Biosynthesis of catecholamines in organotypic cultures of peripheral autonomic nervous system: modifications by biopterin and other agents.

Organotypic cultures of chick-embryo sympathetic ganglion chains maintained in vitro for 3-4 weeks rapidly synthesized catecholamines, as demonstrated by the conversion of L-[U-14C]tyrosine to catechol derivatives and by histofluorescence assay. The biosynthesis of catechols from radioactive L-tyrosine leveled off at 6 hr of incubation and dropped slightly at 10 hr. The addition of DL-alpha-methyl-p-tyrosine to the culture medium did not affect protein synthesis, but produced a complete block in the synthesis of catecholamines from L-tyrosine, with consequent loss of fluorescence in the bodies and proximal processes of adrenergic neurons in 2 hr, and essentially complete loss in 6 hr. Our observations suggest that a major portion of the catecholamines were synthesized in the perikarya and transported via neuronal processes to their terminals. The addition of monoamine oxidase inhibitors to the incubation medium produced a moderate to pronounced increase in fluorescence; reserpine caused a rapid and profound loss of catecholamines. When added to the culture medium, crude biopterin produced an increase in the synthesis of catechol derivatives from radioactive L-tyrosine and a marked increase in fluorescence, beginning in the neuronal perikarya. This effect was completely blocked by DL-alpha-methyl-p-tyrosine. The mechanism of biopterin's action in the synthesis of catecholamines in cultures of sympathetic ganglia is not completely elucidated from these studies, but may be related to the role it plays as cofactor for tyrosine hydrocylase.     

Microfluorimetric quantitation of catecholamine turnover in the sympathetic neurons of rat

Summary The quantitative aspects of the formaldehydeinduced fluorescence and the turnover of catecholamines in the sympathetic neuronal perikaryon of different sympathetic ganglia were studied after a blockade of the amine synthesis with -methyltyrosine. The concentration of catecholamines was determined by microfluorimetric quantitation method. The half-life of catecholamines in sympathetic neuronal perikarya was short and depended on the ganglion studied. The turnover rate of catecholamines in sympathetic neurons was highest in superior cervical and lowest in coeliac ganglion. Brightly fluorescent fibers were still seen five hours after the amine synthesis blockade, whereas almost all cell bodies had lost their fluorescence. Also small intensely fluorescent cells were still brightly fluorescent after the follow-up period. Microfluorimetrically determined turnover of catecholamines gave more detailed information about the turnover of catecholamines in sympathetic nervous system when compared to the biochemical methods used earlier.     

Effect of Block of Catecholamine Synthesis on the Functional State of Vasopressinergic Neurons of Hypothalamus in Dehydrated Rats

Effect of catecholamines (CA) on the functional state of vasopressin (VP)-ergic neurons of hypothalamus at their stimulation produced by dehydration (salt diet and water deprivation) was studied in in vivo experiments on adult male Wistar rats. Quantitative assessment of VP-immunopositive substance and digoxigenin-labeled VP mRNA (hybridization in situ) in neurosecretory cells of supraoptic (SON) and paraventricular (PVN) nuclei was performed using measurements of optical density of the stained substance in perykaria and a computer digital television analyzer with PhotoM software. Hybridization in situ technique allowed evaluating intensity of VP synthesis, while comparison of the amount of VP mRNA and VP-immunoreactive substance in neurons of SON and PVN made it possible to evaluate release of VP from perykaria. In PVN, repeated saline administration (0.25 ml per 100 g weight) and severe dehydration led to activation both of synthesis and of release of VP from cell perikarya. Use of -methyl-p-tyrosine, an inhibitor of catecholamine (CA) synthesis on the background of dehydration was not accompanied by changes of the functional state of VP-ergic neurons of PVN as compared with dehydrated animals. No changes in functional state of VP-ergic neurosecretory cells in SON were found after saline administration, whereas dehydration activated synthesis and release of VP from perykaria, like in VP-ergic neurons of PVN. Inhibition of CA synthesis on the background of dehydration led to activation of VP release by SON neurons without affecting the level of VP synthesis. The data obtained indicate that CA is able to suppress the VP release from neurons of SON, which is produced caused by activation of the VP-ergic system under conditions of dehydration.     

Influence of Catecholamines on Migration and Differentiation of GnRH Neurons in Rats

The GnRH producing neurons are the key link of neuroendocrine regulation of the adult reproductive system. Synthesis and secretion of GnRH are, in turn, under the afferent catecholaminergic control. Taking into account that catecholamines exert morphogenetic effects on target cells during ontogenesis, this study was aimed at investigation of the effects of catecholamines on development of GnRH neurons in rats during ontogenesis. We carried out comparative quantitative and semiquantitative analyses of differentiation and migration of GnRH neurons in fetuses of both sexes under the conditions of normal metabolism of catecholamines (administration of saline) or their pharmacologically induced deficiency (administration of -methyl-para-tyrosine). The inhibition of catecholamine synthesis from day 11 of embryogenesis led to an increasing number of GnRH neurons in rostral regions of the trajectory of their migration over the brain: in the area of olfactory tubercles on day 17 and in the area of olfactory bulb on days 18 and 21. In addition, the optical density of GnRH neurons located in the rostral regions of migration was higher in the fetuses after administration of -methyl-para-tyrosine during embryogenesis, as compared to the control. It has been concluded that catecholamines stimulate the migration of GnRH neurons and affect their differentiation.     

Tyrosine availability prevents tyramine-induced tachyphylaxis in the isolated rat heart

Isolated rat hearts perfused with Chenoweth-Koelle solution exhibit tachyphylaxis to tyramine. This tachyphylaxis was prevented if tyrosine was included in the perfusion solution. Other amino acids, including glycine, serine, phenylalanine, valine, tryptophan, glutamate, aspartate, arginine, lysine and -tyrosine, failed to prevent the tyramine-induced tachyphylaxis. Hearts from reserpinized animals showed increased chronotropy after tyramine only when tyrosine was present in the medium. This response could be blocked by , m-hydroxybenzyl-hydrazine and d,l-propanolol, indicating that it was mediated by the synthesis and release of catecholamines. These experiments suggest that sympathetic nerves in the isolated rat heart exhibit a requirement for tyrosine when catecholamine release is enhanced.     

Light and electron microscopic histochemistry of the monoamines in the human foetal sympathetic ganglion in culture

Synopsis Sympathetic ganglia of 13 to 19-week-old human foetuses were cultured in small pieces with and without nerve growth factor for up to 5 weeksin vitro. The cultures were studied using phase-contrast, fluorescence and electron microscopy. Monoamines were demonstrated with the formaldehyde-induced fluorescence method, with and without pretreatment of the cultures with catecholamines or monoamine oxidase inhibitor.In the long-term cultures, primitive sympathetic cells, sympathicoblasts of types I and II, and young sympathetic neurons showed a fine structure identical to that described earlierin vivo. There were virtually no satellite or Schwann cells in the cultures. The neurons showed a considerable capacity to grow new nerve fibres in culture, even without nerve growth factor. Nerve terminals with accumulations of other nervous structures. Large granular vesicles were regularly found in the sympathicoblasts after glutaraldehyde-osmium tetroxide fixation. After permanganate fixation, dense-cored vesicles typical of adrenergic neurons were not seen, either in the perikarya, or in the processes, although it was possible to demonstrate specific fluorescence. No small intensely fluorescent (SIF) cells were observed.Variable formaldehyde-induced fluorescence was observed in the nerve cell perikarya and nerve fibres. The intensity of the fluorescence increased after treatment of the cultures with monoamine oxidase inhibitor and after incubation with catecholamines.     

PRIMARY CULTURES OF DISSOCIATED SYMPATHETIC NEURONS : II. Initial Studies on Catecholamine Metabolism

Initial studies are reported on the catecholamine metabolism of low-density cultures of dissociated primary sympathetic neurons. Radioactive tyrosine was used to study the synthesis and breakdown of catecholamines in the cultures. The dependence of catecholamine synthesis and accumulation on external tyrosine concentration was examined and a concentration which is near saturation, 30 µM, was chosen for further studies. The free tyrosine pool in the nerve cells equilibrated with extracellular tyrosine within 1 h; the total accumulation of tyrosine (free tyrosine plus protein, catecholamines, and metabolites) was linear for more than 24 h of incubation. Addition of biopterin, the cofactor of tyrosine hydroxylase, only slightly enhanced catecholamine biosynthesis by the cultured neurons. However, addition of reduced ascorbic acid, the cosubstrate for dopamine β-hydroxylase, markedly stimulated the conversion of dopamine (DA) to norepinephrine (NE). Phenylalanine, like tyrosine, served as a precursor for some of the DA and NE produced by the cultures, but tyrosine always accounted for more than 90% of the catecholamines produced. The DA pool labeled rapidly to a saturation level characteristic of the age of the culture. The NE pool filled more slowly and was much larger than the DA pool. The disappearance of radioactive NE and DA during chase experiments followed a simple exponential curve. Older cultures showed both more rapid production and more rapid turnover of the catecholamines than did younger cultures, suggesting a process of maturation.     

The effect of various amino acids and drugs on thepara-andmeta-hydroxyphenylacetic acid concentrations in the mouse caudate nucleus

Injection ofl-p-tyrosine (800 mg/kg, 2 h) increased the mouse striatalpara-hydroxyphenylacetic acid (p-HPAA) concentrations. A smaller dose ofd,l-m-tyrosine (20 mg/kg, 2h) produced a larger increase in mouse striatalmeta-hydroxyphenylacetic acid (m-HPAA) concentrations. The administration ofl-phenylalanine to mice caused a slight increase in thep-HPAA concentrations in the corpus striatum after 2h while a larger dose ofl-phenylalanine (800 mg/kg) produced a greater increase. Eight hours followingl-phenylalanine injection,p-HPAA concentrations were still elevated. Withd-phenylalanine a significant increase was observed at eight hours after drug administration.Two drugs which reduce dopamine synthesis, -methyl-para-tyrosine and apomorphine, decreasedm-HPAA striatal concentrations without affectingp-HPAA concentrations. From these results, it is proposed that tyrosine hydroxylase activity determinesp-HPAA concentrations by regulatingp-tyrosine availability. This enzyme may also synthesizem-tyrosine which is subsequently decarboxylated to formm-tyramine and then oxidatively deaminated to formm-HPAA.     

Chronic depolarization stimulates norepinephrine transporter expression via catecholamines

Chronic depolarization increases norepinephrine (NE) uptake and expression of the norepinephrine transporter (NET) in sympathetic neurons, but the mechanisms are unknown. Depolarization of sympathetic neurons stimulates catecholamine synthesis, and several studies suggest that NET can be regulated by catecholamines. It is not clear if the depolarization-induced increase in NET is because of nerve activity per se, or is secondary to elevated catecholamines. To determine if induction of NET mRNA was a result of increased catecholamines, we used pharmacological manipulations to (i) inhibit tyrosine hydroxylase activity in neurons depolarized with 30 mm KCl, thereby preventing increased catecholamines, or (ii) stimulate tyrosine hydroxylase activity in the absence of depolarization. Inhibiting the depolarization-induced increase in catecholamines prevented the up-regulation of NET mRNA, but did not block the increase in tyrosine hydroxylase (TH) mRNA. Furthermore, stimulating catecholamine production in the absence of depolarization elevated NE uptake, NET protein, and NET mRNA in sympathetic neurons. Similarly, elevating endogenous catecholamines in SK-N-BE2M17 neuroblastoma cells increased NE uptake and NET expression. These data suggest that chronic depolarization of sympathetic neurons induces NET expression through increasing catecholamines, and that M17 neuroblastoma cells provide a model system in which to investigate catechol regulation of NET expression.     

On the induction of tyrosine aminotransferase in rat liver by α-methyl-p-tyrosine

Hepatic tyrosine aminotransferase (l-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5) is known to be induced by α-methyl-p-tyrosine, a well-known catecholamine depletor, in both intact and adrenalectomized rats. The authors have studied this subject further and their results show that α-methyl-p-tyrosine does not influence the activity of tyrosine aminotransferase in the isolated, perfused liver and that hypophysectomy totally abolishes the induction of the enzyme by this agent. The involvement of hypophyseal hormones is discussed.     

Effect of divalent cations of the amphetamine-induced stimulation of [3H]catechol synthesis in the striatum.

Abstract— The effects of divalent cations on the stimulation of [³H]catechol formation in striatal slices induced by d-amphetamine was studied in order to determine the role of calcium in this action of amphetamine. In the absence of any divalent cations in the medium, amphetamine did not significantly stimulate [³H]catechol synthesis in striatal slices, but it produced a marked stimulation of synthesis when calcium (1.25 mm ) was added to the medium. In the presence of calcium (1.25 mm ), high concentrations of magnesium (15mm ), other divalent cations (2.5 mm ) such as barium, strontium, manganese and cobalt, as well as verapamil, inhibited the amphetamine-induced stimulation. When the slices were incubated in medium containing no divalent cations, the addition to the medium of either strontium, cobalt, zinc, or magnesium (2.5 mm ) could not support the amphetamine-induced stimulation of [³H]catechol synthesis, while the addition of barium resulted in a significant stimulation of synthesis. In contrast, the stimulation produced by amphetamine in the presence of manganese was comparable to that observed when calcium had been added to the medium. Since amphetamine did not alter the specific activity of [³H]tyrosine in the tissue in the presence of any of the divalent cations tested, the amphetamine-induced stimulation of [³H]catechol synthesis was probably due to an increase in tyrosine hydroxylase activity. Calcium and manganese were also able to support the stimulation of [³H]catechol synthesis in striatal slices induced by high potassium concentration. However, compared to the effects with amphetamine, manganese was much less effective than calcium in supporting the stimulation induced by high potassium concentration. These results show that specific divalent cations can support the stimulation of catechol synthesis induced by amphetamine in striatal slices, and suggest that the entry of these specific ions into cells, presumably dopamine neurons, is involved in this action.     

Catecholamines in Regulation of Development of GnRH Neurons of Rat Fetuses

The contents of dopamine, serotonin, and noradrenaline in rat fetuses developing under the conditions of their deficiency induced by administration of α-methyl-para-tyrosine to females during 11th to 16th or 20th day of pregnancy and in fetuses, whose mothers were given saline at the same time, were determined using HPLC with subsequent electrochemical detection. Administration of α-methyl-para-tyrosine led to decreased levels of dopamine and noradrenaline in the areas of migration of GnRH-neurons in fetuses on days 17 and 21 of prenatal development. The concentration of serotonin remained unchanged, except in the head nasal area in males on day 21. The areas of interaction between the brain catecholaminergic systems and migrating and differentiating GnRH-neurons were determined by double immunohistochemical labeling. Close topographical location of GnRH-immunoreactive neurons and tyrosine hydroxylase-immunoreactive in the area of nucleus accumbens on days 17 and 20, as well as in the median eminence on day 20. The GnRH concentration in the caudal areas of migration of GnRH-neurons under the normal conditions and in the case of catecholamine deficiency was determined using radioimmunoassay. After administration of α-methyl-para-tyrosine the GnRH concentration in the anterior hypothalamus decreased in females. The data obtained suggest the involvement of catecholamines in the regulation of development of GnRH-Neurons during prenatal development. In addition, the adequacy and efficiency of the used model of catecholamine deficiency for studying the development of such neurons was confirmed.     

PRIMARY CULTURES OF DISSOCIATED SYMPATHETIC NEURONS : I. Establishment of Long-Term Growth in Culture and Studies of Differentiated Properties

Rat sympathetic ganglia were disrupted by mechanical agitation to yield dissociated primary neurons, and the conditions for long-term growth in culture of the isolated neurons were examined. The neurons were grown with or without non-neural cells, simply by the addition or deletion of bicarbonate during growth in culture. Fluorescence histochemistry indicated that the isolated neurons contained catecholamines; incubations with radioactive precursors were used to verify the synthesis and accumulation of both dopamine and norepinephrine. The neurons also produced octopamine using tyramine as precursor, but not with tyrosine as the precursor. In the presence of eserine, older cultures synthesized and stored small amounts of acetylcholine. The cultures did not synthesize and accumulate detectable levels of radioactive γ-aminobutyric acid, 5-hydroxytryptamine, or histamine.     

PRIMARY CULTURES OF DISSOCIATED SYMPATHETIC NEURONS : III. Changes in Metabolism with Age in Culture

Several biochemical parameters of dissociated sympathetic neurons from superior cervical ganglia of the newborn rat were monitored as a function of age in culture. The neurons, which were grown in the virtual absence of non-neural cells, displayed a striking increase in their ability to synthesize and accumulate catecholamines. This capacity increased 50-fold during a 3-wk period in vitro, after which it appeared to reach a steady level. The major change took place during the second week. The time course of this change was not affected by plating the neurons at a higher cell density. The change in the catecholamine metabolism was far greater in magnitude and quite different in time course from the overall growth of the cells which was monitored by the incorporation of radioactive tyrosine into protein, lipid synthesis from radioactive choline, and incorporation of radioactive uridine into acid-precipitable material. Of the total tyrosine used by the cultures, the proportion devoted to catecholamine synthesis increased to 25% (a 10-fold rise) during the 3-wk period. This changing pattern of metabolism in the cultures suggested a process of maturation which may be similar to neuronal development in vivo.     

Microspectrofluorimetric identification of m-hydroxyphenylethylamines (m-tyramines) in central and peripheral monoamine neurons

Summary Catecholamines and m-tyramine derivatives form strongly fluorescent products on formaldehyde gas treatment according to the method of Falck and Hillarp with maximal emission at 480 and 420 m. respectively. Administration of the m-tyramine compounds m-tyrosine, -methyl-m-tyrosine and 4, -dimethyl-m-tyramine caused a depletion of the green fluorescence (max. 480 m) in both central and peripheral catecholamine neurons with a concomitant appearance of a blue fluorescence (max. 420 m) due to uptake and accumulation of the administered m-tyramine derivatives. Injection of metaraminol gave the same results in sympathetic adrenergic neurons while of the central catecholamine neurons only the dopamine nerves in the median eminence were depleted and showed uptake and accumulalation of metaraminol.Abbreviations used NA noradrenaline - DA dopamine - -MMT -methyl-m-tyrosine - 5-HT 5-hydroxytryptamine     

Are L-tyrosine and L-dopa hormone-like bioregulators?

Some amino acids have bioregulatory functions, which far exceed those of precursors for proteins or of substrates for specific enzymes. Two of these amino acids, L-tyrosine and L-dopa, are precursors to melanin and catecholamines. In vertebrates, they can act as inducers and regulators of the melanogenic apparatus and of MSH receptors--two quite complex functions that could hardly be performed by mere substrates. Focussing on the pigmentary system as a study model, we therefore explore the hypothesis that L-tyrosine and L-dopa act as hormone-like bioregulators in mammals, with melanocytes regulating tyrosine and dopa activity via their metabolic consumption.     

TYROSINE HYDROXYLASE IN BOVINE CAUDATE NUCLEUS

Approximately 80 per cent of tyrosine hydroxylase activity in bovine caudate nucleus was particle-bound. The rest of the activity was found in the soluble fraction. The enzyme activity in crude tissue preparations was inhibited, probably by the presence of endogenous inhibitors. Dilution of crude tissue preparations such as the crude mitochondrial fraction caused an increase in the specific activity. The particle-bound enzyme was solubilized by incubation with trypsin. The presence of deoxycholate increased the degree of solubilization. The activity of the solubilized enzyme from the washed particles was also inhibited, but the subsequent purification by ammonium sulphate could eliminate the inhibition. The solubilized enzyme was partially purified by ammonium sulphate fractionation and Sephadex G-150 chromatography. A tetrahydropteridine and ferrous ion were required as cofactors for the partially purified enzyme. Among various divalent cations, only ferrous ion could activate the partially purified enzyme. The enzyme was inhibited by L-α-methyl-p-tyrosine and catecholamines such as dopamine. The optimum pH was found between 5.5 and 6.0. K_m values toward tyrosine, 2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine and Fe²⁺, were approximately 5 × 10^?5 M, 1 × 10^?4 M and 4 × 10^?4 M, respectively.     

Hydrocortisone-induced increase in the number of small intensely fluorescent cells and their histochemically demonstrable catecholamine content in cultures of sympathetic ganglia of the newborn rat

Synopsis It is known that hydrocortisone causes a great increase in the number of small intensely fluorescent (SIF) cells in the sympathetic ganglia when injected into newborn rats. The effect of hydrocortisone on nervous tissuein vitro has not been studied previously.Pieces of newborn rat sympathetic ganglia were cultivated in Rose chambers. Hydrocortisone was dissolved in the medium in concentrations of 1–9 mg/l. Both control and hydrocortisone-containing cultures were examined daily by phase-contrast microscopy, and the catecholamines were demonstrated histochemically by formaldehyde-induced fluorescence after 7 days in culture.All cultures showed outgrowths of axons and supporting cells elements, although these were less extensive in the groups of cultures with hydrocortisone. After a week, SIF cells with a green fluorescence were observed in the control explants. In all cultures with hydrocortisone, a concentration-dependent increase was observed in the fluorescence intensity and the number of the SIF cells in the explant; numerous SIF cells were also seen in the outgrowth. Some SIF cells showed processes and the longest processes were seen in cultures with the highest concentration of hydrocortisone.It is concluded that hydrocortisone causes an increased synthesis of catecholamines in the SIF cellsin vitro, and an increase in their number by affecting either their division or their differentiation from a more immature form, or both. This effect was a direct one and not mediated by any system other than the ganglion itself. Induction of enzyme synthesis by hydrocortisone is proposed as an explanation of the increase in catecholamine concentration.University of Melbourne Senior Research Fellow, September 1971-August 1972Sunshine Foundation and Rowden White Trust Overseas Research Fellow in the University on Melbourne, September 1971-August 1972     

L-Tyrosine Production by Analog-resistant Mutants Derived from a Phenylalanine Auxotroph of Corynebacterium glutamicum

Mutants resistant to various phenylalanine- or tyrosine-analogs were isolated from a phenylalanine auxotroph of Corynebacterium glutamicum KY 10233 by treatment with N- methyl-N′-nitro-N-nitrose guanidine (NTG) and screened for L-tyrosine production. A mutant, 98–Tx–71, which is resistant to 3-aminotyrosine, p-aminophenylalanine, p-fluoro-phenylalanine, and tyrosine hydroxamate was found to produce L-tyrosine at a concentration of 13.5 mg/ml in the cane molasses medium containing 10% of sugar calculated as glucose. A tyrosine-sensitive mutant, pr–20 which was derived from 98–Tx–71 produced L-tyrosine at a concentration of 17.6 mg/ml. L-Tyrosine formation in the strain pr–20 was found to be still inhibited by L-phenylalanine though it was not inhibited by L-tyrosine. The L-tyrosine formation in the mutant was repressed neither by L-phenylalanine nor by L-tyrosine.     

Tyrosine and the synthesis of melanin in embryonic cells fromAmbystoma mexicanum

Summary Explants comprising about 15 cells were dissected from various regions of the blastula ofAmbystoma mexicanum and cultured in Barth's medium. By addition of L-tyrosine to the culture medium it was possible to induce melanin synthesis in three different cells types: undifferentiated embryonic cells, mesenchyme cells and nerve cells. Tyrosine was found to act as an inductor in a very low concentration (1 M). It is suggested that tyrosine serves both as an inductor and as a substrate for melanin synthesis in the amphibian larva.