Doppler ultrasound of blood flow velocities in ophthalmic and central retinal arteries during the early neonatal period

Dopamine has been implicated as a potential mediating factor in a variety of neurodegenerative disorders. Dopamine can be oxidized to form a reactive dopamine quinone that can covalently modify cellular macromolecules including protein and DNA. This oxidation can be enhanced through various enzymes including tyrosinase and/or prostaglandin H synthase. One of the potential targets in brain for dopamine quinone damage is tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis. The present studies demonstrated that dopamine quinone, the formation of which was enhanced through the activity of the melanin biosynthetic enzyme, tyrosinase, covalently modified and inactivated tyrosine hydroxylase. Dihydroxyphenylalanine (DOPA; the catechol-containing precursor of dopamine) also inactivated tyrosine hydroxylase under these conditions. Catecholamine-mediated inactivation occurred with both purified tyrosine hydroxylase as well as enzyme present in crude pheochromocytoma homogenates. Inactivation was associated with covalent incorporation of radiolabelled dopamine into the enzyme as assessed by immunoprecipitation, size exclusion chromatography, and denaturing sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis. Furthermore, the covalent modification and inactivation of tyrosine hydroxylase was blocked by antioxidant compounds (dithiothreitol, reduced glutathione, or NADH). In addition to kinetic feedback inhibition and the formation of an inhibitory dopamine/Fe+3 complex, these findings suggest that a third mechanism exists by which dopamine (or DOPA) can inhibit tyrosine hydroxylase, adding further complexity to the regulation of catecholamine biosynthesis.     

Prevalence of cryoglobulinemia in chronic hepatitis C virus infection and response to treatment with interferon-alpha

A recombinant Schistosoma mansoni protein has been identified as a useful antigen for the detection of S. mansoni and Schistosoma haematobium antibodies. The purified recombinant protein, Sm22.3, was assayed using an enzyme-linked immunosorbent assay format against a battery of 491 well defined sera, including S. mansoni, S. haematobium, and Schistosoma japonicum infection sera, normal human sera, sera from 9 other parasitic infections, and sera from 2 additional infections. The sensitivity for detecting S. mansoni and S. haematobium infections with this single recombinant protein is 80.1%. The specificity is 94.8%. However, 15 of the 16 cross-reactive sera are malaria infection sera, and we have data suggesting that these malaria sera are actually recognizing an epitope on the vector-derived 6Xhistidine tag of recombinant Sm22.3. If this is the case, then, the actual specificity of the assay is 99.6%.     

Deletion mutagenesis of rat PC12 tyrosine hydroxylase regulatory and catalytic domains

The functional organization of rat tyrosine hydroxylase was investigated by deletion mutagenesis of the regulatory and catalytic domains. A series of tyrosine hydroxylase cDNA deletion mutants were amplified by PCR, cloned into the pET3C prokaryotic expression vector, and the mutant proteins were partially purified from E. coli. The results show that the deletion of up to 157 N-terminal amino acids activated the enzyme, but further deletion to position 184 completely destroyed catalytic activity. On the carboxyl end, the removal of 43 amino acids decreased but did not eliminate activity, suggesting that this region may play a different role in the regulation of the enzyme. These findings place the amino end of the catalytic domain between residues 158 and 184 and the carboxyl end at or prior to position 455. Deletions within the first 157 amino acids in the N-terminus caused an increase in hydroxylating activity, a decrease in the apparent Km for tyrosine and phenylalanine substrates, and a substantial increase in the Ki for dopamine inhibition. The results define this region of the N-terminus as the regulatory domain of tyrosine hydroxylase, whose primary functions are to restrict the binding of amino acid substrates and to facilitate catecholamine inhibition. The results also suggest that the well-established role of the regulatory domain in restricting cofactor binding may be secondary to an increase in catecholamine binding, which in turn lowers the affinity for the cofactor. These findings provide new insight into the functional organization and mechanisms of regulation of tyrosine hydroxylase.     

Role of aromatic L-amino acid decarboxylase for dopamine replacement by genetically modified fibroblasts in a rat model of Parkinson's disease

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.     

Evidence for synthesis and release of catecholamines by human amniotic epithelial cells

The present study investigated the presence, possible synthesis and release of catecholamines (CA) by human amniotic epithelial cells (HAEC) using HPLC with electrochemical detection. The presence of CA was indicated by the detection of norepinephrine (NE), dopamine (DA) and its metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) in extracts of cultured HAEC. Incubation of HAE cells in medium supplemented with 1-tyrosine (CA precursor) and tetrahydrobiopterin (tyrosine hydroxylase cofactor) significantly increased the production of catecholamines, suggesting CA synthesis by HAEC. In contrast, pharmacological inhibition of tyrosine hydroxylase by alpha-methyl-p-tyrosine (MPT) significantly reduced CA production, further confirming CA synthesis by HAEC. Catecholamines were also detected in the cell incubation media, demonstrating the ability of HAEC to spontaneously secrete CA. Moreover, incubation of cells with 50 mM K+ for 10 min increased the amount of CA released into the medium. Additionally, the detection of DOPAC, a primary metabolite of DA, in HAEC strongly indicates that these cells contain DA metabolizing enzymes. The present results suggest that HAEC synthesize and release CA. These cells may be a possible candidate for transplantation therapy of neurodegenerative diseases such as Parkinson's disease and also may serve as a model to study the aspects of catecholaminergic activity.     

Occurrence and metabolism of anandamide and related acyl-ethanolamides in ovaries of the sea urchin Paracentrotus lividus

Cannabinoid receptors have been described in sea urchin sperm and shown to mediate inhibition of sperm acrosome reaction. Anandamide (arachidonoyl-ethanolamide), the mammalian physiological ligand at the cannabinoid CB1 receptor, has been subsequently found to effect this inhibition. Here we present data showing that ovaries from the sea urchin Paracentrotus lividus contain anandamide and two related acyl-ethanolamides, as well as enzymatic activities potentially responsible for their biosynthesis and degradation. Pilot experiments carried out with either ovaries or spermatozoa, extracted from both P. lividus and Arbacea lixula and radiolabelled with [14C]ethanolamine, showed that in sexually mature ovaries of both species significant levels of radioactivity were incorporated into a lipid component with the same chromatographic behaviour as anandamide. Lipid extracts from P. lividus ovaries were purified and analysed by gas chromatography/mass spectrometry which showed the presence of low but measurable amounts of anandamide, palmitoyl- and stearoyl-ethanolamides. The extracts were also found to contain lipid components with the same chromatographic behaviour as the N-acyl-phosphatidyl-ethanolamines, the phospholipid precursors of acyl-ethanolamides in mammalian tissues, and capable of releasing anandamide, palmitoyl- and stearoyl-ethanolamides upon digestion with S. chromofuscus phospholipase D. Accordingly, whole homogenates from P. lividus contained an enzymatic activity capable of converting synthetic [3H]N-arachidonoyl-phosphatidyl-ethanolamine into [3H]anandamide. Finally, mature ovaries of P. lividus were shown also to contain an amidohydrolase activity which catalyses the hydrolysis of anandamide and palmitoyl-ethanolamide to ethanolamine. This enzyme displayed subcellular distribution, pH/temperature dependency profiles and sensitivity to inhibitors similar but not identical to those of the previously described 'anandamide amidohydrolase' from mammalian tissues. These data support the hypothesis, formulated in previous studies, that anandamide or related metabolites may be oocyte-derived cannabimimetic regulators of sea urchin fertility.     

Crystal structure of tyrosine hydroxylase at 2.3 A and its implications for inherited neurodegenerative diseases

Tyrosine hydroxylase (TyrOH) catalyzes the conversion of tyrosine to L-DOPA, the rate-limiting step in the biosynthesis of the catecholamines dopamine, adrenaline, and noradrenaline. TyrOH is highly homologous in terms of both protein sequence and catalytic mechanism to phenylalanine hydroxylase (PheOH) and tryptophan hydroxylase (TrpOH). The crystal structure of the catalytic and tetramerization domains of TyrOH reveals a novel alpha-helical basket holding the catalytic iron and a 40 A long anti-parallel coiled coil which forms the core of the tetramer. The catalytic iron is located 10 A below the enzyme surface in a 17 A deep active site pocket and is coordinated by the conserved residues His 331, His 336 and Glu 376. The structure provides a rationale for the effect of point mutations in TyrOH that cause L-DOPA responsive parkinsonism and Segawa's syndrome. The location of 112 different point mutations in PheOH that lead to phenylketonuria (PKU) are predicted based on the TyrOH structure.     

Inhibition of nicotinic responses of bovine adrenal chromaffin cells by the protein kinase C inhibitor, Ro 31-8220

1. The effects of the protein kinase C inhibitor, Ro 31-8220, on the responses of cultured bovine adrenal chromaffin cells to nicotine, phorbol 12, 13-dibutyrate (PDBu) and K+ have been investigated. 2. Tyrosine hydroxylase activity was measured in situ in intact cells by measuring 14CO2 evolved following the hydroxylation and rapid decarboxylation of [14C]-tyrosine offered to the cells. Secretion of endogenous adrenaline and noradrenaline was measured by use of h.p.l.c. with electrochemical detection. Cyclic AMP levels were measured in cell extracts by RIA. 3. Ro 31-8220 produced a concentration-dependent inhibition of 300 nM PDBu-stimulated tyrosine hydroxylase activity with an IC50 of < 2 microM and complete inhibition at 10 microM. It had no effect on the responses to forskolin. 4. Ro 31-8220 produced a concentration-dependent inhibition of 5 microM nicotine-stimulated tyrosine hydroxylase activity, adrenaline and noradrenaline secretion and cellular cyclic AMP levels, with an IC50 of about 3 microM and complete inhibition by 10 microM. At concentrations up to 10 microM, Ro 31-8220 had little or no effect on the corresponding responses to 50 mm K+. 5. A structural analogue of Ro 31-8220, bisindolylmaleimide V, that lacks activity as a protein kinase C inhibitor, had no effect up to 10 microM on PDBu-stimulated tyrosine hydroxylase activity or on nicotine-stimulated cyclic AMP levels or noradrenaline secretion and only marginal inhibitory effects on nicotine-stimulated tyrosine hydroxylase activity and adrenaline secretion. 6. A structurally related protein kinase C inhibitor, bisindolylmaleimide I, inhibited PDBu-stimulated tyrosine hydroxylase activity with an IC50 of < 1 microM and complete inhibition by 3 microM, but had essentially no effect on nicotine stimulated tyrosine hydroxylase activity or catecholamine secretion. 7. The results suggest that Ro 31-8220 is not only a protein kinase C inhibitor but is also a potent inhibitor of nicotinic receptor responses in adrenal chromaffin cells by a mechanism unrelated to protein kinase C inhibition. The results are consistent with Ro 31-8220 being a nicotinic receptor antagonist.     

Localization of tyrosine hydroxylase in neuronal targets and efferents of the area postrema in the nucleus tractus solitarii of the rat

Catecholamines in the nucleus tractus solitarii (NTS) have been implicated in autonomic responses to circulating hormones that act on neurons in the area postrema, the most caudal circumventricular organ in brain. We combined immunoperoxidase labeling of the anterograde tracer, Phaseolus vulgaris leucoagglutinin (PHAL) with immunogold-silver labeling of tyrosine hydroxylase to determine whether this enzymatic marker for catecholamines was present in efferents from the area postrema or their targets in the rat NTS. At survival periods of 10-12 days after PHAL injections into the area postrema, light microscopy revealed numerous varicose processes containing peroxidase reaction product for PHAL in the dorsomedial, medial, and commissural NTS. Some of these labeled processes were located near neuronal perikarya and processes containing immunogold-silver intensified reaction product for tyrosine hydroxylase. Electron microscopy of the commissural and dorsomedial NTS established that the majority of the labeling for PHAL was in axon terminals, whereas immunogold labeling for tyrosine hydroxylase was mainly in soma and dendrites. Only 3 out of 579 PHAL-labeled terminals also contained detectable tyrosine hydroxylase immunoreactivity. Fifty-eight percent (335/579) of the PHAL-labeled terminals formed synapses with recognized symmetric junctions, whereas the remainder lacked synaptic specializations within the examined series of serial sections. Of those PHAL terminals forming recognized symmetric junctions, 22% were on tyrosine hydroxylase-immunoreactive dendrites, 74% on unlabeled dendrites and 4% on unlabeled axon terminals. From a total of 1,250 observed contacts on tyrosine hydroxylase labeled dendrites, 88 (7%) contained PHAL, 9 (< 1%) contained TH, and 1,180 (93%) lacked detectable immunoreactivity and formed primarily symmetric synapses. We conclude that a few catecholamine, but mainly noncatecholamine efferents from the area postrema provide a monosynaptic, and most likely inhibitory input to target neurons both with and without tyrosine hydroxylase immunoreactivity in the dorsomedial and commissural NTS. Synapses between the efferent terminals from the area postrema and tyrosine hydroxylase labeled and unlabeled dendrites as well as unlabeled axons in these specific subnuclei of the NTS suggest multiple sites for modulation of gastric and cardiovascular reflexes in response to circulating peptides.     

Biochemical hallmarks of tyrosine hydroxylase deficiency

We report the biochemical hallmarks of tyrosine hydroxylase deficiency with emphasis on reliable diagnostic strategies of four new cases of an inborn error of tyrosine hydroxylase (TH). Three of our patients from different parts of the Netherlands were found homozygous for a mutation in exon 6 (G698A) of the TH gene, and one patient was found compound heterozygous for the same mutation and an additional mutation in exon 3. The first clinical symptoms of hypokinesia, rigidity of arms and legs and axial hypotonia, developed between 3 and 7 months of age. Cerebrospinal fluid investigations revealed a characteristic metabolite constellation in every case: low homovanillic acid (HVA) and 3-methoxy-4-hydroxyphenylethyleneglycol concentrations in the presence of normal reference range 5-hydroxyindolacetic acid concentrations. Strict adherence to a standardized lumbar puncture protocol and adequate age-related reference values are essential for diagnosis of this "new" treatable neurometabolic disorder. Urinary measurements of HVA, vanillylmandelic acid, and catecholamines can lead to false-negative conclusions. All patients showed a remarkable clinical improvement on a low dose of L-dihydroxyphenylalanine/ (S)-2-(3,4-dihydroxybenzyl)-2-hydrazinpropionic acid. During treatment, cerebrospinal fluid HVA, and 3-methoxy-4-hydroxy-phenylethyleneglycol increased substantially.     

Escape performance after inescapable shock in selectively bred lines of mice: Reponse maintenance and catecholamine activity.

Examined the effects of inescapable shock on subsequent escape performance and shock-elicited activity in 6 lines of mice (a total of 528 Ss in 6 experiments) selectively bred for differences in general locomotor activity. The line differences in locomotor activity were found to be unrelated to the differences observed on shock-elicited activity. However, escape performance following exposure to inescapable shock was predictable from the levels of shock-elicited activity. Those lines that displayed the greatest decline in motor activity during shock likewise displayed the most pronounced escape deficits. The line differences in escape performance induced by inescapable shock could be mimicked by treatment with a tyrosine hydroxylase inhibitor, alpha-methylparatyrosine. As predicted, the lines that displayed the least interference after tyrosine hydroxylase inhibition exhibited the smallest reduction in levels of catecholamines. The effects on escape performance following inescapable shock are interpreted in terms of the role of response maintenance deficits produced by catecholamine depletion. (18 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Kinetic properties of ovine adipose tissue fructose-1,6-bisphosphatase

The presence of FBPase was confirmed in both human and ovine white adipose tissue in metabolically significant amounts. The partially purified enzyme from ovine adipose tissue exhibited kinetic properties very similar to other mammalian FBPases (pH optimum of 7.5, absolute requirement for divalent metal ions and strong inhibition by both AMP and F-2,6-P2). The micromolar S0.5 value obtained suggests that the enzyme may be of physiological significance.     

Tyrosine hydroxylase: mechanisms of oxygen radical formation

A new mechanism of oxygen radical formation in dopaminergic neurons is proposed, based on the oxidative mechanism of tyrosine hydroxylase. The cofactor (6R,6S)-5,6,7,8-tetrahydrobiopterin can rearrange in solution which allows an autoxidation reaction producing O2.-, H2O2 and HO.. The combination of tyrosine hydroxylase and the cofactor produces more oxygen radicals than does the autoxidation of the cofactor. This production of oxygen radicals could be damaging to dopaminergic neurons. In the presence of tyrosine, the enzyme produces less radicals than it does in the absence of tyrosine. Mechanisms are proposed for the generation of reactive oxygen species during the autoxidation of the cofactor and during enzymatic catalysis. The generation, by tyrosine hydroxylase, of very small amounts of oxygen radicals over the period of 65 years could contribute to the oxidative stress that causes Parkinson's disease.     

The neuropeptide VIP regulates the expression of the tyrosine hydroxylase gene in cultured avian sympathetic neurons

The neuropeptide vasoactive intestinal polypeptide (VIP) increases the activity of tyrosine hydroxylase (TH), the rate-limiting enzyme in the biosynthesis of catecholamines, in cultured chicken sympathetic neurons. We report here that VIP acts by increasing TH mRNA levels in these cells. Induction of TH mRNA is transient and reaches maximal values 6-8 h after the addition of the peptide to the cultures. TH mRNA levels return to control values after 1-2 days. The quail cDNA probe detects a single mRNA species of approximately 9 kb in RNA extracted both from embryonic chicken sympathetic neurons and adult quail adrenal medulla.     

Enzymatic basis for the lack of oxamniquine activity in Schistosoma haematobium infections

The notion that oxamniquine is active against Schistosoma mansoni but inactive against S. haematobium was confirmed using in vitro cultures of adult worms. Since oxamniquine and hycanthone have been shown to become effective upon activation by a schistosome enzyme, enzymatic tests were carried out to detect possible differences between the enzyme of S. mansoni and that of S. haematobium. It was found that the S. mansoni enzyme could activate hycanthone and, to a lesser extent, oxamniquine. The S. haematobium enzyme, on the other hand, was capable of activating hycanthone but virtually incapable of activating oxamniquine. It is concluded that the different activity of oxamniquine in the two species is due to differences in the drug-activating enzyme.     

Conformational properties and stability of tyrosine hydroxylase studied by infrared spectroscopy. Effect of iron/catecholamine binding and phosphorylation

The conformation and stability of recombinant tetrameric human tyrosine hydroxylase isoenzyme 1 (hTH1) was studied by infrared spectroscopy and by limited tryptic proteolysis. Its secondary structure was estimated to be 42% alpha-helix, 35% beta-extended structures (including beta-sheet), 14% beta-turns, and 10% nonstructured conformations. Addition of Fe(II) or Fe(II) plus dopamine to the apoenzyme did not significantly modify its secondary structure. However, an increased thermal stability and resistance to proteolysis, as well as a decreased cooperativity in the thermal denaturation transition, was observed for the ligand-bound forms. Thus, as compared with the apoenzyme, the ligand-bound subunits of hTH1 showed a more compact tertiary structure but weaker intersubunit contacts within the protein tetramer. Phosphorylation of the apoenzyme by cyclic AMP-dependent protein kinase did not change its overall conformation but allowed on iron binding a conformational change characterized by an increase (about 10%) in alpha-helix and protein stability. Our results suggest that the conformational events involved in TH inhibition by catecholamines are mainly related to modifications of tertiary and quaternary structural features. However, the combined effect of iron binding and phosphorylation, which activates the enzyme, also involves modifications of the protein secondary structure.     

Structure of tetrameric human phenylalanine hydroxylase and its implications for phenylketonuria

Phenylalanine hydroxylase (PheOH) catalyzes the conversion of L-phenylalanine to L-tyrosine, the rate-limiting step in the oxidative degradation of phenylalanine. Mutations in the human PheOH gene cause phenylketonuria, a common autosomal recessive metabolic disorder that in untreated patients often results in varying degrees of mental retardation. We have determined the crystal structure of human PheOH (residues 118-452). The enzyme crystallizes as a tetramer with each monomer consisting of a catalytic and a tetramerization domain. The tetramerization domain is characterized by the presence of a domain swapping arm that interacts with the other monomers forming an antiparallel coiled-coil. The structure is the first report of a tetrameric PheOH and displays an overall architecture similar to that of the functionally related tyrosine hydroxylase. In contrast to the tyrosine hydroxylase tetramer structure, a very pronounced asymmetry is observed in the phenylalanine hydroxylase, caused by the occurrence of two alternate conformations in the hinge region that leads to the coiled-coil helix. Examination of the mutations causing PKU shows that some of the most frequent mutations are located at the interface of the catalytic and tetramerization domains. Their effects on the structural and cellular stability of the enzyme are discussed.     

Plasma metanephrines are markers of pheochromocytoma produced by catechol-O-methyltransferase within tumors

This study examined whether the high sensitivity of plasma free metanephrines for diagnosis of pheochromocytoma may result from production of free metanephrines within tumors. Presence in pheochromocytomas of catechol-O-methyltransferase (COMT), the enzyme responsible for conversion of catecholamines to metanephrines, was confirmed by Western blot analysis, enzyme assay, and immunohistochemistry. Western blot analysis and enzyme assay indicated that membrane-bound and not soluble COMT was the predominant form of the enzyme in pheochromocytoma. Immunohistochemistry revealed colocalization of COMT in the same chromaffin cells where catecholamines are translocated into storage vesicles by the vesicular monoamine transporter. Levels of free metanephrines in pheochromocytoma over 10,000 times higher than plasma concentrations in the same patients before removal of tumors indicated production of metanephrines within tumors. Comparisons of the production of metanephrines in patients with pheochromocytoma with production from catecholamines released or infused into the circulation indicated that more than 93% of the consistently elevated levels of circulating free metanephrines in patients with pheochromocytoma are derived from metabolism before and not after release of catecholamines into the circulation. The data indicate that the elevated plasma levels of free metanephrines in patients with pheochromocytoma are derived from catecholamines produced and metabolized within tumors. Some tumors do not secrete catecholamines, but all appear to metabolize catecholamines to free metanephrines, thus explaining the better sensitivity of plasma free metanephrines over other tests for diagnosis of pheochromocytoma.     

Substantial production of dopamine in the human gastrointestinal tract

Considerable urinary excretion of dopamine metabolites indicates that large amounts of dopamine are produced in unknown locations of the body. This study assessed the contribution of mesenteric organs (gastrointestinal tract, spleen, and pancreas) to the total body production of dopamine in humans and examined the presence of the rate-limiting enzyme for dopamine synthesis, tyrosine hydroxylase, in gastrointestinal tissues. Blood sampled from an artery and portal and hepatic veins in eight subjects and from arterial and renal venous sites in other subjects was analyzed for plasma concentrations of dopamine and its metabolites. The activity and distribution of tyrosine hydroxylase was also examined in tissue samples from the stomach and duodenum. Higher concentrations of dopamine and its metabolites in portal venous than arterial plasma indicated substantial production of dopamine by mesenteric organs (12.0 nmol/min) amounting to 42-46% of the renal removal of circulating dopamine metabolites. Tissue samples showed immunoreactive tyrosine hydroxylase in nonneuronal cell bodies and detectable levels of tyrosine hydroxylase in nonneuronal cell bodies and detectable levels of tyrosine hydroxylase enzyme activity. The results show that mesenteric organs produce close to half of the dopamine formed in the body, most of which is unlikely to be derived from sympathetic nerves but may reflect production in a novel nonneuronal dopaminergic system.