTHIQ was a better competitive inhibitor than its parent, THIQ ($\text{8}$). A tricyclic conformationally defined analogue $\text{9}$ of 6, 7-ADTN was devoid of either substrate or inhibitory activity. From these results it may be concluded that a fully extended side chain conformation is required for NMT substrate activity, and the better substrate activity for 6, 7-D2HX compared to $\text{4}$ is consistent with a proper catechol orientation for interaction with the norepinephrine (NE) binding site of NMT.     

The isomers of cocaine and tropacocaine: Effect on 3H-catecholamine uptake by rat brain synaptosomes

Compared to (+)-$\text{pseudo}$cocaine, (?)-cocaine was 20 times more potent in inhibiting uptake of ³H-norepinephrine (³HNE) by cortical synaptosomes and 66 times more potent with respect to ³H-dopamine (³HDA) uptake by striatal synaptosomes. Although the tropacocaine isomers were equipotent as inhibitors of ³HNE uptake in the cortex, tropacocaine was 3.9 times more potent as an inhibitor of ³HDa uptake in the striatum than $\text{pseudo}$tropococaine. A major known cocaine metabolite, benzoylecgonine failed to inhibit the accumulation of ³HNE and ³HDA by synaptosomes from the cortex and striatum, respectively. The implications of these findings in relation to the motor stimulation seen with (?)-cocaine, (+)-$\text{pseudo}$cocaine and benzoylecgonine in rats are discussed.     

Impaired noradrenergic transmission in schizophrenia?

Recently, increased brain and spinal fluid (CSF) norepinephrine (NE), and a decreased cAMP response to prostaglandin E₁ (PgE₁) stimulation of platelet NE sensitive adenylcyclase were observed in some schizophrenic patients. Low $\text{CSF}$ dopamine-beta-hydroxylase (DBH) activity was related to brain atrophy, whereas high $\text{plasma}$ DBH was associated with tardive dyskinesia. Increased NE (in brain and CSF) and 3-methoxy-4-hydroxy-phenylglycol (MHPG) levels and decreased plasma DBH activity in the brain were associated with a diagnosis of paranoid schizophrenia. Impaired NE transmission in schizophrenia may relate to disturbances in the autonomic nervous system, deficits in attention and information processing and to an impaired ability to deal with stress. Although pharmacological studies have suggested a major role for dopamine (DA) in schizophrenic psychosis, this review indicates the need for further exploration of the NE system. Future studies should address the relationship with DA, the autonomic nervous system (ANS), cerebral blood flow, brain metabolism, stress response, negative and prodromal symptoms.     

Effects of norepinephrine and acetylcholine on 32P incorporation into phospholipids of the rabbit iris muscle following unilateral superior cervical ganglionectomy.

The effect of norepinephrine and acetylcholine on the ³²P incorporation into phospholipids of normal and sympathetically denervated rabbit iris muscle was investigated. (1) In the absence of exogenously added neurotransmitters sympathetic denervation exerted little effect on the incorporation of ³²P into the phospholipids of the excised iris muscle. $\text{In vivo}$ thr iris muscle incorporated ³²P into phosphatidylinositol, phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and sphingomyelin in that order of activity while $\text{in vitro}$ phosphatidylinositol was followed by phosphatidylcholine. (2) Tension responses of iris dilator muscle from denervated irises exhibited supersensitivity to norepinephrine. Furthermore, norepinephrine at concentrations of 3 μM and 30 μM produced 1.6 times and 3 times stimulation of the phosphatidic acid of the denervated muscle respectively. In contrast at 30 μM it stimulated this phospholipid by 1.6 times in the normal muscle. This stimulation was completely blocked by phentolamine. (3) While in the normal muscle acetylcholine stimulated the labelling of phosphatidic acid and phosphatidylinositol by more than 2 times, in the denervated muscle it only stimulated 1.4 to 1.7 times. (4) Similarly when ³²Pi was administered intracamerally, the labelling found in the various phospholipids of the denervated iris was significantly lower than that of the normal. (5) It was concluded that denervation decreases the ³²P labelling in the presence of acetylcholine. (6) The norepinephrine-stimulated ³²P incorporation into phosphatidic acid appears to be post-synaptic.     

3H-dopamine uptake by synaptic storage vesicles of rat whole brain and brain regions

A crude preparation of neurotransmitter storage vesicles was obtained by differential centrifugation and the ability to take up ³H-dopamine was evaluated $\text{in}$$\text{vitro}$. The uptake was highly dependent on temperature, had an absolute requirement for ATP and Mg²⁺ and was inhibited totally by reserpine. The uptake displayed saturation kinetics, with a Km of 0.26 μM at 20°. ³H-dopamine uptake was inhibited competitively by norepinephrine, with a Ki of 0.69 μM. Vesicles derived from a primarily dopaminergic region (corpus striatum) exhibited the same ratio of uptakes of ³H-dopamine/³H-norepinephrine as did those from a primarily noradrenergic region (cerebral cortex). These results indicate that viable rat brain storage vesicles can be readily prepared and used for evaluation of pharmacologic effects on ³H-dopamine uptake, and that dopaminergic and noradrenergic storage vesicles exhibit identical uptake properties.     

Stimulation of ornithine decarboxylase synthesis in the rat thyroid

High titer antiserum to hepatic ornithine decarboxylase was prepared by employing enzyme·monospecific antibody complex as the immunizing antigen. This new antiserum preparation was successfully labeled with ¹²⁵I and was found to retain its specific immune properties. Iodinated antiserum was used to precipitate thyroid ornithine decarboxylase induced by a mixture of thyroid stimulating hormone and methyl xanthine in rat thyroids $\text{in vitro}$. ¹²⁵I-labeled antibody incorporation into the enzyme antibody complex after induction $\text{in vitro}$ showed an increase which paralleled the increase in enzymatic activity and thus suggested $\text{de novo}$ synthesis of thyroid enzyme protein.     

The roles of cytochrome b5 in a reconstituted N-demethylase system containing cytochrome P-450.

Compound 102804 isolated from $\text{Bacillus cereus}$ has been found to be a potent inhibitor of the N⁵-methyltetrahydrofolate-homocysteine transmethylase isolated from $\text{Escherichia coli}$ B. This inhibition was noted when 102804 was added to the enzyme reaction mixture after the reaction started or concurrently with the preparation of the mixture. Chemically inactivated 102804 has no activity as an inhibitor of this enzyme system.