STUDIES OF AMINES IN THE STRIATUM IN MONKEYS WITH NIGRAL LESIONS

The effects of ventromedial tegmental lesions on the biosynthesis and disposition of biogenic amines in the striatum of monkeys were investigated. The concentrations of endogenous dopamine and of the intraventricularly injected [³H]dopamine were distinctly lower in the striatum on the lesion side than on the intact side. The storage of [³H]dopamine in the caudate nucleus was impaired to a much greater extent than the storage of the newly synthesized [³H]norepinephrine. The concentrations of endogenous serotonin and of the intraventricularly injected [¹⁴C]serotonin were lower in the striatum on the lesion side than on the intact side. However following MAO inhibition, the concentration of [¹⁴C]serotonin did not differ significantly on the two sides of the caudate nucleus. The in vivo biosynthesis of dopamine from tyrosine was significantly reduced in the striatum on the lesion side. Tyrosine hydroxylase and DOPA decarboxylase activities were decreased on the lesion side of the striatum as compared with the intact side. Thus, the ventromedial tegmental lesions affect the storage and the synthesis of dopamine and serotonin in the ipsilateral striatum.     

CHANGES IN SPECIFIC ACTIVITY OF DOPAMINE METABOLITES AS EVIDENCE OF A MULTIPLE COMPARTMENTATION OF DOPAMINE IN STRIATAL NEURONS

The functional status of dopaminergic nerve terminals has been studied with a method that allows the simultaneous determination of the specific activities of dopamine (DM), tyrosine (Tyr), 3-methoxytyramine (3-MT) and 3,4-dihydroxyphenylacetic acid (DOPAC), after administration of [³H]tyrosine ([³H]Tyr). Combined fluorimetric, mass fragmentographic and radiometric techniques have been used. [³H]Tyrosine was given intraventricularly to unanaesthetized rats and the animals were killed by exposure for 4 s to high energy microwave radiations. The specific activities of 3-MT and DOPAC measured 5 and 20 min after administration of [³H]Tyr, i. e. at time intervals in which the specific activity of DM is rising, are much higher than those their physiological precursor, suggesting that they are generated by more than one DM compartment. This hypothesis seems to be supported by the finding that in animals killed by decapitation instead of microwave radiations the post mortem accumulation of 3-MT occurs to a much smaller extent for the radioactive fraction than for the endogenous one, indicating that 3-MT formed after death may be mainly derived from DM coming from a compartment where this monoamine has been poorly labeled by the radioactive precursor.     

SHORT-TERM REGULATION OF CATECHOLAMINE BIOSYNTHESIS IN A NERVE GROWTH FACTOR RESPONSIVE CLONAL LINE OF RAT PHEOCHROMOCYTOMA CELLS

Abstract— A clonal cell line (designated PC12) has been previously established from a transplantable rat adrenal medullary pheochromocytoma. Tissue cultures of PC12 cells synthesize, store, release and take up catecholamines. PC12 cells also respond to nerve growth factor (NGF) protein by cessation of mitosis and extension of neurites. The present studies concern the comparison of several aspects of catecholamine metabolism in PC12 cultures with that in normal noradrenergic tissues. One question was why the ratio of dopamine to norepinephrine in PC12 cultures (in contrast to that in normal noradrenergic tissue) is considerably more than one. The presence of exogenous reduced ascorbate (a cofactor for dopamine-β-monooxygenase) enhanced by 5–10-fold the rate at which PC12 cultures converted [³H]tyrosine to [³H]norepinephrine. Under such conditions, the rate of synthesis of [³H]do-pamine was unchanged. It was also found that the ratio of norepinephrine to dopamine increased by 10-fold when the cells were grown in vivo as tumors. Since tissue culture medium is essentially free of reduced ascorbate, it is likely that the absence of this cofactor is responsible for the low norepinephrine to dopamine ratio in PC12 cultures. Experiments were also carried out on short-term regulation of catecholamine synthesis in PC12 cultures. These studies revealed the following: (1) The rate of conversion of [³H]tyrosine to [³H]catechols was increased 2–3-fold (as compared with controls) in the presence of depolarizing levels of K⁺ (51.5 mM), and by 2-fold in the presence of 0.5–2 mM-dibutyryl cyclic adenosine 3′, 5’monophosphoric acid (db-cAMP). (2) Similar increases occurred in cultures which had been treated with (and had responded to) nerve growth factor. (3) The stimulatory effects of 51.5 mM-K⁺ rapidly returned toward control levels when the cultures were returned to control medium and (4) required the presence of Ca²⁺ in the extracellular medium. (5) Stimulation of catechol synthesis by 51.5 mM-K⁺ and db-cAMP also occurred in the presence of an inhibitor of DOPA decar-boxylase. Thus, the ultimate effects of these agents were probably at the level of conversion of tyrosine to dopa by tyrosine 3-monooxygenase. (6) Simultaneous exposure of cultures to 51.5 mM-K⁺ and mM-db-cAMP gave additive levels of stimulation. Such findings demonstrate that catecholamine synthesis in cultures of PC12 cells undergoes short-term regulation which is similar to that previously demonstrated in normal monoaminergic tissues. As a homogeneous tissue culture line, the PC12 bears certain advantages for studying the primary mechanisms of such effects.     

AN ADAPTATION OF THE PUSH-PULL CANNULA METHOD TO STUDY THE IN VIVO RELEASE OF [3H]DOPAMINE SYNTHESIZED FROM [3H]TYROSINE IN THE CAT CAUDATE NUCLEUS: EFFECTS OF VARIOUS PHYSICAL AND PHARMACOLOGICAL TREATMENTS

Abstract— The release of [³H]dopamine ([³H]DA) continuously synthesized from l-[3,5–³H]tyrosine from the caudate nucleus of the cat was estimated in halothane anaesthetized or‘encéphale isolé’animals. For this purpose, an improved superfusion cannula, avoiding tissue damage, was used. The best localization for the tip of the superfusion cannula was found first by determining the topographical distribution of endogenous DA within the caudate nucleus. A rostro-caudal heterogenous distribution of the transmitter was detected. In perfusion experiments, l-[3,5–³H]tyrosine was introduced continuously at a rate of 33μl/min. [³H]DA was the only catecholamine found in serial 15 min fractions as revealed by cochromatography. The spontaneous release of [³H]DA was greater in anaesthetized than in ‘encéphale isolé’ cats; it represented 150 and 100 times the blank value, respectively. Depolarization by K⁺ (30 mm) applied locally in the striatum or by electrical or mechanical stimulation of the substantia nigra caused a transitory increase in [³H]DA release. Conversely, a decrease in nerve activity induced by tetrodotoxin (5 × 10^?-⁷ m) or by electrocoagulation of the substantia nigra was associated with a decline in the amounts of [³H]DA in superfusates. A temporary reduction in [³H]DA release could also be obtained by a short-lasting cooling block of the substantia nigra. As expected, d-amphetamine (10^?-⁵ m) and benzotropine(10^?-⁷ m) added to the superfusing medium increased [³H]DA release. These pharmacological results, as well as the changes in [³H]DA release observed after various manipulations of the activity of dopaminergic neurones, confirms the validity and the high sensitivity of this approach.     

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.     

ACCUMULATION AND DISAPPEARANCE OF NORADRENALINE AND ITS MAJOR METABOLITES SYNTHESIZED FROM INTRAVENTRICULARLY INJECTED [3H]DOPAMINE IN THE RAT BRAIN

Abstract— A new combined ion-exchange and thin-layer-chromatographic procedure is described which separates and measures quantitatively, after intraventricular injection of [³H]dopamine (DA), the rat brain content of labelled noradrenaline (NA) and the following labelled noradrenaline metabolites: free 3-methoxy-4-hydroxyphenylethyleneglycol (MOPEG), conjugated MOPEG, free plus conjugated dihydroxyphenylethyleneglycol (DOPEG), vanillic mandelic acid (VMA) and normetanephrine (NM). Labelled dopamine and its metabolites were also measured. The time-course study performed from 5 min to 24 h after [³H]DA showed that MOPEG and DOPEG, mainly as conjugates, are major NA metabolites whereas VMA is a very insignificant NA metabolite in the rat brain. A very rapid initial increase of [³H]NM, free MOPEG and conjugated MOPEG was found during the time interval where the [³H]NA biosynthesis is very high (0–15 min). This combined with the finding that these metabolites stabilize at lower levels during the [³H]NA ‘storage phase’ (9–24 h) provides a strong indication that newly synthesized NA preferentially is metabolized. Our measurements of endogenous NA, free MOPEG and conjugated MOPEG provide additional support. The injections of various decreasing doses of [³H]DA (3·08–0·0010 μg) showed that the proportions of total [³H]MOPEG and total [³H]DOPEG to [³H]NA were constant after all [³H]DA doses investigated. This finding indicates that the [³H]NA synthesized in situ behaves as a tracer, even after injections of non-tracer doses of [³H]DA. The results seem thus to indicate that the present technique provides a powerful tool for the investigations on central noradrenaline metabolism.     

l-Glutamic acid,a neuromodulator of dopaminergic transmission in the rat corpus striatum

A superfusion system was used to study the effects of neuroexcitatory amino acids upon spontaneous and depolarization-evoked release of exogenously taken up and newly synthesized [³H]dopamine by rat striatal slices. Neither l-glutamate nor other aminoacids such as l-aspartate and d-glutamate (5 × 10^?5 M) modified the spontaneous release of exogenous [³H]dopamine from rat striatal slices. In contrast, these neuroexcitatory aminoacids did potentiate spontaneous release of striatal [³H]dopamine newly synthesized from [³H]tyrosine. A different pattern of effects emerged when depolarization-evoked release of dopamine was studied. Only l-glutamate (5 × 10^?6-1 × 10^?4 M) potentiated dopamine release under these experimental conditions in a rather specific and stereoselective manner. In addition, similar results were obtained regardless of whether depolarization-induced release of exogenous or newly synthesized [³H]dopamine was studied. The effect of l-glutamate on depolarization-induced release depended both upon the degree of neuronal depolarization and upon the presence of external Ca²⁺ in the superfusion medium and it was blocked by l-glutamate diethylester. Furthermore, this effect of l-glutamate seemed quite specific with regard to regional localization within the brain as it was only demonstrated in slices from striatum and not in slices from olfactory tubercle or hippocampus. It is suggested that during depolarization a Ca²⁺-dependent event occurs at the striatal membrane level which changes the sensitivity of the dopamine release process to neuroexcitatory aminoacids in such a way as to render it relatively more specific and stereoselective towards l-glutamate stimulation. The findings reported have led us to propose that l-glutamic acid could play a role as a neuromodulator of dopaminergic transmission in the rat corpus striatum.     

Newly Synthesized Dopamine as the Precursor for Norepinephrine Synthesis in Bovine Adrenomedullary Chromaffin Cells

The precursor pool of dopamine for norepinephrine synthesis was investigated in cultured bovine adrenomedullary chromaffin cells incubated with [14C]tyrosine. Under conditions where the intracellular [14C]tyrosine specific activity was constant and [14C]dopamine synthesis was maximal, [14C]dopamine and [14C]norepinephrine accumulated over time, and the total intracellular dopamine content more than doubled within 120 min. When [14C]norepinephrine synthesis was calculated at different times based on the specific activity of [14C]dopamine, this rate was approximately equal to the rate of [14C]dopamine synthesis and was, thus, inconsistent with the observed dopamine accumulation. However, the rate of [14C]norepinephrine synthesis based on the [14C]tyrosine specific activity accounted for the dopamine accumulation, an observation suggesting that newly synthesized dopamine, i.e., dopamine with a specific activity equivalent to that of its precursor, [14C]tyrosine, is preferentially utilized for norepinephrine synthesis. Further studies showed that the subcellular distribution of [14C]dopamine was identical to that of norepinephrine and epinephrine and that the accumulated [14C]dopamine could be converted to norepinephrine within the chromaffin vesicle if dopamine uptake was blocked. Taken together, these results suggest that a small intravesicular dopamine pool, rapidly replenished by newly synthesized dopamine, serves as the substrate for dopamine beta-hydroxylase. Several mechanisms to account for this observation are discussed.     

THE UPTAKE OF BIOGENIC MONOAMINES BY THE ISOLATED AURICLE OF THE SNAIL (HELIX POMATIA)

—The uptake of [³H]5HT, [³H]dopamine, [³H]noradrenaline and [³H]octopamine into the auricle of Helix pomatia was studied. When tissues were incubated at 25°C in media containing radioactive amines, tissue:medium ratios of about 49:1, 14:1 and 5:1 for 5-HT, dopamine, noradrenaline, and octopamine respectively were obtained after a 20–30 min incubation time. Tissues incubated at 25°C in media containing radioactive amines for 20–30 mins showed that almost all (96%) the radioactivity was present as unchanged [³H]5-HT, [³H]dopamine, [³H]octopamine or [³H]noradrenaline. The high tissue:medium ratios for 5-HT and dopamine, but not for noradrenaline and octopamine, showed saturation kinetics which were dependent upon temperature and sodium ions. From the Lineweaver–Burk plots, two uptake mechanisms for 5-HT at 25°C were resolved; the high affinity uptake process having a K_m1 value of 6.0 ± 10^?8m and a V_m1 value of 0.115 nmol/g/min while the lower affinity process had a K_m2 value of 1.04 ± 10^?6m and a V_m2 value of 0.66nmol/g/min. At 0°C a single uptake mechanism for 5-HT occurred which gave a K_m value of 5.02 ± 10^?8m and a V_m value of 0.0165 nmol/g/min. In the case of dopamine, the Lineweaver–Burk plot at 25°C showed a single uptake process with values for K_m and V_m of 1.55 ± 10^?7m and 0.086 nmol/g/min respectively. This process did not function at 0°C. The effect of various agents and ions upon the accumulation processes for all amines was also studied, and the data indicate that the same neurons probably accumulate more than one amine type. It is concluded that 5-HT and dopamine uptake in the auricle is a mechanism for inactivating these substances at 25°C and that an uptake mechanism for 5-HT also functions at 0°C. The results are discussed from the point of view of 5-HT's being the cardioexcitatory substance in the snail heart.     

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.     

IN VIVO RELEASE OF ENDOGENOUSLY SYNTHESIZED CATECHOLAMINES FROM THE CAT BRAIN EVOKED BY ELECTRICAL STIMULATION AND BY d-AMPHETAMINE

Abstract— The cerebral ventricles of spinal-sectioned cats were perfused with artificial cerebrospinal fluid after the intraventricular administration of [³H]DOPA or [³H]tyrosine. Endogenously synthesized [³H]dopamine or [³H]norepinephrine were identified in the perfusate. Electrical stimulation of catecholaminergic nerve tracts in the hypothalamus increased the efflux of both catecholamines. The addition of d -amphetamine to the perfusing cerebrospinal fluid caused a large increase in [³H]dopamine and a small increase in [³H]norepinephrine appearing in the perfusate. Most of the endogenously synthesized [³H]catecholamines detected in the perfusate following stimuli originated from structures bordering the lateral cerebral ventricle. Thus, norepinephrine and dopamine can be synthesized in and released from catecholaminergic nerve terminals in structures bordering the cerebral ventricles.     

Amphetamine-induced release of dopamine from the substantia nigra in vitro

Incubation of chopped tissue from the substantia nigra of the rat brain with d-amphetamine resulted in a significant release of [³H]dopamine into the incubation medium. This effect was observed with both exogenous [³H]dopamine previously taken up by the tissue and [³H]dopamine endogenously synthesized from L-[3,5-³H]tyrosine. The observed release was greater in magnitude when the apparent conversion of released dopamine to 3-methoxytyramine was taken into account. The relevance of the present results to the previously postulated self-inhibition by dopaminergic neurons of the substantia nigra pars compacta is discussed. The present data also provide support for the concept that catechol-O-methyltransferase (E.C.2.1.1.6.) is located primarily extraneuronally in brain.     

THE UPTAKE AND RELEASE OF [3H]DOPAMINE IN THE GOLDFISH RETINA

Abstract— The uptake and release of [³H]dopamine was studied in the goldfish retina with the following results: (1) when goldfish retinas were incubated with 2 ± 10^-7m -[³H]dopamine for less than 20min and processed for autoradiography. most of the label was associated with dopaminergic terminals that contact certain horizontal cells. Biochemical analysis showed that > 93% of this label was [³H]-dopamine. (2) [³H]dopamine uptake saturated with increasing dopamine concentration and followed Michaelis-Menten kinetics. This uptake could be explained by a single ‘high-affinity’ mechanism with a K_m of 2.61 ± 0.41 ± 10^-7m and a V_max of 66 ± 12 ± 10^-12 mol/min/mg protein. (3) [³H]dopamine uptake was temperature-dependent with a temperature coefficient of 1.7 and an energy of activation of 11.4 kcal/mol. (4) The initial rate of uptake was unaffected by the absence of Ca²⁺ or the presence of Co²⁺; however, more than 85, uptake was blocked in the absence of external Na⁺. (5) Neither 1 mm -cyanide nor 5 mm -iodoacetate blocked more than 30% of uptake individually; however, in combination > 70% of uptake was blocked. (6) Centrally acting drugs benztropine and diphenylpyraline inhibited at least 60–70% of [³H]dopamine uptake. (7) [³H]dopamine in the retina could be released by increasing the external K⁺ concentration. This release was Ca²⁺ -dependent and was blocked by 10mm -Co²⁺ or 2Omm -Mg²⁺. The amount of [³H]dopamine released was not affected by the presence of benztropine, diphenylpyraline or fluphenazine in the incubation medium. These studies add further support for dopamine as a neurotransmitter used by interplexiform cells of the goldfish retina.     

AUTORADIOGRAPHIC STUDIES OF SYNTHESIS AND INTRACELLULAR MIGRATION OF GLYCOPROTEINS IN THE RAT ANTERIOR PITUITARY GLAND

The incorporation of [³H]fucose in the somatotrophic and gonadotrophic cells of the rat adenohypophysis has been studied by electron microscope autoradiography to determine the site of synthesis of glycoproteins and to follow the migration of newly synthesized glycoproteins. The pituitaries were fixed 5 min, 20 min, 1 h, and 4 h after the in vivo injection of [³H]fucose and autoradiographs analyzed quantitatively. At 5 min after [³H]fucose administration, 80–90% of the silver grains were localized over the Golgi apparatus in both somatotrophs and gonadotrophs. By 20 min, the Golgi apparatus was still labeled and some radioactivity appeared over granules. At 1 h and 4 h, silver grains were found predominantly over secretory granules. The kinetic analysis showed that in both protein-secreting cells (somatotrophs) and glycoprotein-secreting cells (gonadotrophs), the glycoproteins have their synthesis completed in the Golgi apparatus and migrate subsequently to the secretory granules. It is concluded from these in vivo studies that glycoproteins which are not hormones are utilized for the formation of the matrix and/or of the membrane of the secretory granules. The incorporation of [³H]fucose in gonadectomy cells (hyperstimulated gonadotrophs) was also studied in vitro after pulse labeling of pituitary fragments in medium containing [³H]fucose. The incorporation of [³H]fucose was localized in both the rough endoplasmic reticulum (ER) and the Golgi apparatus. Later, the radioactivity over granules increased while that over the Golgi apparatus decreased. The concentration of silver grains over the dilated cisternae of the rough ER was not found to be modified at the longest time intervals studied.     

Uptake of (3H)catecholamines by chick embryo sympathetic ganglia in tissue culture

Abstract— Chick embryo sympathetic chains were grown in tissue culture and pulse labelled with tritiated catecholamines. The uptake was restricted to sympathetic nerve cells. The capability of these cells to take up radioactive dopamine and norepinephrine from the culture media was retained after one month in tissue culture. The uptakes of both [³H]norepinephrine and [³H]dopamine were inhibited when nonradioactive DOPA, dopamine, norepinephrine or epinephrine were present in the pulse media.     

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.     

PRODUCTS OF BIOGENIC AMINE METABOLISM IN THE LOBSTER: SULFATE CONJUGATES

Octopamine, dopamine and serotonin, the three biogenic amines found in the lobster nervous system, are each converted by lobster tissues into two principal classes of products, A and B metabolites. In this paper, evidence is presented that the B metabolites are sulfate conjugates of the amines and their A metabolites. Two double-labelled conjugates were formed from each of the three amines during incubations of lobster nerves with tritiated amine and ³⁵SO₄. When the two octopamine conjugates were hydrolyzed by mild acid, one of the conjugates was converted to a mixture of ³⁵SO₄ and [³H]-octopamine, and the other to a mixture of ³⁵SO₄, [³H]octopamine, and [³H]metabolite A. [³H]Metabolite A was also converted to octopamine by acid hydrolysis. The results indicated that one of the double-labelled conjugates was octopamine-sulfate, and the other metabolite-A-sulfate. An enzyme fraction prepared from nerve homogenates catalyzed the synthesis of double-labelled sulfate conjugates from the tritiated amines and [³⁵S]3′-phosphoadenosine-5′-phospho-sulfate. Double-labelled conjugates formed in this way contained 1 mol of sulfate per mol of amine. Indirect evidence suggested that the sulfate was in ester linkage with the ring hydroxyls of the amines. Neither monoamine oxidase, nor catechol-O-methyl transferase is found in lobster tissues; therefore, in these animals, sulfation may be a major means of inactivation of the biogenic amines following their release from nerve endings.     

THE RELEASE IN VIVO OF DOPAMINE SYNTHESIZED FROM LABELLED PRECURSORS IN THE CAUDATE NUCLEUS OF THE CAT

Abstract— To study the release of dopamine (DA) evoked in vivo from the caudate nucleus, a push-pull cannula was inserted into the head of the caudate nucleus of cats anaesthetised with pentobarbitone sodium (Nembutal), and the tissue in the vicinity of the cannula tip was continuously irrigated with either l -[¹⁴C]tyrosine or DL-[¹⁴C]3,4-dihydroxyphenylala-nine (DOPA). The contents of [¹⁴C]DA and of the [¹⁴C]acidic metabolites in the perfusates were determined after separation from the labelled precursors by column chromatography, TLC and solvent partition. During perfusion with radioactive tyrosine, only small quantities of [¹⁴C]DA appeared in the effluent while the concentrations of the [¹⁴C]acidic metabolites gradually increased during the course of the experiment. When [¹⁴C]DOPA was substituted for [¹⁴C]tyrosine, the proportion of precursor that was converted to DA and released into the effluent as the amine or as its acidic metabolites was increased ten-fold. In an attempt to increase the resting release of [¹⁴C]DA, D-amphetamine, tropolone or pheniprazine were individually added to the perfusion fluid. Each drug increased the content of [¹⁴C]DA in the perfusate, but the enhanced release was maintained only when pheniprazine was added during perfusion with [¹⁴C]DOPA. Stimulation of the rostral substantia nigra (A5-5) and the medial forebrain bundle caused, in a majority of experiments, a two-to five-fold increase in the concentration of labelled DA in the effluent. Stimulation of the substantia nigra at A4-0 did not enhance the release of [¹⁴C]DA from the caudate nucleus but did enhance the release from the putamen. Since the increase in the output of [¹⁴C]DA was independent of changes in the output of labelled acidic metabolites, the evoked release was apparently not attributable to changes in extracellular fluid dynamics.     

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.     

DYNAMIC CHARACTERISTICS OF THE ''FUNCTIONAL COMPARTMENT'' OF DOPAMINE IN DOPAMINERGIC TERMINALS OF THE RAT STRIATUM

Rats were injected with α-methyl-p-tyrosine and the disappearance of dopamine in the striatum was examined as a function of time. Two main phases of dopamine decline were detected which probably correspond to the amine utilization in a ‘functional’ and a ‘main storage compartment’ in dopaminergic terminals. Similar observations were made when the turnover of dopamine had been previously accelerated by thioproperazine. In control rats the half-lives of dopamine were about 9 and 120 min in the ‘functional’ and ‘main storage’ compartments respectively. The rate of dopamine synthesis was estimated to be not less than 13-2 μg/g/h and the rate of dopamine utilization in the ‘functional compartment’ was calculated to be about four times that of the ‘main storage compartment'.