Characterization of Tritiated Noradrenaline Release from the Rat Preoptic Area with Microdialysis In Vivo

Abstract: Present techniques are unable to provide a sensitive and accurate index of noradrenergic activity in the rat preoptic area. In this study, we have examined the brainstem A₁ noradrenergic input to the preoptic area using a new technique whereby [³H]noradrenaline is preloaded into the preoptic area and release of radioactivity from this region is measured subsequently using microdialysis in vivo. Electrical stimulation of the ipsilateral A₁ area for 20 min at 5, 10, and 15 Hz evoked significant increases in dialysate radioactivity that were repeatable and frequency-dependent. After removal of calcium from the perfusion medium, basal release of radioactivity was markedly reduced and the effect of A₁ stimulation abolished. Changing to a 100 mM K⁺ medium evoked an increase in the release of radioactivity that was sixfold greater than that seen after A₁ stimulation. Separation of the dialysate with HPLC showed that 33% of the increase in measured radioactivity after A₁ stimulation was directly attributable to [³H]noradrenaline and the remainder to the metabolites vanillylmandelic acid, 3,4-dihydroxymandelic acid, and 3,4-dihydroxyphenylglycol. In contrast, the increase in radioactivity after K⁺ depolarization was due almost completely to [³H]noradrenaline. Addition of 10 μM clonidine to the perfusion medium markedly reduced basal release of radioactivity, but had no effect on evoked release following A₁ stimulation. Conversely, perfusion with 10 μM yohimbine had no effect on basal release, but significantly increased evoked release after A₁ stimulation. These results now provide a characterization of noradrenergic activity in the preoptic area and indicate the importance of the A₁ noradrenergic input to this region. The technique of measuring radioactivity with microdialysis after preloading with [³H]noradrenaline provides a relatively simple, sensitive index of noradrenergic activity in vivo with good temporal resolution.     

Antagonism of 5-Hydroxytryptamine by Methiothepin shown in Micro-electrophoretic Studies of Neurones in the Lateral Geniculate Nucleus

THERE has been little success in the search for a specific antagonist of the actions of 5-hydroxytryptamine (5-HT) on central neurones, although several compounds reduce the effects of both tryptamine derivatives and catecholamines in the central nervous system^1,2. The recent report that lysergic acid diethylamide blocked the excitant action of 5-HT, but not that of noradrenaline, on medullary reticular neurones³ has not been confirmed⁴. Moreover, an earlier investigation of olfactory bulb neurones indicated that lysergic acid diethylamide blocked the action of noradrenaline more readily than that of 5-HT⁵.     

Release of [3H]noradrenaline by α1-adrenoceptor agonists

Mouse isolated vas deferens preincubated with [³-H]noradrenaline was superfused and the effect of ₁-adrenoceptor agonists was studied on the release of total radioactivity ([³H]noradrenaline +³H-metabolites) and [³H]noradrenaline. Reverse phase high pressure liquid chromatography (HPLC) combined with scintillation spectrometry was used to separate [³H]noradrenaline from its metabolites. Among the ₁-adrenoceptor agonists (1-phenylephrine, ST-587(2-(2-chloro-5-trifluoromethyl phenylimino)-imidazole), (–)-amidephrine, methoxamine, cirazoline and l-noradrenaline) studied l-phenylephrine, ST-587 and l-noradrenaline were capable of releasing³H-noradrenaline. The effect of noradrenaline was stereospecific. As determined by HPLC combined with scintillation spectrometry the release of total radioactivity in response to l-noradrenaline is mainly due to [³H]noradrenaline. It is suggested that l-noradrenaline, l-phenylephrine, and ST-587 in addition to their direct effect on different receptors they also have indirect action through the release of noradrenaline which might be partly involved in the pharmacological responses. The mechanisms whereby l-noradrenaline and l-phenylephrine release noradrenaline would appear to involve a saturable Ca-independent and a cocaine and temperature sensitive process. On the basis of our findings among the ₁-adrenoceptor agonist studied (–)-amidephrine, methoxamine and cirazoline is a better choice than l-phenylephrine or ST-587 for selective stimulation of postjunctional ₁-adrenoceptor, they do not release noradrenaline.     

Pharmacological study of inhibited frog olfactory bulb glomerular input produced by a puff of air on the olfactory mucosa

The effects of applying 4-aminopyridine (10^–2 M), aminooxyacetic acid (AOAA — 10^–4–10^–3 M), -alanine (10^–3–10^–2 M), and bicuculline (10^–5, 10^–4 M) to the intact frog olfactory bulb were investigated. Having measured inhibition of orthodromic potential postsynaptic components produced either by a puff of air on the olfactory mucosa (OB input inhibition) or by single electrical stimulation of the olfactory nerve (postsynaptic inhibition) or by single electrical stimulation of the olfactory nerve (postsynaptic inhibition), it was found that 4-aminopyridine greatly intensified postsynaptic inhibition but strongly reduced that of OB input; inhibition of the latter was raised by AOAA or bicuculline and decreased by -alanine. These substances failed to exert any consistent, clear-cut effects on postsynaptic inhibition. Findings would support the hypothesis that OB input inhibition produced by a puff of air on the olfactory mucosa could occur as a result of GABA release from glial cells and subsequent binding of GABA to presynaptic GABA_B-receptors in glomeruli.M. V. Lomonosov Moscow State University. Translated from Neirofiziologiya, Vol. 19, No. 1, pp. 12–20, January–February, 1987.     

The release of 3H-adenosine and its derivatives from cat sensorimotor cortex.

The release of tritiated adenosine and its derivatives from the surface of the pericruciate sensorimotor cortex of cats has been measured. Most of the radioactive material was released as adenosine or as adenosine monophosphate. Direct electrical stimulation of the cortex (either monopolar or bipolar) increased the level of release during and immediately following the period of stimulation. ¹⁴C-labelled urea release was not enhanced by electrical stimulation. It is suggested that adenosine and its derivatives may be released from presynaptic nerve terminals. The results support the view that adenosine and its nucleotides may be inhibitory transmitters in the cerebral cortex.     

Accumulation et excrétion de substances organiques par les cellules à chlorure de la branchie d'Anguilla anguilla L. adaptée à l'eau de mer

Résumé Les sites branchiaux d'accumulation de certaines substances (bleu de méthylène, urée, paraaminohippurate et inuline) injectées dans la circulation générale ont été localisés sur des anguilles adaptées à l'eau de mer. Le bleu de méthylène est accumulé et excrété par les cellules à chlorure. Les autoradiographies des corps marqués au ¹⁴C: inuline, PAH et urée, faites après cryodessication montrent que les cellules à chlorure accumulent ces substances. La signification de ces résultats est discutée.

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Accumulation and excretion of organic substances by the branchial chloride cells in sea-water adapted eel (anguilla anguilla L.)

Summary The branchial site of accumulation of various organic substances (methylene blue, urea, inulin and paraaminohippurate) following intracardiac injection has been localised in sea-water adapted eels. Methylene blue is concentrated and excreted by chloride cells. By combining freeze-drying and radioautographic technique it was possible to demonstrate that chloride cells concentrate urea, inulin and paraaminohippurate. The signification of these results is discussed in relation with the branchial excretion of organic substances.

     

Function of Catecholamine-containing Neurones in Mammalian Central Nervous System

SEVERAL chemical substances are involved in synaptic transmission in the mammalian central nervous system^1–3. The Falck-Hillarp technique⁴ has demonstrated noradrenaline, dopamine and 5-hydroxytryptamine within nerve cell bodies and terminals^5,6 and the belief that these amines act as neurohumours is strengthened by observations that nerve fibre activation leads to their release from the terminals^7,8. Histo-chemical evidence suggests that discrete systems of neurones can be identified by their content of particular amines and it seems possible that such neurohumorally homogeneous systems have a functional as well as a chemical identity. Before the anatomical distribution of amine-containing neurones had been described, Brodie and Shore⁹ proposed that noradrenaline functions as the central neurohumour of the sympathetic and 5-hydroxytryptamine of the parasympathetic system. This suggestion has not been supported by anatomical evidence; the amine-containing neurones form systems of small diameter fibres of very diffuse terminal distribution, which do not correspond to recognized ascending or descending pathways^5,6, although amine-containing neurones in invertebrates have been identified as sensory systems¹⁰.     

Elevation of Serum Dopamine-β-hydroxylase Activity with Forced Immobilization

THE formation of the neurotransmitter noradrenaline from 3,4-dihydroxyphenylethylamine (dopamine) is catalysed by the enzyme dopamine-β-hydroxylase (DBH)¹. This enzyme is associated both with the catecholamine-containing chromaffin granules in the adrenal medulla^2,3 and with the vesicular structures in sympathetic nerve terminals which contain catecholamines⁴. Furthermore, DBH activity is released with catecholamines into the perfusate after stimulation of either the isolated perfused adrenal gland⁵ or the isolated perfused spleen^6–8. DBH activity has been reported in the serum of both man and the rat^9,10. This activity is similar to adrenal and sympathetic nerve DBH activity with regard to cofactor requirements, oxygen requirement and kinetic characteristics^9,10. It has been suggested that serum DBH activity might be present as a result of release of enzyme with catecholamines from the adrenal glands and sympathetic nerves. If this is the case, serum DBH activity might be a useful and convenient index of sympathetic-adrenal activity. The work described here was undertaken to investigate both the source of the serum DBH and the effect on this activity of forced immobilization, a procedure which has been used as a model of stress and which has been shown to release catecholamines from the adrenal gland and increase catecholamine excretion¹¹.     

On the significance of the influx of calcium ions into stimulated human blood platelets

Blood platelets, upon stimulation with various substances, take up calcium ions from the suspending medium. This influx occurs simultaneously with the release reaction, i.e. the specific secretion of a variety of substances from storage organelles and the second wave of aggregation. Various inhibitors of the release reaction inhibit this Ca²⁺ influx. Platelets previously loaded with ⁴⁵Ca show an increased efflux of the cation upon stimulation by thrombin. These results suggest that the plasma membrane acquires an increased permeability to Ca²⁺ only in a later phase of platelet activation, in most cases after the earlier release of Ca²⁺ into the cytoplasm from Ca-storing organelles. Rapid shape change and release proceed independently of external calcium, whereas clot retraction depends upon a prolonged increased permeability of the plasma membrane to this cation.     

RELEASE OF RADIOACTIVE GLUTAMIC ACID FROM THIN SECTIONS OF GUINEA-PIG OLFACTORY CORTEX IN VITRO

Slices from the guinea-pig olfactory cortex were incubated in the medium containing [¹⁴C]glutamate and release of radioactive compounds was subsequently studied in the standard or high potassium media or during repetitive stimulation of the lateral olfactory tract (LOT) while electrical activity of the tissue was monitored. In 50 mm -potassium concentration, the pre- and postsynaptic potentials were completely suppressed and effluxes of total ¹⁴C and [¹⁴C]glutamate increased. No significant increase in [¹⁴C]glutamine was found. When Ca²+ concentration was reduced from 2·4 to 0·12 mm , the postsynaptic potential disappeared and release of [¹⁴C]glutamate in 50 mm -potassium decreased to about a third of that in 2·4 mm -Ca²⁺. Repetitive LOT stimulation enhanced release of total ¹⁴C in thinner slices but caused no significant increase in [¹⁴C]glutamate efflux. These findings were discussed in relation to the possibility that glutamate is a mediator between the LOT fibres and cortical neurons.     

On the Recovery of [3H]Noradrenaline from Different Metabolic Compartments of Rat Brain with Respect to the Role of Catechol-O-Methyltransferase

Abstract: Rats were treated with reserpine, desmethylimipramine, or carrier, either alone or in combination with tropolone. Either 10 min (t₁) or 1 h (t₂) after intraventricular injection of [³H]noradrenaline, they were decapitated. The total ³H activity and the recovery of [³H]noradrenaline were determined in tissue extracts from various brain regions. Maximum total ³H activity was measured at t₁ in all tropolone-treated rats; the mean sum of these results served as an estimate of the initial tissue concentration of [³H]noradrenaline. At t₁, 40–50% of the sum of [³H]noradrenaline and its metabolites was recovered unchanged in normal rats; reserpine and DMI reduced the recovery to 18–27%. In all groups, the decline of [³H]noradrenaline was retarded after t₁. Inhibition of catechol-O-methyltransferase by tropolone caused consistently elevated [³H]noradrenaline levels, but did not affect the metabolic rate after t₁ when compared with similarly pretreated, but tropolone-free rats. Thus, if catechol-O-methyltransferase was inhibited during the injection of [³H]noradrenaline, a higher percentage of the amine had been taken up into spaces with a slow noradrenaline turnover. The maximum increase was seen when the neuronal uptake, was inhibited by desmethylimipramine. This supported the hypothesis that an additional extraneuronal space exists, in addition to the known intraneuronal and extraneuronal compartments, which has a slow noradrenaline turnover. The tropolone effect on the noradrenaline recovery possibly shows that there might be a saturable “methylating system,” similar to that described for the periphery, in which catechol-O-methyltransferase is linked to the extraneuronal uptake₂. By affecting the access of noradrenaline to non-neuronal cells it might influence the rate of noradrenaline elimination from the intercellular space.     

Ureide Catabolism of Soybeans : II. Pathway of Catabolism in Intact Leaf Tissue

Allantoin catabolism studies have been extended to intact leaf tissue of soybean (Glycine max L. Merr.). Phenyl phosphordiamidate, one of the most potent urease inhibitors known, does not inhibit ¹⁴CO₂ release from [2,7-¹⁴C]allantoin (urea labeled), but inhibits urea dependent CO₂ release ≥99.9% under similar conditions. Furthermore, ¹⁴CO₂ and [¹⁴C] allantoate are the only detectable products of [2,7-¹⁴C]allantoin catabolism. Neither urea nor any other product were detected by analysis on HPLC organic acid or organic base columns although urea and all commercially available metabolites that have been implicated in allantoin and glyoxylate metabolism can be resolved by a combination of these two columns. In contrast, when allantoin was labeled in the two central, nonureido carbons ([4,5-¹⁴C]allantoin), its catabolism to [¹⁴C]allantoate, ¹⁴CO₂, [¹⁴C]glyoxylate, [¹⁴C]glycine, and [¹⁴C]serine in leaf discs could be detected. These data are fully consistent with the metabolism of allantoate by two amidohydrolase reactions (neither of which is urease) that occur at similar rates to release glyoxylate, which in turn is metabolized via the photorespiratory pathway. This is the first evidence that allantoate is metabolized without urease action to NH₄⁺ and CO₂ and that carbons 4 and 5 enter the photorespiratory pathway.     

Direct measurement of the effect of potassium,calcium, veratridine,and and amphetamine on the rate of release of dopamine from superfused brain tissue

The rate of release of endogenous DA from rat brain striatal minces has been measured using a rapid superfusion apparatus. The apparatus provides immediate, continuous readout of easily oxidized substances in the perfusate using an amperometric detector. Subsequent analysis of the perfusate (which contains pargyline) by liquid chromatography shows that the major substance detected is DA. DA release is induced by a 30 s exposure to 60 mM K⁺ and is Ca²⁺-dependent. Similar results are obtained with veratridine (10^?4 M). The time resolution of the perfusion system permits discrimination of the decreased rate of release induced by veratridine (10^?4 M) and amphetamine (10^?5 M) as opposed to 60 mM K⁺. Repetitive stimulation of the striatal mince with 60 mM K⁺ results in a decreased amount and rate of DA release. Subsequent exposure of the striatal mince to exogenous DA results in a restoration of the K⁺-induced, Ca²⁺-dependent release, indicating uptake of DA is operant under these conditions.     

Prostaglandin controls Neuromuscular Transmission in Guinea-pig Vas Deferens

PROSTAGLANDINS of the E type (PGE₁, PGE₂) inhibit sympathetic neurotransmission in several tissues and species^1–4. On the basis of their natural occurrence and availability for release, as well as observations on the pharmacological actions of the PGs, endogenous PGE₁ and PGE₂ are postulated to operate on sympathetic neurotransmission by a feedback mechanism and thereby modulate the effector responses to nerve activity^{1, 5}. Inhibition by 5,8,11,14-eicosatetraynoic acid (ETA) of PG synthesis in the cat spleen and in the rabbit heart increases the release of noradrenaline (NA) in response to nerve stimulation, thus strongly supporting the hypothesis^{6, 7}. We report here that guinea-pig vas deferens releases PG in response to nerve stimulation and that the neuromuscular transmission is facilitated after inhibition of PG synthesis. PG synthesis was irreversibly inhibited using ETA⁸.     

Diphasic Glucagon Release Induced by Arginine in the Perfused Rat Pancreas

A DIPHASIC pattern has been recorded for insulin release after glucose stimulation in rat pancreas¹ and in man² and also after arginine stimulation in man³: this led to the concept of a two-compartmental storage system for insulin¹. A diphasic pattern of secretion might be expected in some other endocrine cell types. To check this possibility for alpha-2 cells, perfused rat pancreas was submitted to both continuous infusion and pulses of a demonstrated glucagon stimulator, arginine⁴; glucagon responses were then measured.     

Monitoring the specific activities of dopamine and its metabolites in striatum and olfactory tubercle after intravenous administration of l-[3H]tyrosine: Complex relations,indicating more than two compartments

It is still a matter of debate whether in dopaminergic nerve endings dopamine (DA) is present in different functional and/or metabolic compartments. To investigate this, DA metabolism was studied in vivo by measuring the specific activity of DA and its metabolites after intravenous administration of l-[3,5-³H]tyrosine (200 μCi/rat) to freely moving animals. The incorporation of ³H into DA and metabolites was determined in striatum and olfactory tubercle at 5, 10, 20, 40, 60 and 80 min after [³H]tyrosine administration. In both structures the level of [³H]tyrosine initially declined monoexponentially, but deviated from that pattern later on. The curves representing the formation in time of [³H]DA and [³H]metabolites were very similar in both structures, although as a whole, the levels in the olfactory tubercle were higher. The relative patterns of the specific activities of DA and those of its metabolites, a possible clue to DA compartmentation, neither indicated a clearcut metabolic one-compartment, nor a two-compartment system. The flow of radioactivity through DA metabolism could in fact only be explained by assuming more complex metabolic relations.     

Modification of chemoreactive properties of neurons in the neocortex and hippocampus under the effect of electric stimulation of locus coeruleus

Based on the findings of alterations in the chemoreactivity of neurons under conditioning and its possible significance for the formation of transient neuronal junctions, the authors investigated the influence of electrical stimulation of the Locus coeruleus upon the transmitter sensitivity of cortical and hippocampal neurons. The majority of neurons showed immediately after stimulation of the Locus coeruleus a different reaction to microiontophoretic application of acetylcholine, dopamine and glutamate than under normal conditions. Obviously there must have occurred qualitative changes because a superposition effect of the reactions following stimulation of the Locus coeruleus and application of the substances has rarely been observed. The release of noradrenaline provoked by the stimulation of the Locus coeruleus evidently leads to qualitative changes in the neurone texture, which may occur both inter- and intraneuronally.     

Mechanisms of electromechanical and electrochemical coupling in olfactory cilia of the frog (<Emphasis Type="Italic">Rana temporaria</Emphasis>)

The mechanisms of electromechanical and electrochemical coupling in olfactory cilia of the frog (Rana temporaria) have been investigated. High-resolution optical television microscopy of live tissue and pharmacological analysis have been used to reveal the regulation of the motility of olfactory cilia in the absence of odorants; the entry of Ca²⁺ ions mediated by three types of ion channels (mechanosensitive, cyclic nucleotide gated, and voltage-gated) was shown to determine the motility of cilia. Stimulation of the olfactory adenylate cyclase by movements of the cilia in the absence of odors has been demonstrated and the regulation of cilia motility by membrane potential has been revealed. Membrane potential can affect olfactory acuity and the ability to perceive weak olfactory stimuli in the absence of adequate stimulation.     

Actions of prostacyclin (PGI2) on adrenergic neuroeffector transmission in the rabbit kidney

In the Tyrode's perfused rabbit kidney PGI₂ (1.3 × 10⁻⁸-3.3 × 10^−7M) dose-dependently inhibited vasoconstrictor responses to sympathetic nerve stimulation, as did PGE₂. The dose-effect curve of the two compounds differed, making PGI₂ the less potent in the low concentration and the more potent in the high. PGI₂ also inhibited the vasoconstrictor response to exogenous noradrenaline, but it had no effect on transmitter release. The main metabolite of PGI₂, 6-keto-PGF_1α, was ineffective both on noradrenaline release and on vascular responses to nerve stimulation or exogenous noradrenaline. It is suggested that PGI₂,if a significant renal prostaglandin, may modulate renal neuroeffector transmission post-junctionally, thereby forming a complement to the prejunctional action of PGE₂.