ESTIMATION OF NORADRENALINE AND ITS MAJOR METABOLITES SYNTHESIZED FROM [3H]TYROSINE IN THE RAT BRAIN

Abstract— A new procedure is described for the estimation of [³H]noradrenaline (NA) and its major metabolites free and conjugated 3-methoxy-4-hydroxyphenylglycol (MOPEG) and free and conjugated 3,4-dihydroxyphenylglycol (DOPEGI in the rat brain. The procedure involves adsorption on to alumina, cation exchange chromatography. enzymatic hydrolysis of conjugates and thin-layer-chromatography after intraventricular (IVT) or intravenous injection of [³H]tyrosine. In a time-course study the formation and accumulation of the metabolites have been measured from 15min to 23h after IVT injection of [³H]tyrosine. [³H]MOPEG and [³H]DOPEG were found in almost equal amounts during the synthesis phase of [³H]NA as well as during the storage and disappearance phase of [³H]NA. The maximum levels of conjugated [³H]MOPEG and conjugated [³H]DOPEG were found 2 h after IVT [³H]tyrosine. At this time interval the levels of free [³H]MOPEG and free [³H]DOPEG amounted to 25% and 11%, respectively of the corresponding conjugates. Increasing doses of IVT injected [³H]tyrosine (10-90 °Ci) revealed that the accumulation of [³H]NA and metabolites was linear up to about 50 °Ci. Following intravenous instead of IVT injection of [³H]tyrosine. much higher doses (325 °Ci) were needed to obtain measurable amounts of total [³H]MOPEG and [³H]DOPEG-SO₄ in the rat brain. The formation of labelled NA metabolites from [³H]NA in the rat brain in vim measured as total [³H]MOPEG and [³H]DOPEG-SO₄ was influenced by drugs affecting [³H]NA synthesis, release and metabolism. Synthesis inhibition with a-methyltyrosine (250mg-kg^?1) or FLA-63 (30mg-kg^?1) and inhibition of monoamine oxidase with pargyline (75mg-kg^?1) or clorgyline (2mg-kg^?1) strongly decreased the accumulation of total [³H]MOPEG and [³H]DOPEG-SO₄. Noradrenaline receptor blockade with phenoxybenzamine (20mg-kg^?1) increased both total [³H]MOPEG and [³H]DOPEG-SO₄ to about 160% of the control values. NA release and uptake inhibition induced by d-amphetamine (10mg-k^?1) or phenylethylamine (two doses of 80mg-kg^?1) decrease strongly the levels of [³H]NA and [³H]DOPEG-SO₄. whereas total [³H]MOPEG was only very slightly decreased or even increased as compared to controls.     

Determination of Normetanephrine, 3,4-Dihydroxyphenylethyleneglycol (Free and Total), and 3-Methoxy-4-Hydroxyphenylethyleneglycol (Free and Total) in Rat Brain by High-Performance Liquid Chromatography with Electrochemical Detection and Effects of Drugs on Regional Concentrations

A new, fast and sensitive assay for normetanephrine (NM), free and total 3,4-dihydroxyphenylethyleneglycol (DOPEG), and free and total 3-methoxy-4-hydroxyphenylethyleneglycol (MOPEG) in brain tissue is described. The method is based on high-performance liquid chromatography with electrochemical detection. Small Sephadex G 10 columns were used for prepurification. This permitted the additional isolation and quantification of tyrosine, 3,4-dihydroxyphenylalanine, noradrenaline, dopamine, 3-methoxytyramine, 3,4-dihydroxyphenylacetic acid, homovanillic acid, and 5-hydroxyindoleacetic acid. The compounds were determined in six brain areas (striatum, cortex, hippocampus, hypothalamus, brainstem, and cerebellum). Most DOPEG and MOPEG in rat brain was present in the conjugated form, except for the cerebellum, where about 80% of MOPEG was nonconjugated. No postmortem effects on MOPEG levels were observed; a slight increase in DOPEG in certain brain areas was found in microwave-killed rats. The effects of clonidine, yohimbine, N,N-dipropyl-5,6-ADTN, and chlorpromazine on the concentrations of the five noradrenaline (NA) metabolites were determined. Free and total DOPEG and MOPEG provide similar information on NA metabolism, whereas NM (after monoamine oxidase inhibition) reflects a different type of interaction of drugs with NA metabolism. The similarity in the pattern of drug-induced changes in NA metabolism in the various brain areas suggests that adrenoreceptors mediating NA metabolism are homogeneously distributed throughout the brain.     

Accumulation and metabolism of norepinephrine in rat hypothalamus after exhaustive stress

—Exhaustive stress in rats is followed by a temporary reduction of hypothalamic norepinephrine (NE) together with a persistent increase in turnover during recovery. To test for persistent alterations of NE storage and metabolism produced by stress, rats were subjected to 3 h of forced running and were then injected intraventricularly with [³H]NE or [³H]dopamine (DA). The hypothalamus was assayed for [³H]NE and its metabolites at various intervals after injection. The effects of stress were compared with those of reserpine (7·5 mg/kg) or α-methyltyrosine (AMT, 300 mg/kg) pretreatment. It was found that the stress-induced reduction of endogenous NE was not accompanied by a change in the accumulation of exogenous [³H]NE either 10 or 30 min after injection, whereas the NE depletions produced by reserpine or AMT were associated with decreased or increased accumulation, respectively. However, stress did produce an increased accumulation of [³H]NE endogenously synthesized from [³H]DA. These results indicate that exhaustive stress does not adversely affect the storage of NE. They also suggest that stores of NE depleted by stress are replenished chiefly with newly synthesized NE and not through an increased uptake and binding or decreased metabolism of extraneuronal NE. The latter factors may play a role in the maintenance of brain NE stores when biosynthesis is low, i.e. after AMT. The major metabolites of exogenous [³H]NE, at 30 min after injection, were identified as conjugates of 3,4-dihydroxyphenylglycol (DOPEG) and 3-methoxy-4-hydroxyphenylglycol (MOPEG) in approximately equal amounts. The finding of high levels of conjugated DOPEG confirms a recent report (Slgden and Eccleston , 1971) that this compound is a major metabolite of brain NE. Reserpine produced marked elevations of both conjugates; AMT slightly reduced each. Prior stress increased only conjugated MOPEG, an observation suggesting that CNS levels of this metabolite may reflect NE released by nervous activity.     

A method for the assay of conjugated 3,4-dihydroxyphenylglycol,a major noradrenaline metabolite in the rat brain

We present the first published procedure for the measurement of endogenous conjugated 3,4-dihydroxyphenylglycol (DOPEG) in the rat brain. Conjugated DOPEG is estimated from brain extracts after enzymic hydrolysis, isolation of hydrolysed DOPEG on alumina, methylation of DOPEG to 3-methoxy-4-hydroxyphenylglycol (MOPEG) and gas chromatographic quantification of MOPEG. The level of conjugated DOPEG in the CNS of rats (65.7 +/- 0.7 ng/g whole brain tissue corrected for recovery) almost equals the level of conjugated MOPEG. The sensitivity of the method is about 6 ng/g brain tissue. After inhibition of monoamine oxidase with clorgyline (30 mg/kg) conjugated DOPEG and MOPEG both disappeared from the brain with a half-life of about 1 h. Turnover calculations indicate that conjugated DOPEG and MOPEG are the two major noradrenaline end-metabolites in the rat brain. The method of estimating conjugated DOPEG also allows the measurement of noradrenaline, dopamine and total MOPEG in an extract from one half of a rat brain.     

ON THE ORIGIN OF VANILLYLMANDELIC ACID AND 3-METHOXY-4-HYDROXY-PHENYLGLYCOL IN THE RAT BRAIN

Abstract— The effects of electrothermic destruction and electrical stimulation of the locus coeruleus on the brain levels of vanillylmandelic acid (VMA), 3-methoxy-4-hydroxyphenylglycol (MOPEG) and noradrenaline (NA) were studied in the rat. Fourteen days after destruction of the locus coeruleus, the content of NA in the hippocampus and that of MOPEG in the rest of the brain were decreased by more than 70% and 50% respectively.
Stimulation of the locus coeruleus induced a decrease in hippocampal levels of NA of 38%, while MOPEG levels were found to be increased more than 3-fold After intraventricular injection of 1 μg of adrenaline (A), the levels of MOPEG were Substantially increased In none of these experiments was any variation in VMA levels found, These results suggest that in the rat brain endogenous VMA is not a metabolite of either NA or A The formation of MOPEG from A as well as from NA appears to be possible.     

Release of ( 3 H)noradrenaline and ( 3 H)dopamine from field stimulated cerebral cortex slices. Effect of tyrosine hydroxylase and dopamine- -hydroxylase inhibition

Rat cerebral cortex slices were incubated in vitro with [³H]dopamine (DA) or [³H]noradrenaline (NA) (10^?7M), superfused by fresh buffer and stimulated by an electric field. The stimulation-induced overflow of [³H]DA and [³H]NA was determined. In slices from untreated rats about 16 ng [³H]NA/g tissue was formed from [³H]DA, corresponding to about 5 per cent of the endogenous NA concentration. Stimulation markedly enhanced the overflow of [³H]NA. The [³H]NA newly formed from [³H]DA was overflowing to a greater extent than [³H]NA previously taken up from the incubation medium, indicating a preferential release of newly synthesized transmitter. The stimulation-induced overflow of [³H]DA and [³H]NA was increased in slices of rats pretreated with a tyrosine hydroxylase inhibitor (H44/68). It seems that depletion of the endogenous NA stores of central NA neurons by tyrosine hydroxylase inhibition makes the [³H]cate-cholamines more available for release. Pretreatment of the rats with the DA-β-hydroxylase inhibitors FLA63 or FLA69 considerably diminished the formation of [³H]NA from [³H]DA. Stimulation markedly enhanced the overflow of [³H]DA indicating that DA can act as a ‘false transmitter’ in central NA neurons after DA-β-hydroxylase inhibition.     

POTASSIUM-INDUCED RELEASE OF [3H]GABA AND OF [3H]NORADRENALINE FROM NORMAL AND RESERPINIZED RAT BRAIN CORTEX SLICES. DIFFERENCES IN CALCIUMDEPENDENCY, AND IN SENSITIVITY TO POTASSIUM IONS

The release of [³H]GABA induced by elevated extracellular potassium (K)_o, from thin rat brain cortex slices, has been compared with that of [³H]noradrenaline ([³H]NA), released by the same procedures, both from normal slices, and from slices pre-treated with reserpine and nialamide, [³H]NA being predominantly a vesicular component in the former situation, and a soluble substance in the latter one. 46 mM-(K)_o released considerably more [³H]NA from normal than from drug-treated slices, while the release of GABA was about two thirds of the latter. When 4min ‘pulses’ of increasing concentrations of potassium were applied, it was observed that the release of GABA and of [³H]NA from drug-treated slices increased in proportion to (K)_o, up to 36-46 mM and then declined considerably with higher (K)_o. The dependency of potassium-induced release on the concentration of calcium in the medium, indicated that release of [³H]NA from normal slices was proportional to calcium up to 1.5-2 mM, while that of [³H]NA from drug-treated slices increased up to 0.5 mM-Calcium, and then declined with higher concentrations. GABA release also increased up to 0.5 mM-calcium, but no further changes were observed at higher concentrations. The calcium antagonist D-600 inhibited high (K)_o-induced release of [³H]NA from normal slices to a greater extent than that of [³H]GABA or of [³H]NA from drug-treated slices. These results, in which elevated (K)_o-induced release of [³H]GABA resembles considerably that of soluble NA, but differs from that of NA present in synaptic vesicles, suggest that release of [³H]GABA also occurs from the soluble cytoplasmic compartment, and that the partial calcium requirement that is found is unrelated to that of transmitter secretion. These findings are also a further indication of the lack of specificity of elevated (K)_o as a stimulus for inducing transmitter secretions.     

Effects of the neoclerodane Hardwickiic acid on the presynaptic opioid receptors which modulate noradrenaline and dopamine release in mouse central nervous system

We have comparatively investigated the effects of Hardwickiic acid and Salvinorin A on the K⁺-evoked overflow of [³H]noradrenaline ([³H]NA) and [³H]dopamine ([³H]DA) from mouse hippocampal and striatal nerve terminals, respectively. The K⁺-evoked overflow of [³H]DA was inhibited in presence of Salvinorin A (100 nM) but not in presence of Hardwickiic acid (100 nM). Hardwickiic acid (100 nM) mimicked Salvinorin A (100 nM) in facilitating K⁺-evoked hippocampal [³H]NA overflow and the two compounds were almost equipotent. Facilitation of [³H]NA overflow caused by 100 nM Hardwickiic acid was prevented by the κ-opioid receptor (KOR) antagonist norbinaltorphimine (norBNI, 100 nM) and by the selective δ-opioid receptor (DOR) antagonist naltrindole (100 nM), but was not altered by 100 nM D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP), a selective μ-opioid receptor (MOR) antagonist. We conclude that Hardwickiic acid modulates hippocampal [³H]NA overflow evoked by a mild depolarizing stimulus by acting at presynaptic opioid receptor subtypes.     

Distribution of [3H]noradrenaline and [3H]serotonin in photophores of Porichthys notatus

Summary Photophores of Porichthys notatus were examined by electron-microscopic radioautography following incubation in tritiated noradrenaline ([³H]NA) or serotonin ([³H]5-HT). Nerve varicosities surrounding the photocytes were found to accumulate [³H]NA but not [³H]5-HT, providing compelling evidence for the catecholaminergic nature of the monoaminergic innervation of photophores. The photocytes themselves appeared selectively labelled with both tracers, but the intensity of labelling after [³H]5-HT incubation was considerably greater than after [³H]NA. Stereological sampling of organelle content in photocytes showed ultrastructural differences between [³H]NA- and [³H]5-HT-labelled cells, probably related to light emission induced by NA. The main changes noted after incubation with [³H]NA were mitochondrial swelling and disorganization, increased coalescence of photocytic vesicles and extrusion of vesicular material into the extracellular matrix. With respect to the subcellular localization of [³H]NA and [³H]5-HT within the photocytes, statistical analysis of the distribution of silver grains disclosed a preferential affinity of both labels for appositional zones between mitochondria and coalescent vesicles. Moreover, in the case of 5-HT, selective affinity was also exhibited by sites comprising vesicular membrane and adjacent cytoplasm, suggesting binding of this biogenic amine to the entire membrane of photocytic vesicles.Supported by grants from the Natural Sciences and Engineering Research Council (M.A.), and Medical Research Council of Canada (L.D.). Dr. Pierre Legendre kindly provided advice on statistical methods     

CATECHOLAMINE METABOLISM IN THE DOG: COMPARISON OF INTRAVENOUSLY AND INTRAVENTRICULARLY ADMINISTERED [14C]DOPAMINE AND [3H]NOREPINEPHRINE

—The urinary excretion of labelled metabolites was measured in dogs which had been injected intravenously or intraventricularly with [³H]norepinephrine or [¹⁴C]dopamine. [³H]Norepinephrine injected by either route produced more labelled 3-methoxy-4-hydroxy-phenylglycol than 3-methoxy-4-hydroxymandelic acid, as did [¹⁴C]dopamine after intravenous administration. In contrast, following the intraventricular injection of [¹⁴C]dopamine, more [¹⁴C]3-methoxy-4-hydroxymandelic acid was formed than [¹⁴C]3-methoxy-4-hydroxyphenylglycol. These observations suggest that the metabolism of exogenously-administered and endogenously-formed norepinephrine may proceed through different routes and that the predominant metabolite of norepinephrine in canine brain may be 3-methoxy-4-hydroxymandelic acid rather than 3-methoxy-4-hydroxyphenylglycol.     

On the uptake and storage of 5-hydroxytryptamine, 5-hydroxytryptophan and catecholamines by adrenal chromaffin cells and nerve endings

Summary Light-microscopic autoradiographs of the adrenal medulla at various intervals after the intravenous injection of [³H] 5-HTP, [³H] 5-HT, [³H] noradrenaline and [³H] adrenaline have been studied. The distribution of silver grains following [³H] 5-HTP uptake was found to be uniform over each of the two main cell populations, adrenaline-storing (A) cells and noradrenaline-storing (NA) cells in the adrenal medulla, but A cells were twice as active as NA cells in incorporating the isotope, a situation very similar to that found after [³H] dopa uptake. 5-HT administration resulted in a pattern resembling the distribution of [³H] noradrenaline uptake, with A cells being 4 or 5 times more active than NA cells and a gradient of activity from the periphery of the medulla inwards. However, the time-course for the loss of radioactivity was not the same for both amines: levels of 5-HT activity were not significantly reduced after one week whereas the degree of [³H] noradrenaline labelling after one week was less than 10% of that at one hour. Thus 5-HT may be bound to sites in the adrenal medulla normally occupied by noradrenaline but it would appear that the release mechanism is different. There was no evidence of 5-HT uptake by adrenal nerve endings.     

Effect of S-adenosyl-L-homocysteine of dopamine catabolism]

1.--The administration of SAH to rats, at physiologically active dose on the sleep, does not change the urinary level of MD and NM. On the other hand, the excretion of DA and NA decreases. 2.--In the brain, SAH does not modify neither the concentration of NA and NM in hypothalamus and thalamus, nor the concentration of DA and MD in corpus striatum. 3.--After intracisternally injection of [14C]DA or [3H]NA, SAH increases the level of [14C]MD and [3H]NM. 4.--Contrary to the studies in vitro, where SAH is an inhibitor of COMT, on the rat it does not seem prevent the methylation of DA and NA.     

Roles Played by the Na+/Ca2+ Exchanger and Hypothermia in the Prevention of Ischemia-Induced Carrier-Mediated Efflux of Catecholamines into the Extracellular Space: Implications for Stroke Therapy

The release of [³H]dopamine ([³H]DA) and [³H]noradrenaline ([³H]NA) in acutely perfused rat striatal and cortical slice preparations was measured at 37 °C and 17 °C under ischemic conditions. The ischemia was simulated by the removal of oxygen and glucose from the Krebs solution. At 37 °C, resting release rates in response to ischemia were increased; in contrast, at 17 °C, resting release rates were significantly reduced, or resting release was completely prevented. The removal of extracellular Ca²⁺ further increased the release rates of [³H]DA and [³H]NA induced by ischemic conditions. This finding indicated that the Na⁺/Ca²⁺ exchanger (NCX), working in reverse in the absence of extracellular Ca²⁺, fails to trigger the influx of Ca²⁺ in exchange for Na⁺ and fails to counteract ischemia by further increasing the intracellular Na⁺ concentration ([Na⁺]_i). KB-R7943, an inhibitor of NCX, significantly reduced the cytoplasmic resting release rate of catecholamines under ischemic conditions and under conditions where Ca²⁺ was removed. Hypothermia inhibited the excessive release of [³H]DA in response to ischemia, even in the absence of Ca²⁺. These findings further indicate that the NCX plays an important role in maintaining a high [Na⁺]_i, a condition that may lead to the reversal of monoamine transporter functions; this effect consequently leads to the excessive cytoplasmic tonic release of monoamines and the reversal of the NCX. Using HPLC combined with scintillation spectrometry, hypothermia, which enhances the stimulation-evoked release of DA, was found to inhibit the efflux of toxic DA metabolites, such as 3,4-dihydroxyphenylacetaldehyde (DOPAL). In slices prepared from human cortical brain tissue removed during elective neurosurgery, the uptake and release values for [³H]NA did not differ from those measured at 37 °C in slices that were previously maintained under hypoxic conditions at 8 °C for 20 h. This result indicates that hypothermia preserves the functions of the transport and release mechanisms, even under hypoxic conditions. Oxidative stress (H₂O₂), a mediator of ischemic brain injury enhanced the striatal resting release of [³H]DA and its toxic metabolites (DOPAL, quinone). The study supports our earlier findings that during ischemia transmitters are released from the cytoplasm. In addition, the major findings of this study that hypothermia of brain slice preparations prevents the extracellular calcium concentration ([Ca²⁺]_o)-independent non-vesicular transmitter release induced by ischemic insults, inhibiting Na⁺/Cl^?-dependent membrane transport of monoamines and their toxic metabolites into the extracellular space, where they can exert toxic effects.

     

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.     

Serotonin,dopamine, noradrenaline and their metabolites: Levels in the brain of the house cricket (Acheta domesticus L.) during a 24-hour period and after administration of quipazine—a 5-HT2 receptor agonist

1. The levels of 5-HT, DA, NA and DA metabolites (NADA, DOPAC) measured by HPLC (with electrochemical detection) in the brain of the house cricket did not change over a 24-hr period. The level of 5-HIAA, a 5-HT metabolite, was below the limit of detection.2. The 5-HT and DOPAC levels decreased and NADA increased after quipazine injection but DA and NA levels did not change after it.3. [³H]Ketanserin was used to identify serotonin receptors bound to sites in the house cricket brain with a K_d of 5 nM and a concentration of B_max 180 fmol/mg protein.     

METHYLHISTAMINE: EVIDENCE FOR SELECTIVE DEAMINATION BY MAO B IN THE RAT BRAIN IN VIVO

Abstract— A new method has been developed for the separation of histamine and its metabolites after intracisternal injection of [³H]histamine into the rat brain, involving solvent extraction and subsequent thin-layer chromatography. The effect of graded doses of the MAO inhibitors deprenil and pargyline, which at relatively low doses inhibit preferentially the B form (phenethylamine deaminating) of the enzyme, and clorgyline, which mainly inhibits the A form (serotonin, noradrenaline and dopamine deaminating) on the brain levels of intracisternally injected [³H]histamine and its labelled metabolites was studied and compared to MAO A and B activity as determined with the substrates serotonin and phenethylamine, respectively. In addition, the time-course of the effects of a single dose of pargyline (50mg/kg subcutaneously) was investigated. No [³H]imidazoleacetic acid could be detected in any of the control or treated animals. [³H]Histamine accounted for 9–12% of the total extracted radioactivity and this was not altered significantly by pretreatment with any of the MAO inhibitors up to high doses, at which both MAO A and B activities were completely inhibited. In the controls, 40–43% of the total extracted radioactivity was [³H]methylhistamine and 28–30% was [³H]methylimidazoleacetic acid. Deprenil and pargyline caused [³H]methylhistamine levels to increase in a dose-dependent manner up to about 150% of control levels and those of [³H]methylimida-zoleacetic acid to decrease concomitantly to about 10% of control levels. Clorgyline in doses up to 10 mg/kg subcutaneously (s.c.) had no effect on the levels of these two metabolites. The dose-response curves of the effects of deprenil and pargyline on [³H]methylimidazoleacetic acid levels were congruent with those of the MAOI effects on MAO B activity and not with those on MAO A activity. Pargyline (50 mg/kg s.c.) had a long lasting effect on the accumulation of [³H]methylhistamine and [³H]methylimidazoleacetic acid. Recovery occurred within 21 days, and the half-lives observed were 5.3 and 5.6 days, respectively. This compares well to the half-life for the recovery of MAO B activity reported earlier after the same dose of pargyline (5.5 days). These results suggest that methylhistamine is metabolized selectively by MAO B in rat brain. Moreover, the fact that clorgyline, at doses where phenethylamine deamination is already considerably inhibited, did not affect the deamination of methylhistamine, suggests that the latter is an even more selective substrate for MAO B than phenethylamine itself. Therefore, small doses of deprenil (0.3–3 mg/kg s.c.) or pargyline (1–3 mg/kg) can be used to influence histamine catabolism without interfering with catecholamine or serotonin deamination.     

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.     

Roles for Nitric Oxide as an Intra- and Interneuronal Messenger at NMDA Release-Regulating Receptors: Evidence from Studies of the NMDA-Evoked Release of [3H]Noradrenaline and d-[3H]Aspartate from Rat Hippocampal Slices

Abstract: N-Methyl-d -aspartate (NMDA) receptors regulating the release of [³H]noradrenaline ([³H]NA) and d -[³H]aspartate (d -[³H]Asp) were investigated in superfused slices of rat hippocampus in the presence and absence of nitrergic drugs to examine a possible role for nitric oxide (NO) in the release process. In Mg²⁺-free Krebs-Henseleit buffer, the NMDA-evoked release of [³H]NA and d -[³H]Asp was Ca²⁺ dependent and inhibited by the NMDA antagonist (±)-3-(2-carboxypiperazin-4-yl)propenyl-1-phosphonic acid. NMDA-stimulated release of [³H]NA was tetrodotoxin (TTX; 0.1–2 µM) sensitive, whereas that for d -[³H]Asp was TTX insensitive, indicating that the NMDA receptors involved are differentially localized; those for d -[³H]Asp appear to be presynaptic, whereas those for [³H]NA are extrasynaptic in location. l -Arginine (100 µM), the natural precursor of NO synthesis, enhanced NMDA-evoked release of [³H]NA (100%) and d -[³H]Asp (700%). Exogenous NO donors—sodium nitroprusside, 3-morpholinosyndnomine, and S-nitroso-N-acetylpenicillamine (all 100 µM)—stimulated the NMDA-evoked release. An exception was the inhibition by nitroprusside of NMDA-evoked release of [³H]NA, where the presence of antioxidants may influence channel activity. Inhibitors of NO synthase (N^G-nitro-, N^G-methyl-, and N^G-amino-l -arginine, all 100 µM) attenuated (50–80%) the NMDA-stimulated release of [³H]NA and d -[³H]Asp, as did KN-62 (10 µM), a specific inhibitor of calmodulin kinase II. Our data support roles for the NO transducing system subsequent to the activation of NMDA release-regulating receptors as both an intraneuronal (presynaptically) and an extraneuronal messenger.     

EFFECTS OF 6-HYDROXYDOPAMINE ON CATECHOLAMINE CONTAINING NEURONES IN THE RAT BRAIN

After the intraventricular injection of 6-hydroxydopamine (6-OHDA), there was a long lasting reduction in the brain concentrations of noradrenaline (NA) and dopamine (DA). The brain concentration of NA was affected by lower doses of 6-OHDA than were required to deplete DA. A high dose of 6-OHDA which depleted the brain of NA and DA by 81 per cent and 66 per cent respectively, had no significant effect on brain concentrations of 5-hydroxytryptamine (5-HT) or γ-aminobutyric acid (GABA). The fall in catecholamines was accompanied by a long lasting reduction in the activities of tyrosine hydroxylase and DOPA decarboxylase in the hypothalamus and striatum, areas in the brain which are rich in catecholamine containing nerve endings. There was, however, no consistent effect on catechol-O-methyl transferase or monamine oxidase activity in these brain regions. The initial accumulation of [³H]NA into slices of the hypothalamus and striatum was markedly reduced 22–30 days after 6-OHDA treatment. These results are consistent with the evidence in the peripheral sympathetic nervous system that 6-OHDA causes a selective destruction of adrenergic nerve endings and suggest that this compound may have a similar destructive effect on catecholamine neurones in the CNS.     

Assay of catecholamines and dihydroxyphenylethyleneglycol in human plasma and its application in orthostasis and mental stress

A high performance liquid chromatographic method involving electrochemical detection is described which permits the assay of noradrenaline (NA), adrenaline (A), dopamine (DA), and dihydroxyphenylethyleneglycol (DOPEG) in human plasma and brings about analytical recoveries of 70% and more. This method was used to assess the effects of graded orthostasis and mental stress on the plasma levels of these catechols. Orthostasis elicited increases in plasma NA and DOPEG, but did not cause any change in plasma A and DA. The increases in NA and DOPEG were dependent on the degree of orthostasis and correlated closely (rs = 0.724; n = 30, P less than 0.001). Pretreatment with desipramine abolished the DOPEG response to standing, indicating that orthostasis - induced increases in plasma DOPEG are presynaptic in origin. Mental stress evoked pronounced increases in plasma A, less pronounced increases in plasma NA and no changes in plasma DA and DOPEG. Hence, the simultaneous measurement of plasma NA and DOPEG may help to distinguish between various types of activation of the sympathetic nervous system.