Presence of dopamine-immunoreactive cell bodies in the catecholaminergic group A15 of the sheep brain

Summary Antisera were raised in rabbits against dopamine or noradrenaline conjugated to thyroglobulin with glutaraldehyde. These antisera, tested in enzyme linked immunosorbent assay and immunohistochemistry specifically recognized their homologous antigens.With the aid of anti-tyrosine hydroxylase, anti-aromatic aminoacid decarboxylase, anti-dopamine--hydroxylase, anti-dopamine, and anti-noradrenaline antisera, immunohistochemical reactions were performed on glutaraldehyde fixed sections of sheep diencephalon in order to determine the presence of dopamine in the catecholaminergic group A15. Perikarya of this nucleus were stained with anti-tyrosine hydroxylase, anti-aromatic aminoacid decarboxylase and anti-dopamine, but not with anti-dopamine--hydroxylase or anti-noradrenaline. Both of these latter antisera stained fibers within this area. So as recently found in the rat, we could conclude that dopamine is present in group A15 of the sheep.     

Isolation of Monoaminergic Synaptosomes from Rat Brain by Immunomagnetophoresis

Monoaminergic synaptosomes have been isolated and purified from rat brain by immunomagnetophoresis. This novel technique uses magnetic beads to which Protein A is bound. Noradrenergic, dopaminergic, and serotonergic synaptosomes (previously cell-surface labelled with anti-dopamine-beta-hydroxylase, anti-tyrosine hydroxylase, and anti-tryptophan hydroxylase, respectively) may be isolated in a highly purified state. The synaptosomal subpopulations are recovered in a viable metabolic state and show glucose-stimulated respiration and Ca2(+)-dependent neurotransmitter release. A novel subtype of dopamine-beta-hydroxylase was found in dopaminergic terminals. No evidence for glutamate corelease from monoaminergic synaptosomes was obtained.     

Catecholaminergic nuclei of sheep encephalon. Immunocytochemical study. I. Myelencephalon and metencephalon

Using an anti-tyrosine hydroxylase antiserum, the whole of catecholaminergic perikarya of the myelencephalon and metencephalon of the sheep were visualized immunocytochemically. Compared with those of the rat, these neuronal groups in the sheep were featured, firstly, by a greater dispersion relative to their perikarya and, secondly, by the absence of A3 and A4 nuclei described by Dahlstrom and Fuxe.     

Efferent projections from the retrochiasmatic area to the median eminence and to the pars nervosa of the hypophysis with special reference to the A15 dopaminergic cell group in the sheep

Anterograde tracers, viz. Phaseolus vulgaris leucoagglutinin and fluorescein dextran, were used in conjunction with tyrosine hydroxylase immunohisto-chemistry to study the projections of the A15 dopaminergic cell group towards the median eminence and pituitary in sheep. After injection of the tracers in the retrochiasmatic area, which contains the cell group A15, fibres containing anterograde tracer were observed in the internal zone of the median eminence and in the pars nervosa of the pituitary. Numerous tyrosine hydroxylase immunoreactive fibers were present in the external zone of the median eminence and in the pars intermedia and the pars nervosa of the pituitary, with characteristic patterns of organisation in each area. Most tyrosine hydroxylase-immunoreactive fibres containing fluorescein dextran were located in the pars nervosa, whereas only a few were observed in the internal zone of the median eminence. It was concluded that at least part of the dopaminergic innervation of the pars nervosa originated from the A15 group. These results provide morphological evidence for (1) the role of dopaminergic neurons of the A15 cell group in the seasonal control of prolactin secretion via the release of dopamine in the pars nervosa, and (2) putative physiological interactions between dopamine and the secretion of neurohypophysial hormones in sheep.     

Role of Aromatic l-Amino Acid Decarboxylase for Dopamine Replacement by Genetically Modified Fibroblasts in a Rat Model of Parkinson's Disease

Abstract: Investigations of gene therapy for Parkinson's disease have focused primarily on strategies that replace tyrosine hydroxylase. In the present study, the role of aromatic l -amino acid decarboxylase in gene therapy with tyrosine hydroxylase was examined by adding the gene for aromatic l -amino acid decarboxylase to our paradigm using primary fibroblasts transduced with both tyrosine hydroxylase and GTP cyclohydrolase I. We compared catecholamine synthesis in vitro in cultures of cells with tyrosine hydroxylase and aromatic l -amino acid decarboxylase together versus cocultures of cells containing these enzymes separately. l -DOPA and dopamine levels were higher in the cocultures that separated the enzymes. To determine the role of aromatic l -amino acid decarboxylase in vivo, cells containing tyrosine hydroxylase and GTP cyclohydrolase I were grafted alone or in combination with cells containing aromatic l -amino acid decarboxylase into the 6-hydroxydopamine-denervated rat striatum. Grafts containing aromatic l -amino acid decarboxylase produced less l -DOPA and dopamine as monitored by microdialysis. These findings indicate that not only is there sufficient aromatic l -amino acid decarboxylase near striatal grafts producing l -DOPA, but also the close proximity of the enzyme to tyrosine hydroxylase is detrimental for optimal dopamine production. This is most likely due to feedback inhibition of tyrosine hydroxylase by dopamine.     

Gene Expression of Tyrosine Hydroxylase in the Developing Fetal Brain

Tyrosine hydroxylase, aromatic L-amino-acid decarboxylase, and dopamine beta-hydroxylase activities were studied in the developing fetal rat brain. A delay of 2-3 days between the detection of the tyrosine hydroxylase and the aromatic L-amino-acid decarboxylase and dopamine beta-hydroxylase activities was observed. For this reason, the expression of tyrosine hydroxylase mRNA was studied. Tyrosine hydroxylase mRNA was visualized in the whole brain from 13 days of gestation, but the largest increase of the expression was observed in the hypothalamus. These results are discussed in terms of the relative gene expressions of the three enzymes involved in the biosynthesis of catecholamines and phenolamines in nervous tissues.     

An investigation of a sympathetic innervation of the vertebral artery in primates: is there a neurogenic substrate for vasoconstriction?

Vasoconstriction of the vertebral artery may be neurogenic in origin. Although the existence of a perivascular sympathetic plexus of the vertebral artery is not in doubt, no method used to date has conclusively demonstrated a direct sympathetic innervation of the vascular smooth muscle cells and, hence, vasomotor function. It was the aim of this study, therefore, to visualise and localise noradrenergic fibres in the wall of the vertebral artery. Intracranial vertebral artery specimens (10 vervet monkeys and 10 baboon vessels) were sectioned (40 mm serial sections) and treated with anti-tyrosine hydroxylase, anti-dopamine b-hydroxylase, and anti-chromogranin-A antibodies. Some evidence of catecholaminergic fibres in the tunica adventitia but not penetrating the external elastic lamina or tunica media of the vertebral artery wall was seen. These findings were confirmed by electron microscopy. It was concluded that although a perivascular sympathetic plexus exists, the vertebral artery of primates was not shown to have a direct sympathetic innervation and a neurogenic vasoconstrictor function is unlikely.     

Mast cells express tyrosine hydroxylase and store dopamine in a serglycin-dependent manner

Here we show that mast cells contain dopamine and that mast cell activation causes dopamine depletion, indicating its presence within secretory granules. Dopamine storage increased during mast cell maturation from bone marrow precursors, and was dependent on the presence of serglycin. Moreover, the expression of tyrosine hydroxylase, the key enzyme in dopamine biosynthesis, was induced during mast cell maturation; histidine decarboxylase and tryptophan hydroxylase 1 were also induced. Mast cell activation caused a robust induction of histidine decarboxylase, but no stimulation of tyrosine hydroxylase or tryptophan hydroxylase 1 expression. The present study points toward a possible role of dopamine in mast cell function.     

Concomitant elevation of tyrosine hydroxylase and dopamine beta-hydroxylase by cyclic AMP in cultured mouse neuroblastoma cells.

The effects of cyclic AMP analogues and of phosphodiesterase inhibitors were investigated in neuroblastoma cells (NBD-2) cloned from the C-1300 tumor. 8Br-cAMP and phosphodiesterase inhibitors that elevated cAMP induced large (greater than 15 fold) and specific increases in tyrosine hydroxylase and dopamine beta-hydroxylase activity. In contrast, catechol O-methyltransferase, monoamine oxidase and aromatic-l -amino-acid decarboxylase were unaffected by the cAMP altering drugs. Similarly, AChE was unaffected and only a small increase in choline acetyltransferase (3 fold) was observed. The increases in tyrosine hydroxylase and dopamine beta-hydroxylase were similar with respect to dose response relationships and with respect to time course of onset. Only those phosphodiesterase inhibitors that elevated cAMP (papaverine and Ro20-1724 as opposed to theophylline) were effective in elevating tyrosine hydroxylase and dopamine beta-hydroxylase. Further, the doses optimal for elevating cAMP coincided with the optimal doses for elevating the two enzymes. Theophylline had no influence either upon NBD-2 cell cAMP levels or upon tyrosine hydroxylase and dopamine beta-hydroxylase activity. The changes in protein synthesis rates produced by the cAMP altering drugs were temporally distinct from the changes in either tyrosine hydroxylase or dopamine beta-hydroxylase. These results suggest that the intracellular messenger compound cAMP is involved in the specific regulation of both tyrosine hydroxylase and dopamine beta-hydroxylase in adrenergic cells.     

Effect of gonadal steroids and gamma-aminobutyric acid on LH release and dopamine expression and activity in the zona incerta in rats

A dopaminergic system in the zona incerta stimulates LH release and may mediate the positive feedback effects of the gonadal steroids on LH release. In this study the mechanisms by which steroids might increase dopamine activity in the zona incerta were investigated. In addition, experiments were conducted to determine whether the inhibitory effects of gamma-aminobutyric acid (GABA) on LH release in the zona incerta are due to suppression of dopamine activity in this area or conversely whether the stimulatory effects of dopamine on LH release are due to suppression of a tonic inhibitory GABAergic system. Ovariectomized rats were treated s.c. with oil, 5 micrograms oestradiol benzoate or 5 micrograms oestradiol benzoate followed 48 h later by 0.5 mg progesterone, and killed 54 h after the oestradiol benzoate injection. At this time the LH concentrations were suppressed in the oestradiol benzoate group and increased in the group treated with oestradiol benzoate and progesterone. The ratio of tyrosine hydroxylase:beta-actin mRNA in the zona incerta was significantly increased by the oestradiol benzoate treatment, but the addition of progesterone resulted in values similar to those in the control group. At the same time, the progesterone treatment increased tyrosine hydroxylase activity in the zona incerta as indicated by an increase in L-dihydroxyphenylalanine (L-DOPA) accumulation after 100 mg 3-hydroxybenzylhydrazine hydrochloric acid (NSD1015) kg-1 and an increase in dopamine release as indicated by a increase in dihydroxyphenylacetic acid (DOPAC) concentrations (one of the major metabolites of dopamine). Ovariectomized rats treated with oestradiol benzoate plus progesterone were also injected i.p. with 75 mg gamma-acetylenic GABA kg-1 (a GABA transaminase inhibitor) to increase GABA concentrations in the brain. This treatment had no effect on the ratio of tyrosine hydroxylase:beta-actin mRNA but decreased L-DOPA accumulation and DOPAC concentrations in the zona incerta, indicating a post-translational inhibition of dopamine synthesis and release. Treatment of ovariectomized rats with oestradiol benzoate followed by 100 mg L-DOPA i.p. to increase dopamine concentrations in the whole brain had no effect on glutamic acid decarboxylase mRNA expression in the zona incerta, although it increased the glutamic acid decarboxylase:beta-actin mRNA ratio in other hypothalamic areas (that is, the medical preoptic area, ventromedial nucleus and arcuate nucleus). In conclusion, the steroids act to increase dopamine activity in different ways: oestrogen increases tyrosine hydroxylase mRNA expression and progesterone acts after translation to increase tyrosine hydroxylase activity and dopamine release (as indicated by increases in DOPAC concentrations). This latter effect may be due to progesterone removing a tonic GABAergic inhibition from the dopaminergic system.     

Demonstration of dopamine in electron-dense synaptic vesicles in the pars intermedia of Xenopus laevis, by freeze substitution and postembedding immunogold electron microscopy.

The presence of dopamine in the pituitary of the clawed toad Xenopus laevis was studied by light and electron microscope immunocytochemistry, using pre- and postembedding techniques. Light microscopy showed the presence of an intricate, anti-dopamine-positive fibre network throughout the pars intermedia. In preembedded stained material, dopamine appeared to occur in varicosities which make synaptic contacts with both folliculo-stellate cells and melanotrope cells. Post-embedding immunogold staining of freeze-substituted material permitted the localization of anti-dopamine reactivity in electron-dense vesicles in these varicosities. This finding supports the hypothesis that dopamine is involved in the (inhibitory) control of melanotrope cell activity in X. laevis.     

Demonstration of dopamine in electron-dense synaptic vesicles in the pars intermedia of Xenopus laevis,by freeze substitution and postembedding immunogold electron microscopy

Summary The presence of dopamine in the pituitary of the clawed toad Xenopus laevis was studied by light and electron microscope immunocytochemistry, using pre- and postembedding techniques. Light microscopy showed the presence of an intricate, anti-dopamine-positive fibre network throughout the pars intermedia. In preembedded stained material, dopamine appeared to occur in varicosities which make synaptic contacts with both folliculo-stellate cells and melanotrope cells. Postembedding immunogold staining of freeze-substituted material permitted the localization of anti-dopamine reactivity in electron-dense vesicles in these varicosities. This finding supports the hypothesis that dopamine is involved in the (inhibitory) control of melanotrope cell activity in X. laevis.     

Enzymes Related to Monoamine Transmitter Metabolism in Brain Microvessels

The activities of tyrosine hydroxylase, aromatic L-aminoacid decarboxylase, monoamine oxidase, and catechol-O-methyltransferase were measured in microvessel (capillaries and venules), parenchymal arterioles, and pial vessels from rat brains, and the decarboxylase activity was compared in brain microvessels from rabbit, cat, dog, pig, cow, baboon, and man. Cranial sympathectomy was performed to estimate the neuronal contribution to the enzyme activities. All vascular regions had substantial activities of the various enzymes studied. The activity of aromatic L-aminoacid decarboxylase in cerebral microvessels was high in rat, dog, pig, cow, and man; intermediate in rabbit and cat; and low in baboon. In addition to this enzyme, cerebral microvessels also contained tyrosine hydroxylase and monoamine oxidase. Aromatic aminoacid decarboxylase and monoamine oxidase serve an enzymatic barrier function at the microvascular level, whereas the main function of tyrosine hydroxylase is probably to synthesize monoamines within nerve terminals that remain in close association with microvessels under the conditions used for preparation of the microvascular fraction. In larger intracerebral and pial vessels monoamine oxidase was present both in the wall itself and in perivascular sympathetic nerves; the remaining two enzymes had a primarily neuronal localization. The latter types of vessels also contained catechol-O-methyltransferase in their walls.     

Effect of glutaraldehyde treatment of human melanoma cells on the expression of melanoma-associated antigens

Summary Human melanoma cells were treated with different concentrations of glutaraldehyde, and retention of serological reactivity with antisera against melanoma-associated antigens, HLA antigen, and 2-microglobulin was assessed by quantitative absorption analysis in mixed hemadsorption microassays. Glutaraldehyde concentrations of 0.025% or greater significantly impaired binding to melanoma cells of antibody against melanoma-associated antigens. At a concentration of 0.0025% antibody binding was not decreased although plating efficiency was reduced to less than 1%. Glutaraldehyde concentrations of 0.25% or greater significantly reduced binding to the same melanoma cells of antisera against HLA antigen and 2-microglobulin. Glutaraldehyde treatment (up to 2.5%) of HT-29 colon carcinoma cells failed to reduce reactivity of antisera against CEA and blood group A isoantigen, which are present on these cells. These studies indicate that the effect of glutaraldehyde treatment of cells on retention of surface antigens is critically dependent on the concentration of glutaraldehyde used and the type of antigens involved. Abbreviations used in this paper: MAA, melanoma-associated antigens; GA, glutaraldehyde; FCS, fetal calf serum; RPMI, Roswell Park Memorial Institute; 2M, 2-microglobulin; CEA, carcinoembryonic antigen; PBS, phosphate-buffered saline; NGP, normal glycoprotein cross-reacting with CEA; SRBC, sheep red blood cells     

Adrenergic enzymes in cultured mouse neuroblastoma: absence of detectable aromatic-L-amino-acid decarboxylase.

The relative activities of tyrosine hydroxylase, aromatic-l -amino-acid decarboxylase and dopamine beta-hydroxylase were established in a number of clones of neuroblastoma cells isolated from the uncloned mouse C-1300 tumor. One clone, NBD-2, was chosen for further analysis on the basis of its relatively high activities of tyrosine hydroxylase and dopamine beta-hydroxylase. The levels of these enzymes, and monoamine oxidase and catechol O-methyltransferase, were at least 20-80 fold lower in the neuroblastoma culture than in mouse superior cervical ganglion. More importantly, aromatic-l -amino-acid decarboxylase activity was not even detectable in any neuroblastoma clone examined. Based on the relative sensitivities of the tyrosine hydroxylase and aromatic-l -amino-acid decarboxylase assays and on the ratio of these two enzymes in the mouse ganglion, decarboxylase activity is more than 10 fold lower in the cultured cells than would be predicted on the basis of tyrosine hydroxylase activity. Dialysis and mixing studies with neuroblastoma extracts and partially purified aromatic-l -amino-acid decarboxylase did not reveal the presence of any endogenous inhibitors that could account for the low level of decarboxylase activity in the cultured cells. During growth of the neuroblastoma cells to confluency, only one enzyme, monoamine oxidase, exhibited an elevated specific activity on the basis of cell number. However, when based on the amount of protein, the specific activity of all measurable enzymes increased in culture-because cell protein decreased 5 fold during growth to confluency. These findings are discussed with respect to individual cell function.     

RELATIONSHIP BETWEEN THE RATE OF AXOPLASMIC TRANSPORT AND SUBCELLULAR DISTRIBUTION OF ENZYMES INVOLVED IN THE SYNTHESIS OF NOREPINEPHRINE

The net rate of proximo-distal transport of tyrosine hydroxylase, dopamine β-hydroxylase, DOPA decarboxylase and choline acetyltransferase was determined by measuring the accumulation of these enzymes proximal to a ligature of the rat sciatic nerve. The rate of accumulation was constant for at least 12 h. For the enzymes involved in the biosynthesis of norepinephrine the rate of transport was correlated to their subcellular distribution and a close correlation between these two parameters was found. Dopamine β-hydroxylase, an enzyme mainly localized in the particulate fraction of the sciatic nerve, showed the fastest rate of transport (1·94 mm/h) whereas DOPA decarboxylase, exclusively located in the high-speed supernatant fluid, gave the slowest (0·63 mm/h) rate of transport. Tyrosine hydroxylase, predominantly located in the non-particulate fraction of the sciatic nerve was transported much slower (0·75 mm/h) than dopamine β-hydroxylase but still significantly (P < 0.005) faster than DOPA decarboxylase. The subcellular distribution of dopamine β-hydroxylase in ganglia did not differ significantly (0·45 > P > 0·40) from that in the sciatic nerve, but in nerve endings a greater proportion of dopamine β-hydroxylase was localized in particulate fractions. Tyrosine hydroxylase and DOPA decarboxylase were found exclusively in the non-particulate fractions of ganglia. In the nerve endings of the effector organs a small but consistent portion of tyrosine hydroxylase was found in particulate fractions, whereas DOPA decarboxylase was exclusively localized in the high-speed supernatant fluid.     

Design and synthesis of a photocleavable biotin-linker for the photoisolation of ligand–receptor complexes based on the photolysis of 8-quinolinyl sulfonates in aqueous solution

The ability of avidin (Avn) to form strong complex with biotin (Btn) is frequently used in the detection and isolation of biomolecules in biochemical, analytical, and medicinal research. The fact that the binding is nealy irreversible, however, constitutes a drawback in term of the isolation and purification of intact biomolecules. We recently found that 8-quinolinyl esters of aromatic or aliphatic sulfonic acids undergo photolysis when irradiated at 300–330 nm in aqueous solution at neutral pH. In this work, a biotin–dopamine (BD) conjugate containing a photocleavable 8-quinolinyl benzenesulfonate (QB) linker, BDQB, was designed and synthesized for use in the efficient recovery of dopamine–protein (e.g., antibody) complexes from an Avn–Btn system. The complexation of BDQB with a primary anti-dopamine antibody (anti-dopamine IgG₁ from mouse) on an Avn-coated plate was confirmed by an enzyme-linked immunosorbent assay (ELISA) utilizing a secondary antibody (anti-IgG₁ antibody) conjugated with horseradish peroxidase (HRP). Upon the photoirradiation (at 313 nm) of the BDQB–IgG₁ complex, the release of dopamine–IgG₁ complex was confirmed by ELISA. Characterization of the resulting photoreleased dopamine–anti-dopamine IgG₁ complex was performed by SDS–PAGE and Western blot.     

DOPAMINERGIC PROPERTIES OF A SOMATIC CELL HYBRID LINE OF MOUSE NEUROBLASTOMA X SYMPATHETIC GANGLION CELLS

Abstract— Studies were made on the regulation of dopamine metabolism in a cell line derived by hydridization of a non-tyrosine-hydroxylase-containing line of murine neuroblastoma cells with a neur-onally-enriched population of murine embryonic sympathetic ganglion cells. Hybrid subclones with tyrosine hydroxylase activity were selected by exposure to tyrosine-free medium. The cells also exhibited DOPA decarboxylase activity and the subclone (named T28) with the highest specific activities of both enzymes was further characterized. The hybrid T28 line did not contain dopamine-β-hydroxylase activity. The specific activity of tyrosine hydroxylase as well as of DOPA decarboxylase increased significantly in T28 cultures when the cells entered the stationary phase of growth. Both of these enzymes were also induced after several days of exposure to 1 m m -dibutyryl cyclic AMP in culture medium containing either 5% or 0.8% serum. However, maintenance in medium containing 0.8% serum alone, which inhibited cell multiplication, did not induce either enzyme. The dopamine content of T28 cells was also regulated as a function of cell density. High density (stationary phase) cultures of T28 cells contained about 300 pmol dopamine per mg protein and at least half of this endogenous amine appeared to he stored in vesicles or granules (as judged by depletion with reserpine or α-methyl- m -tyramine). The T28 and other neuronal hybrid lines appear to be useful model systems for neuro-chemical studies.