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.     

Effects of 5-(3,3-dimethyl-1-triazeno) imidazole-4-carboxamide [NSC 45388, DTIC] on neuroblastoma cells in culture.

The effects of 5-(3,3-dimethyl-1-triazeno) imidazole-4-carboximide (DTIC) on morphological and biochemical parameters of differentiation were studied in mouse neuroblastoma cells in culture. DTIC (10 μg/ml) did not induce formation of neurites in the cells but inhibited cell division, and produced a marked increase in cell size and in activity of three enzymes (tyrosine hydroxylase, choline acetyltransferase and acetylcholinesterase) involved in neurotransmitter metabolism. These effects were apparently not related to an increase in the intracellular level of cyclic AMP.     

Multiple Forms of Tyrosine Hydroxylase in Human Neuroblastoma Cells: Quantitation with Isoform-Specific Antibodies

Abstract: Human tyrosine hydroxylase (HTH) RNA undergoes alternative splicing, and four different forms of HTH mRNA have been previously identified. Rabbit antibodies were raised against octapeptides unique to each of the four isoforms of HTH predicted from these mRNAs. Blot immunolabeling of human adrenal medulla, pheochromocytoma, and several neuroblastoma cell lines with affinitypurified anti-HTH peptide antibodies demonstrated the presence of all four HTH isoforms in each of these tissues. Quantitative immunolabeling assays for HTH-1, -2, and -4 were established, and HTH isoform levels were determined in several human neuroblastoma cell lines. Whereas total HTH levels differed up to fourfold among the HTH-positive neuroblastoma cell lines studied [LA-N-1, LA-N-5, CHP-234, BE(2)-C, and BE(2)-M17], the relative abundances of HTH isoforms in each of the cell lines were similar. Immunocytochemical analyses demonstrated that HTH immunoreactivity was distributed unequally among the cells in each of these neuroblastoma lines, and morphological interconversion did not account for this heterogeneity. A direct relationship between the percentage of HTH-positive cells and overall HTH levels was also observed. This relationship, in the absence of an apparent clonal basis for the heterogeneity, suggests that HTH expression in neuroblastoma cells may be controlled in a relatively "all-ornone"(bimodal) fashion.     

Calcium dependent regulation of catecholamine and serotonin metabolism in human neuroblastoma cells

Three human neuroblastoma cell lines were shown to have markedly different contents of catecholamines and serotonin. Two of the cell lines (CHP-134 and IMR-5) have higher levels of dopamine and its metabolites, while CHP-404 cells have higher levels of serotonin and its metabolites. Each cell line responded to the addition of D,L-2-amino-5-phosphonovalerate, an agent which increases plasma membrane permeability to Ca2+ (Pastuszko and Wilson, 1988; with striking changes in the metabolism of the neurotransmitters. These changes were dependent on the extracellular calcium concentration and include activation of dopamine synthesis (tyrosine hydroxylase), increased levels of dihydroxyphenylacetic acid and increased formation of N-methylated dopamine derivatives. Catabolism of serotonin to 5-hydroxyindole acetic acid was inhibited while that to 5-hydroxytryptophol was stimulated. These data clearly identify several important sites for regulation of neurotransmitter metabolism by calcium. The mechanisms, direct or indirect, by which the enzyme activities are modulated by calcium remain to be established.     

An Analysis of the Effects of Retinoic Acid and Other Retinoids on the Development of Adrenergic Cells from the Avian Neural Crest

In the present work, we have investigated the role of all-trans-retinoic acid (all-transRA), and several other natural and synthetic retinoids, in the development of adrenergic cells in quail neural crest cultures. Dose response studies using all-transRA and 13-cisRA revealed a dose-dependent increase in the number of adrenergic cells in neural crest cultures. Similar dose response studies using RA isomers and other natural retinoids did not result in the same increases. In order to determine the receptor mediating the effects of all-transRA in the neural crest, we tested several synthetic analogs which specifically bind to a particular RA receptor (RAR) subtype. We found that the compound AM 580, which activates the RAR-α, produced an increase in adrenergic cells similar to that seen with all-transRA. The compound TTNPB, which activates all RAR subtypes, also resulted in an increase in adrenergic cells. We conclude that the increase in adrenergic cells seen with all-transRA is mediated by RAR-α and possibly RAR-β. To further define the actions of all-transRA on the neural crest we incubated cultures with 5-bromo-2′-deoxyuridine (BrdU) to determine whether all-transRA could affect the rate of proliferation. The results show that while all-transRA did not increase the fraction of cells incorporating BrdU into their nuclei at early time points (24 h), it did increase BrdU incorporation by tyrosine hydroxylase (TH) positive cells at 5 days in culture. These findings demonstrate that the increase in adrenergic cells seen with all-transRA in neural crest cultures is likely due to an increase in the proliferation of cells already expressing TH.     

Tissue effects on the expression of serotonin, tyrosine hydroxylase and GABA in cultures of neurogenic cells from the neuraxis and branchial arches

The phenotypically diverse neurones of the enteric nervous system are developmentally derived from precursors that migrate to the bowel from the vagal and sacral regions of the neuraxis. In order to gain insight into the generation of enteric neuronal diversity, we examined the expression of serotonin (5-HT), tyrosine hydroxylase and GABA in vitro. In the mature avian intestine, intrinsic neurones contain 5-HT or GABA but not tyrosine hydroxylase. These markers were demonstrated immunocytochemically, singly or simultaneously. All three phenotypic markers developed in cultures of cranial, vagal or truncal neural crest when the cultures were grown in enriched medium, containing horse serum and chick embryo extract; however, 5-HT and GABA, but not tyrosine hydroxylase-immunoreactive cells, also developed in cultures that were grown in partially defined medium. Tyrosine hydroxylase immunoreactivity was seen when partially defined medium was supplemented with nerve growth factor (NGF). Cultures of branchial arches (III and IV) contained cells that displayed tyrosine hydroxylase immunoreactivity, but not that of 5-HT- or GABA-; however, 5-HT immunoreactivity was seen when branchial arches were cocultured with aneuronal hindgut (from 4-day chick embryos). Cultures of cells from chick gut dissociated at 7 days contained tyrosine hydroxylase as well as 5-HT and GABA immunoreactivities; however, no cultures of bowel dissociated at 8 days or later expressed tyrosine hydroxylase immunoreactivity. When neuraxial cells were cocultured with branchial arches or heart instead of gut, no 5-HT-immunoreactive cells were seen; nevertheless, the further addition of explants of gut to the heart/crest cocultures did permit the expression of 5-HT immunoreactivity. These results are consistent with the hypotheses that precursors with the potential to give rise to cells that express 5-HT, GABA and tyrosine hydroxylase are found at several levels of the neuraxis; however, the ability to express these phenotypes may be suppressed either while the crest cells are migrating (for example, 5-HT and GABA expression by crest cells passing through the branchial arches) or in their final destination (for example, tyrosine hydroxylase in the gut). This suppression may be transient and reversed by the microenvironment of the target organs.     

Stimulation of DOPA Synthesis in the Superior Cervical Ganglion by Veratridine

We have investigated the effect of veratridine on DOPA (3,4-dihydroxyphenylalanine) accumulation by the superior cervical ganglion of the rat. Incubation of the ganglion with veratridine (50 microM) causes a 10-fold increase in the rate of DOPA accumulation. Veratridine-stimulated DOPA accumulation is blocked by tetrodotoxin, but not by cholinergic or adrenergic antagonists or by decentralization of the ganglion. The cyclic nucleotide 8-bromo cyclic GMP does not increase DOPA accumulation, and 8-bromo cyclic AMP causes only a 2-fold increase in DOPA accumulation, which is additive with the effect of veratridine. Thus, the action of veratridine appears to be independent of these cyclic nucleotides. The effect of veratridine on DOPA accumulation is probably due to a stable modification of tyrosine hydroxylase, since an increase in tyrosine hydroxylase activity can be measured in cell-free extracts of veratridine-treated ganglia. Both the increase in DOPA accumulation and the stable activation of tyrosine hydroxylase are dependent upon extracellular Ca2+. The activation of tyrosine hydroxylase by veratridine may be mediated by the depolarization of, and the subsequent entry of Ca2+ into, ganglionic neurons.     

A cross-talk between TrkB and Ret tyrosine kinases receptors mediates neuroblastoma cells differentiation

Understanding the interplay between intracellular signals initiated by multiple receptor tyrosine kinases (RTKs) to give the final cell phenotype is a major pharmacological challenge. Retinoic acid (RA)-treatment of neuroblastoma (NB) cells implicates activation of Ret and TrkB RTKs as critical step to induce cell differentiation. By studying the signaling interplay between TrkB and Ret as paradigmatic example, here we demonstrate the existence of a cross-talk mechanism between the two unrelated receptors that is needed to induce the cell differentiation. Indeed, we show that TrkB receptor promotes Ret phosphorylation by a mechanism that does not require GDNF. This reveals to be a key mechanism, since blocking either TrkB or Ret by small interfering RNA causes a failure in NB biochemical and morphological differentiation. Our results provide the first evidence that a functional transactivation between distinct tyrosine kinases receptors is required for an important physiological process.     

Coexpression of sensory and autonomic neurotransmitter traits by avian neural crest cells in vitro

This study shows that explants of quail neural crest cultured in a medium containing serum and chick embryo extract give rise to large numbers of cells expressing immunoreactivity for substance P (SP), a neuropeptide found in sensory neurons. These cells arise from cycling precursors, but do not appear to divide after expressing SP. The SP-positive cells in cranial neural crest cultures express both neurofilament and the Q211 antigen, but those in trunk cultures express only the Q211 antigen. In both cranial and trunk cultures, large subpopulations of the SP-positive cells express tyrosine hydroxylase and/or choline acetyltransferase, neurotransmitter markers characteristic of autonomic neurons. This finding argues against the idea that SP expression necessarily indicates commitment to the sensory neuron lineage. I further show that embryonic dorsal root ganglion (DRG) cells retain the ability to coexpress SP and tyrosine hydroxylase in vitro although to a lesser extent than do neural crest cells.     

Coexpression of sensory and autonomic neurotransmitter traits by avian neural crest cells in vitro.

This study shows that explants of quail neural crest cultured in a medium containing serum and chick embryo extract give rise to large numbers of cells expressing immunoreactivity for substance P (SP), a neuropeptide found in sensory neurons. These cells arise from cycling precursors, but do not appear to divide after expressing SP. The SP-positive cells in cranial neural crest cultures express both neurofilament and the Q211 antigen, but those in trunk cultures express only the Q211 antigen. In both cranial and trunk cultures, large subpopulations of the SP-positive cells express tyrosine hydroxylase and/or choline acetyltransferase, neurotransmitter markers characteristic of autonomic neurons. This finding argues against the idea that SP expression necessarily indicates commitment to the sensory neuron lineage. I further show that embryonic dorsal root ganglion (DRG) cells retain the ability to coexpress SP and tyrosine hydroxylase in vitro, although to a lesser extent than do neural crest cells.     

L-tyrosine, L-dopa, and tyrosinase as positive regulators of the subcellular apparatus of melanogenesis in Bomirski Ab amelanotic melanoma cells

In cultured cells of the Bomirski Ab amelanotic hamster melanoma line, the substrates of tyrosinase, L-tyrosine, and L-DOPA induce the melanogenic pathway. In this report, we demonstrate that these substrates regulate the subcellular apparatus involved in their own metabolism and that this regulation is under the dynamic control of one of the components of this apparatus, tyrosinase, via tyrosine hydroxylase activity. Culturing cells with nontoxic but melanogenically inhibitory levels of phenylthiourea (PTU; 100 microM) strongly inhibits induction of both the tyrosine hydroxylase and DOPA oxidase activities of tyrosinase by L-tyrosine (200 microM) but has no effect on the induction of either activity by L-DOPA (50 microM). De novo synthesis of premelanosomes precedes the onset of tyrosine-induced melanogenesis. Thereafter, increases in the population of melanosomes (likewise inhibited by PTU) correlate positively with increases in tyrosinase activity induced by L-tyrosine. Melanogenesis induced by L-DOPA in the absence of L-tyrosine is rate-limited not by tyrosinase but by inadequate melanosome synthesis. Our findings indicate that in Bomirski Ab amelanotic hamster melanoma cells the synthesis of the subcellular apparatus of melanogenesis is initiated by L-tyrosine and is regulated further by tyrosinase and L-DOPA, which serves as a second messenger subsequent to tyrosine hydroxylase activity.     

Human neuroblastoma tumor cell lines correspond to the arrested differentiation of chromaffin adrenal medullary neuroblasts

We have examined the hypothesis that nonhematopoietic malignancies may contain cells corresponding to those which occur during the differentiation of tissue precursors. Neuroblastoma, an embryonal tumor of the adrenal medulla, was studied because of its well described ability to differentiate both in vivo and in vitro. We examined the expression of four genes during development of the human adrenal medulla: tyrosine hydroxylase, chromagranin A, pG2, and beta-2-microglobulin. The sequential expression of these genes by adrenal neuroblasts marks successive stages during maturation of the chromaffin lineage. We also observed a population of neuroblasts during adrenal medullary development that did not express any of these four genes, suggestive of adrenal medullary cells differentiating along nonchromaffin lineage(s). We then evaluated 27 neuroblastoma cell lines for the expression of these genes and found that 24 expressed chromaffin markers, with 19 of these mimicking the pattern of gene expression found during development. Three cell lines did not express tyrosine hydroxylase, chromogranin A, or pG2, consistent with either a very undifferentiated neural crest cell or maturation along a nonchromaffin lineage. These data indicate that neuroblastoma tumor cells correspond to adrenal neuroblasts arrested during morphogenesis of the adrenal medulla and raise the possibility that malignant transformation of cells at different stages of tissue maturation may contribute to the diversity that characterizes tumors of solid tissues.     

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.     

Cholinergic Receptor-Mediated Phosphorylation and Activation of Tyrosine Hydroxylase in Cultured Bovine Adrenal Chromaffin Cells

We have identified a 56-kilodalton protein in cultured bovine adrenal chromaffin cells that is phosphorylated when catecholamine secretion is stimulated. Immunodetection on Western blots from both one- and two-dimensional polyacrylamide gels indicated that this protein was tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis. Two-dimensional polyacrylamide gel electrophoresis of proteins from unstimulated cells revealed small amounts of phosphorylated protein with a molecular weight of 56K and pI values of 6.37 and 6.27 which were subunits of tyrosine hydroxylase. Nicotinic stimulation of chromaffin cells caused the phosphorylation of three proteins of 56 kilodaltons with pI values of approximately 6.37, 6.27, and 6.15 which were tyrosine hydroxylase. The immunochemical analysis also revealed that there was unphosphorylated tyrosine hydroxylase 56 kilodaltons with a pI of 6.5 which may have decreased on nicotinic stimulation. The phosphorylation of tyrosine hydroxylase was associated with an increase in in situ conversion of [3H]tyrosine to [3H]dihydroxyphenylalanine ([3H]DOPA). Muscarinic stimulation also caused phosphorylation of tyrosine hydroxylase, but to a smaller extent than did nicotinic stimulation. The secretagogues, elevated K+ and Ba2+, stimulated phosphorylation of tyrosine hydroxylase and [3H]DOPA production. The effects of nicotinic stimulation and elevated K+ on tyrosine hydroxylase phosphorylation and [3H]DOPA production were Ca2+-dependent. Nicotinic agonists also raised cyclic AMP levels in chromaffin cells after 2 min. Dibutyryl cyclic AMP and forskolin, which have little effect on catecholamine secretion, also caused phosphorylation of tyrosine hydroxylase. These stimulators of cyclic AMP-dependent processes caused the appearance of two phosphorylated subunits of tyrosine hydroxylase with pI values of 6.37 and 6.27. There was also a small amount of phosphorylated subunit with a pI of 6.15. Both agents stimulated [3H]DOPA production. The experiments indicate that tyrosine hydroxylase is phosphorylated and activated when chromaffin cells are stimulated to secrete. The data suggest that the earliest phosphorylation of tyrosine hydroxylase induced by a nicotinic agonist occurs through stimulation of a Ca2+-dependent protein kinase. After 2 min phosphorylation by a cyclic AMP-dependent protein kinase may also occur. Phosphorylation of tyrosine hydroxylase is associated with an increase in in situ tyrosine hydroxylase activity.     

Plasticity and regulatory mechanisms of Hox gene expression in mouse neural crest cells

In amniotes, the developmental potentials of neural crest cells differ between the cranium and the trunk. These differences may be attributable to the different expression patterns of Hox genes between cranial and trunk neural crest cells. However, little is known about the factors that control Hox genes expression in neural crest cells. The present data demonstrate that retinoic acid (RA) treatment and the activation of Wnt signaling induce Hoxa2 and Hoxd9 expression, respectively, in mouse mesencephalic neural crest cells, which never express Hox genes in vivo. Furthermore, Wnt signaling suppresses the induction of Hoxa2. We also demonstrate that these factors participate in the maintenance of Hoxa2 and Hoxd9 expression in mouse trunk neural crest cells. Our results suggest that RA and Wnt signaling function as environmental factors that regulate the expression of Hoxa2 and Hoxd9 in mouse neural crest cells.     

Activin receptor-like kinase 2 can mediate atrioventricular cushion transformation

The development of enteric and sympathetic neurons from neural crest precursor cells is regulated by signals produced by the embryonic environments to which the cells migrate. Bone morphogenetic proteins (BMPs) are present in the developing embryo and act to induce neuronal differentiation and noradrenergic properties of neural crest cells. We have investigated the role of BMP2 in regulating the appearance of distinct populations of autonomic neurons from postmigratory, HNK-1-positive neural crest precursor cells. BMP2 promotes neuronal differentiation of sympathetic and enteric precursor cells isolated from E14.5 rat. The effects of BMP2 change over time, resulting in a decrease in neuron number that can be attributed to apoptotic cell death. BMP2-dependent neuron death is rescued by gut-derived factors that provide trophic support to maturing neurons, indicating that BMP2 regulates the acquisition of trophic dependence of developing peripheral neurons. In addition to regulating neuron number, BMP2 promotes both panneuronal maturation and the acquisition of an enteric phenotype, as measured by lineage-specific changes in the expression of tyrosine hydroxylase and MASH-1. While BMP2 is sufficient to induce neuronal differentiation and panneuronal development, these results suggest that additional factors in the environment must collaborate with BMP2 to promote the final noradrenergic phenotype of sympathetic neurons.     

Low-Na+ Medium Increases the Activity and the Phosphorylation of Tyrosine Hydroxylase in the Superior Cervical Ganglion of the Rat

Incubation of the rat superior cervical ganglion in Na+-free or low-Na+ medium increased the rate of synthesis of 3,4-dihydroxyphenylalanine (DOPA) in the ganglion fourfold and caused a concomitant stable activation of tyrosine hydroxylase. DOPA synthesis was half-maximal in medium containing about 20 mM Na+. Low-Na+ medium also increased the incorporation of 32Pi into tyrosine hydroxylase; the dependence of tyrosine hydroxylase phosphorylation on the Na+ concentration resembled that of DOPA synthesis. The stimulatory effects of low-Na+ medium on DOPA production and on tyrosine hydroxylase activity in vitro were dependent on extra-cellular Ca2+. The stimulation of DOPA synthesis in low-Na+ medium was inhibited by methoxyverapamil, an inhibitor of Ca2+ uptake, and was partially blocked by tetrodotoxin, but it was not affected by the cholinergic antagonists hexamethonium and atropine. Ionomycin, a calcium ionophore, stimulated DOPA synthesis to about the same extent as low-Na+ medium and also increased the incorporation of 32Pi into tyrosine hydroxylase. 8-Bromo cyclic AMP (1 mM) also stimulated DOPA production in the ganglion, and this stimulation was more than additive with that produced by low-Na+ medium. These data support the hypothesis that low-Na+ medium stimulates DOPA synthesis by raising intracellular Ca2+, which then promotes the phosphorylation of tyrosine hydroxylase.