Step In Catecholamine Biosynthesis Research Articles

Overview of the 2022 WHO Classification of Paragangliomas and Pheochromocytomas.

This review summarizes the classification of tumors of the adrenal medulla and extra-adrenal paraganglia as outlined in the 5th series of the WHO Classification of Endocrine and Neuroendocrine Tumors. The non-epithelial neuroendocrine neoplasms (NENs) known as paragangliomas produce predominantly catecholamines and secrete them into the bloodstream like hormones, and they represent a group of NENs that have exceptionally high genetic predisposition. This classification discusses the embryologic derivation of the cells that give rise to these lesions and the historical evolution of the terminology used to classify their tumors; paragangliomas can be sympathetic or parasympathetic and the term pheochromocytoma is used specifically for intra-adrenal paragangliomas that represent the classical sympathetic form. In addition to the general neuroendocrine cell biomarkers INSM1, synaptophysin, and chromogranins, these tumors are typically negative for keratins and instead have highly specific biomarkers, including the GATA3 transcription factor and enzymes involved in catecholamine biosynthesis: tyrosine hydroxylase that converts L-tyrosine to L-DOPA as the rate-limiting step in catecholamine biosynthesis, dopamine beta-hydroxylase that is present in cells expressing norepinephrine, and phenylethanolamine N-methyltransferase, which converts norepinephrine to epinephrine and therefore can be used to distinguish tumors that make epinephrine. In addition to these important tools that can be used to confirm the diagnosis of a paraganglioma, new tools are recommended to determine genetic predisposition syndromes; in addition to the identification of precursor lesions, molecular immunohistochemistry can serve to identify associations with SDHx, VHL, FH, MAX, and MEN1 mutations, as well as pseudohypoxia-related pathogenesis. Paragangliomas have a well-formed network of sustentacular cells that express SOX10 and S100, but this is not a distinctive feature, as other epithelial NENs also have sustentacular cells. Indeed, it is the presence of such cells and the association with ganglion cells that led to a misinterpretation of several unusual lesions as paragangliomas; in the 2022 WHO classification, the tumor formerly known as cauda equina paraganglioma is now classified as cauda equina neuroendocrine tumor and the lesion known as gangliocytic paraganglioma has been renamed composite gangliocytoma/neuroma and neuroendocrine tumor (CoGNET). Since the 4th edition of the WHO, paragangliomas have no longer been classified as benign and malignant, as any lesion can have metastatic potential and there are no clear-cut features that can predict metastatic behavior. Moreover, some tumors are lethal without metastatic spread, by nature of local invasion involving critical structures. Nevertheless, there are features that can be used to identify more aggressive lesions; the WHO does not endorse the various scoring systems that are reviewed but also does not discourage their use. The identification of metastases is also complex, particularly in patients with germline predisposition syndromes, since multiple lesions may represent multifocal primary tumors rather than metastatic spread; the identification of paragangliomas in unusual locations such as lung or liver is not diagnostic of metastasis, since these may be primary sites. The value of sustentacular cells and Ki67 labeling as prognostic features is also discussed in this new classification. A staging system for pheochromocytoma and extra-adrenal sympathetic PGLs, introduced in the 8th Edition AJCC Cancer Staging Manual, is now included. This paper also provides a summary of the criteria for the diagnosis of a composite paragangliomas and summarizes the classification of neuroblastic tumors. This review adopts a practical question-answer framework to provide members of the multidisciplinary endocrine oncology team with a most up-to-date approach to tumors of the adrenal medulla and extra-adrenal paraganglia.

Personalized Medicine to Improve Treatment of Dopa-Responsive Dystonia-A Focus on Tyrosine Hydroxylase Deficiency.

Dopa-responsive dystonia (DRD) is a rare movement disorder associated with defective dopamine synthesis. This impairment may be due to the fact of a deficiency in GTP cyclohydrolase I (GTPCHI, GCH1 gene), sepiapterin reductase (SR), tyrosine hydroxylase (TH), or 6-pyruvoyl tetrahydrobiopterin synthase (PTPS) enzyme functions. Mutations in GCH1 are most frequent, whereas fewer cases have been reported for individual SR-, PTP synthase-, and TH deficiencies. Although termed DRD, a subset of patients responds poorly to L-DOPA. As this is regularly observed in severe cases of TH deficiency (THD), there is an urgent demand for more adequate or personalized treatment options. TH is a key enzyme that catalyzes the rate-limiting step in catecholamine biosynthesis, and THD patients often present with complex and variable phenotypes, which results in frequent misdiagnosis and lack of appropriate treatment. In this expert opinion review, we focus on THD pathophysiology and ongoing efforts to develop novel therapeutics for this rare disorder. We also describe how different modeling approaches can be used to improve genotype to phenotype predictions and to develop in silico testing of treatment strategies. We further discuss the current status of mathematical modeling of catecholamine synthesis and how such models can be used together with biochemical data to improve treatment of DRD patients.

A SENSITIVE ASSAY FOR NON-SPECIFIC N-METHYLTRANSFERASE ACTIVITY IN RAT TISSUES BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY ELECTROCHEMICAL DETECTION

Phenylethanolamine N-methyltransferase (PNMT) and non-specific N -methyltransferase (EC 2.1.1.28) catalyze the N-methylation of aromatic amines. PNMT is specific for phenylethanolamines such as noradrenaline (NA). and catalyzes the step in catecholamine biosynthesis, forming adrenaline (AD) from NA. PNMT activity is high in adrenal gland, whereas non-specific N-methyltransferase is distributed in various tissues such as the lungs. Borchardt et al. first reported a method to detect PNMT activity by high-performance liquid chromatography electrochemical detection (HPLC-EICD), which could demonstrate the activity only in the adrenal medulla and hypothalamus. Recently, Troeewicz et al. reported a highly sensitive assay method for PNMT using HPLC-EICD by which the activity in all regions of rat brains could be measured. The activity of non-specific N-methyltransferase in brain regions and peripheral tissues of the rat could be detected by a radioassay. However, there has been no repot on an assay method for non-specific N-methyltransferase using HPLC-EICD. In this paper, we describe a highly sensitive assay procedure for the activity of non-specific N-methyltransferase by high-performance reversed-phase ion pair chromatography with electrochemical detection. By this method, the non-specific N-methyltransferase activity could be determined in various rat brain regions and peripheral tissues.

A Novel Model of Dexamethasone-Induced Hypertension: Use in Investigating the Role of Tyrosine Hydroxylase.

Our objective was to study hypertension induced by chronic administration of synthetic glucocorticoid, dexamethasone (DEX), under nonstressful conditions and examine the role of catecholamine biosynthesis. To achieve this, we did the following: 1) used radiotelemetry to record mean arterial pressure (MAP) and heart rate (HR) in freely moving rats, and 2) administered different doses of DEX in drinking water. To evaluate the involvement of tyrosine hydroxylase (TH), the rate-limiting step in catecholamine biosynthesis, we treated rats with the TH inhibitor, α-methyl-para-tyrosine (α-MPT), for 3 days prior to administration of DEX and assessed TH mRNA and protein expression by quantitative real-time polymerase chain reaction and Western blot in the adrenal medulla. We observed a dose-dependent elevation in blood pressure with a DEX dose of 0.3 mg/kg administered for 10 days, significantly increasing MAP by +15.0 ± 1.1 mm Hg, while concomitantly reducing HR. Although this DEX treatment also significantly decreased body weight, pair-fed animals that showed similar decreases in body weight due to lowered food intake were not hypertensive, suggesting that body weight changes may not account for DEX-induced hypertension. Chronic DEX treatment significantly increased the TH mRNA and protein levels in the adrenal medulla, and α-MPT administration not only reduced DEX pressor effects, but also inhibited TH (serine(40)) phosphorylation. Our study thus validates a novel model to study hypertension induced by chronic intake of DEX in freely moving rats not subject to the confounding factors of previous models and establishes its dependence on concomitant activation of peripheral catecholamine biosynthesis.

Kinetic and pH studies on human phenylethanolamine N-methyltransferase

Phenylethanolamine N-methyltransferase (PNMT) catalyzes the conversion of norepinephrine (noradrenaline) to epinephrine (adrenaline) while, concomitantly, S-adenosyl-l-methionine (AdoMet) is converted to S-adenosyl-l-homocysteine. This reaction represents the terminal step in catecholamine biosynthesis and inhibitors of PNMT have been investigated, inter alia, as potential antihypertensive agents. At various times the kinetic mechanism of PNMT has been reported to operate by a random mechanism, an ordered mechanism in which norepinephrine binds first, and an ordered mechanism in which AdoMet binds first. Here we report the results of initial velocity studies on human PNMT in the absence and presence of product and dead end inhibitors. These, coupled with isothermal titration calorimetry and fluorescence binding experiments, clearly shown that hPNMT operates by an ordered sequential mechanism in which AdoMet binds first. Although the logV pH-profile was not well defined, plots of logV/K versus pH for AdoMet and phenylethanolamine, as well as the pKi versus pH for the inhibitor, SK&F 29661, were all bell-shaped indicating that a protonated and an unprotonated group are required for catalysis.

Comparative anatomy of the locus coeruleus in humans and nonhuman primates

The locus coeruleus (LC) is a dense cluster of neurons that projects axons throughout the neuroaxis and is located in the rostral pontine tegmentum extending from the level of the inferior colliculus to the motor nucleus of the trigeminal nerve. LC neurons are lost in the course of several neurodegenerative disorders, including Alzheimer's and Parkinson's diseases. In this study we used Nissl staining and tyrosine hydroxylase (TH) immunoreactivity to compare the human LC with that of closely related primate species, including great and lesser apes, and macaque monkeys. TH catalyzes the initial and rate-limiting step in catecholamine biosynthesis. The number of TH-immunoreactive (TH-ir) neurons was estimated in each species using stereologic methods. In the LC of humans the mean total number of TH-ir neurons was significantly higher compared to the other primates. Because the total number of TH-ir neurons in the LC was highly correlated with the species mean volume of the medulla oblongata, cerebellum, and neocortical gray matter, we conclude that much of the observed phylogenetic variation can be explained by anatomical scaling. Notably, the total number of LC neurons in humans was most closely predicted by the nonhuman allometric scaling relationship relative to medulla size, whereas the number of LC neurons in humans was considerably lower than predicted according to neocortex and cerebellum volume.

Biochemical and molecular characterization of tyrosine hydroxylase deficiency in Hong Kong Chinese

Tyrosine hydroxylase deficiency is a rare neurotransmitter disorder affecting the rate-limiting step in catecholamine biosynthesis. There are about 40 cases reported worldwide. Here, we report the biochemical and molecular findings of eight unrelated Chinese patients with tyrosine hydroxylase deficiency. We have identified eight novel mutations with 5 missense, 2 nonsense and 1 splicing mutations in the TH gene, namely p.R153X, p.R169X, p.G294R, p.G315S, p.A385V, p.I394T, p.G408R, and c.1163+5G>C. The mutations of the TH gene in Chinese are heterogeneous.

Stimulation of catecholamine synthesis via activation of p44/42 MAPK in cultured bovine adrenal medullary cells by milnacipran

Milnacipran is a serotonin noradrenaline reuptake inhibitor (SNRI) and is used clinically as an antidepressant. We report here the effect of milnacipran on catecholamine synthesis in cultured bovine adrenal medullary cells. Incubation of adrenal medullary cells with milnacipran (300 ng/ml, 1,065 nM) for 20 min resulted in a significant increase in 14C-catecholamine synthesis from [14C]tyrosine, but not from [14C]DOPA, whereas the selective serotonin reuptake inhibitors (SSRIs), paroxetine (300 ng/ml, 800 nM) and fluvoxamine (300 ng/ml, 691 nM), had little effect. Milnacipran, but not paroxetine or fluvoxamine, increased the activity of tyrosine hydroxylase, the rate-limiting step of catecholamine biosynthesis, in a concentration-dependent manner (100-300 ng/ml, 355-1,065 nM). U0126 (1 microM), an inhibitor of p44/42 mitogen-activated protein kinase (MAPK) kinase, abolished the stimulatory effects of milnacipran on tyrosine hydroxylase activity. Furthermore, incubation of cells with milnacipran (30-100 ng/ml) for 5 min activated p44/42 MAPK, whereas paroxetine and fluvoxamine did not. The present findings suggest that milnacipran activates tyrosine hydroxylase and then stimulates catecholamine synthesis through a p44/42 MAPK-dependent pathway in cultured bovine adrenal medullary cells.

A developmental role for catecholamines in Drosophila behavior

Tyrosine hydroxylase (TH), the enzyme which catalyzes the conversion of tyrosine to l-DOPA and is the rate limiting step in catecholamine biosynthesis, is genetically expressed during development in Drosophila. Null mutant alleles of the single copy gene which codes for this enzyme are developmentally lethal as is a conditional TH mutant at its restrictive temperature. In adult flies, inhibition of TH by alpha-methyl- p-tyrosine (αMT) decreases locomotor activity in a dose-dependent manner. This behavioral effect is accompanied by reductions in brain levels of dopamine, the primary CNS catecholamine in Drosophila, and can be prevented by the coadministration of l-DOPA. Similar effects are found with reserpine and at the restrictive temperature in flies with a temperature conditional mutation for TH. In agreement with published studies in mammals, inhibition of TH by αMT during Drosophila development results in enhanced expression of this enzyme in the progeny of surviving adults. This biochemical outcome is accompanied behaviorally by increased sensitivity to the locomotor effects of both αMT and reserpine, drugs which act via depletion of brain catecholamines. Since TH is the rate limiting enzyme responsible for the conversion of tyrosine to l-DOPA and l-DOPA is converted to dopamine by aromatic amino acid decarboxylase (AAAD), the results indicate that depletion of catecholamine levels in the fly embryo results in increased dopamine biosynthesis in the next generation accompanied by alterations in behavior.

Effects of phosphorylation by protein kinase A on binding of catecholamines to the human tyrosine hydroxylase isoforms.

Tyrosine hydroxylase (TyrH), the catalyst for the key regulatory step in catecholamine biosynthesis, is phosphorylated by cAMP-dependent protein kinase A (PKA) on a serine residue in a regulatory domain. In the case of the rat enzyme, phosphorylation of Ser40 by PKA is critical in regulating the enzyme activity; the effect of phosphorylation is to relieve the enzyme from inhibition by dopamine and dihydroxyphenylalanine (DOPA). There are four isoforms of human tyrosine hydroxylase (hTyrH), differing in the size of an insertion after Met30. The effects of phosphorylation by PKA on the binding of DOPA and dopamine have now been determined for all four human isoforms. There is an increase of about two-fold in the Kd value for DOPA for isoform 1 upon phosphorylation, from 4.4 to 7.4 microM; this effect decreases with the larger isoforms such that there is no effect of phosphorylation on the Kd value for isoform 4. Dopamine binds more much tightly, with Kd values less than 3 nM for all four unphosphorylated isoforms. Phosphorylation decreases the affinity for dopamine at least two orders of magnitude, resulting in Kd values of about 0.1 microM for the phosphorylated human enzymes, due primarily to increases in the rate constant for dissociation of dopamine. Dopamine binds about two-fold less tightly to the phosphorylated isoform 1 than to the other three isoforms. The results extend the regulatory model developed for the rat enzyme, in which the activity is regulated by the opposing effects of catecholamine binding and phosphorylation by PKA. The small effects on the relatively high Kd values for DOPA suggest that DOPA levels do not regulate the activity of hTyrH.

Quantitative evaluation of catecholamine enzymes gene expression in adrenal medulla and sympathetic Ganglia of stressed rats.

Stress-induced changes in mRNA levels of tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH), and phenylethanolamine N-methyltransferase (PNMT) have been expressed as relative arbitrary units compared with a control group. The aim of this study was to quantify basal and stress-induced levels of TH, DBH, and PNMT mRNAs in rat adrenal medulla (AM) and stellate ganglia (SG) by the RT-competitive PCR method using corresponding competitors of known concentration. In rats stressed by immobilization (IMO) once for 2 h, the concentration of mRNAs was determined in various intervals after the end of stress stimulus. In SG, the basal concentration of TH mRNA was 0.017 amol/ng of total RNA, which is approximately 30 times lower than in the AM (0.460 amol/ng RNA). The basal concentration of DBH mRNA in SG was 2.60 amol/ng of total RNA, which is about 150 times more than TH mRNA in SG but only two times less than DBH mRNA in the AM in which PNMT mRNA is present in the highest concentration. After a single 2-h IMO, the peak elevation of TH and DBH mRNA concentration in SG occurred 24 h after the termination of stress stimulus, when their AM mRNA concentrations were already at control values. Presence of PNMT mRNA levels in the SG, of control and stressed rats has been demonstrated for the first time. Repeated IMO (7 days, 2 h daily) did not produce further increase in the mRNA concentrations compared with the elevated values found in adapted control groups. Levels of TH protein were significantly increased only after repeated IMO in SG and AM. Thus, our data show for the first time the exact concentrations of TH, DBH, and PNMT mRNA in SG and AM of rats under control and stress conditions. The lowest concentration of TH mRNA in the AM and SG supports the hypothesis that tyrosine hydroxylation is the rate-limiting step in catecholamine biosynthesis.

Regulation of the tyrosine hydroxylase gene promoter by histone deacetylase inhibitors

Tyrosine hydroxylase (TH) catalyzes the conversion of l-tyrosine to 3,4-dihydroxy- l-phenylalanine, which is the first and rate-limiting step in catecholamine biosynthesis. In the present study, we report that treatment with the histone deacetylase (HDAC) inhibitors, trichostatin A (TSA) or sodium butyrate, prominently induces the TH promoter activity in both non-neuronal and neuronal cell lines. By analyzing a series of deletional reporter constructs, we also determined that the proximal 151 bp region of the TH promoter is largely responsible for TSA-mediated activation. Finally, we found that mutation of the Sp1 or CRE site, residing in the proximal area, abolishes TSA-mediated activation, strongly suggesting that the Sp1 and CRE sites may mediate TH promoter activation by inhibition of HDAC. In summary, our results provide a novel regulatory frame in which modulation of chromatin structure by histone deacetylase may contribute to transcriptional regulation of the TH via the Sp1 and/or CRE site.

Effects of pharmacological agents upon a transgenic model of Parkinson's disease in Drosophila melanogaster.

The human gene that codes for the protein alpha-synuclein has been transferred into the Drosophila melanogaster genome. The transgenic flies recapitulate some of the essential features of Parkinson's disease. These include the degeneration of certain dopaminergic neurons in the brain accompanied by the appearance of age-dependent abnormalities in locomotor activity. In the present study, we tested the locomotor response of these transgenic flies to prototypes of the major classes of drugs currently used to treat this disorder. A time course study was first conducted to determine when impaired locomotor activity appeared relative to normal "wild-type" flies. A climbing or negative geotaxis assay measuring the ability of the organisms to climb up the walls of a plastic vial was used. Based on the results obtained, normal and transgenic flies were treated with each of the drugs in their food for 13 days and then assayed. The activity of transgenic flies treated with L-DOPA was restored to normal. Similarly, the dopamine agonists pergolide, bromocriptine, and 2,3,4,5-tetrahydro-7,8-dihydroxy- 1-phenyl-1H-3-benzazepine (SK&F 38393) were substantially effective. Atropine, the prototypical muscarinic cholinergic receptor antagonist, was also effective but to a lesser extent than the other antiparkinson compounds. p-Chlorophenylalanine, an inhibitor of serotonin synthesis, was without beneficial effect as was alpha-methyl-p-tyrosine, an inhibitor of tyrosine hydroxylase, the rate-limiting step in catecholamine biosynthesis. This behavioral study further demonstrates the utility of this model in studying Parkinson's disease and reinforces the concept that inhibition of the action of alpha-synuclein may be useful in its treatment as may dopamine D(1) receptor agonists.

Identification and characterization of potential cis-regulatory elements governing transcriptional activation of the rat tyrosine hydroxylase gene.

Tyrosine hydroxylase (TH) catalyzes the conversion of L-tyrosine to 3,4-dihydroxy-L-phenylalanine, which is the first and rate-limiting step in catecholamine biosynthesis. We have previously shown that the cyclic AMP response element (CRE), an essential promoter element for both basal and cyclic AMP-inducible TH transcription, activates the promoter activity in a distance-dependent manner. To identify further cis-regulatory elements controlling TH gene expression, we analyzed the potential regulatory sequences by several approaches. First, using transient transfection assays, we examined the cell-specific promoter activities of TH-reporter gene constructs and a dopamine beta-hydroxylase (DBH)-reporter construct containing the 5' upstream sequences of the rat TH and human DBH genes, respectively, that had been shown to direct tissue-specific reporter expression in transgenic mice experiments. Second, DNase I footprinting analysis of the 503-bp proximal area of the rat TH gene identified seven footprinted regions that encompass the putative cis-regulatory motifs, including the CRE (domain 1), Sp1 (domain III), Octamer (domain IV), AP1 (domain V), AP2 (domain VI), and two potentially novel sequence motifs (domains II and VII). Footprinting patterns at these sites by nuclear proteins from TH-positive and -negative cell lines appeared to be similar. Third, site-directed mutagenesis demonstrates that domain III, but not domain II, critically contributes to the TH promoter activity. Furthermore, electrophoretic mobility shift, competition, and supershift assays demonstrate that domain III is an authentic Sp1 site and that the transcription factor Sp1 interacts with it. This and previous results suggest that the CRE and Sp1 site may synergistically activate TH transcription in a promoter context-dependent manner.

A stress-activated kinase cascade can mediate the activation of tyrosine hydroxylase in chromaffin cells.

Tyrosine hydroxylase (TH) catalyses the initial, rate-limiting step in catecholamine biosynthesis and is therefore responsible for replenishing the stocks of adrenalin and noradrenalin in the adrenal medulla which are depleted following release of these hormones into the circulation. TH is activated in vitro and in vivo by phosphorylation and various kinases have been shown to phosphorylate four sites on this enzyme [l-41. We were interested to investigate whether a novel stress-activated kinase cascade [5 ] could mediate the phosphorylation and activation of TH. This cascade is triggered by exposure of cells to cellular stresses and inflammatory cytokines (Fig 1) and is analogous to, but distinct from, the ‘classical’ growth factor-activated MAP kinase cascade. Study of the roles of the novel cascade is facilitated by the use of SB 203580 [6], a specific inhibitor of SAPK-2, a MAP kinase homologue in the novel cascade. Physiological targets for the cascade identified using this inhibitor include the small heat shock protein HSP27 and the transcription factor CREB.

Regulation of Tyrosine Hydroxylase by Neuropeptides

The rate-limiting step in catecholamine biosynthesis, the hydroxylation of tyrosine (TH), is regulated primarily by two processes, one involving activation of preexisting TH molecules and the other involving the synthesis of new enzyme molecules. Activation of TH occurs rapidly after a stimulus, is quickly reversible, and involves post-translational modification of preexisting TH molecules. Increasing both cholinergic antagonists by an order of magnitude does not further diminish the increase in TH activity. This unexpected finding eventually led to the discovery that certain neuropeptides cause the activation of TH. Of the large number of neuropeptides that were tested as possible mediators of the noncholinergic transsynaptic regulation of TH, only a subgroup of the peptides of the secretin-glucagon family were found to be candidates. Five peptides from this family, namely secretin, vasoactive intestinal peptide (VIP), peptide histidine isoleucineamide (PHI), rat growth hormone-releasing hormone, and helodermin, increase TH activity acutely. With regard to the mechanisms underlying this peptidergic activation of TH, an important observation was the finding that those peptides that caused TH activation also cause increased levels of cyclic adenosine monophosphate (cAMP) in the SCG, whereas those peptides that have no effect on TH activity have no effect on cAMP levels. To determine whether neuropeptides of the secretin-glucagon family might influence catecholamine biosynthesis not only in adrenergic cells but also in adrenergic nerve terminals, tissues innervated by the SCG were examined, namely, the iris, pineal gland, and submaxillary gland. Incubation of these target tissues with secretin and VIP leads to TH activation.

Testis of Prepubertal Rhesus Monkeys Receives a Dual Catecholaminergic Input Provided by the Extrinsic Innervation and an Intragonadal Source of Catecholamines1

The mammalian testis is innervated by extrinsic catecholaminergic nerves and responds to catecholamines with steroid secretion. Although the primate testis has also been shown to be innervated, potential differences in the density of this innervation between immature and sexually developed individuals have not been described. A recent study demonstrated that the primate ovary contains a network of neuron-like cells and that some of these cells are catecholaminergic. It is thus possible that the male gonad is also endowed with a similar intragonadal source of catecholamines. The present study addresses these two issues. Catecholaminergic nerves were identified as such by their content of immunoreactive tyrosine hydroxylase (TH; the rate-limiting step in catecholamine biosynthesis), and in some cases by glyoxylic acid histochemistry. Fibers containing TH were abundant in testes from juvenile animals (1-2 yr of postnatal life), but the density of this innervation was not maintained in adult animals, whose testis showed only a few TH-positive fibers scattered in the interstitial tissue. Testicular norepinephrine (NE) concentration was much lower in adult than in juvenile animals, suggesting that the marked increase in testicular weight that occurs with the attainment of sexual maturity is not accompanied by corresponding changes in NE content. At the ultrastructural level, testicular nerve fibers contained pleiomorphic, dense-core and clear vesicles, suggesting the presence of catecholamines and other neurotransmitters. In addition to this extrinsic catecholaminergic innervation, prepubertal testes, but not adult gonads, contained an intrinsic population of TH-immunopositive neuron-like elements, identified as cells by confocal scanning laser microscopy. To determine whether the prepubertal monkey testis indeed expresses the TH gene, testicular RNA was subjected to reverse transcriptase polymerase chain reaction to amplify the 5' end of TH mRNA, which encodes the regulatory domain of the enzyme. The cDNA that was obtained predicts an amino acid sequence similar, but not identical, to that encoded by the alternatively spliced type 1 TH mRNA form present in the adrenal gland. These results indicate 1) that the primate testis receives a dual catecholaminergic input, one provided by the extrinsic innervation and the other by neuron-like cells located within the gonad itself, and 2) that the influence exerted by both sources on testicular function may be more prominent during the prepubertal period than in adulthood. The presence in the testis of a TH mRNA variant encoding amino acid substitutions in its 5' end suggests that regulation of testicular TH enzyme activity may include a gonad-specific component.

Effects of hypercortisolemia or hyperinsulinemia on neurochemical indices of catecholamine release and synthesis in conscious rats

Glycocorticoids and insulin (INS) complexly affect sympathoneural and adrenomedullary outflows. This study assessed effects of chronic hypercortisolemia and effects of INS independent of INS-induced hypoglycemia on neurochemical indices of different aspects of catecholaminergic function in conscious rats. Since l-DOPA is the precursor of the endogenous catecholamines and the immediate product of the rate-limiting enzymatic step in catecholamine biosynthesis, alterations in rates of appearance (spillover) of l-DOPA in arterial plasma may reflect alterations in catecholamine synthesis. The study therefore included examination of whether cortisol (CORT) or INS affects l-DOPA spillover or renal excretion of dopamine (DA) derived from plasma l-DOPA. Arterial plasma levels and urinary excretion rates of endogenous catechols and radiolabelled l-DOPA and DA were measured during systemic intravenous infusions of [ 3H] l-DOPA. CORT was administered via subcutaneous minipump reservoir for one week prior to [ 3H] l-DOPA infusion, and INS was infused with glucose to examine effects of hyperinsulinemia independently of hypoglycemia. CORT decreased plasma levels and urinary excretion of norepinephrine (NE). INS did not. Neither CORT nor INS affected levels of other catechols, l-DOPA spillover, or the rate of urinary excretion of [ 3H]DA for a given plasma level of [ 3H] l-DOPA. The results suggest that CORT inhibits sympathetically-mediated NE release without altering overall rates of catecholamine turnover or synthesis in sympathetic nerves in vivo and that INS effects on catecholaminergic function depend entirely on INS-induced hypoglycemia.

Catalytic core of rat tyrosine hydroxylase: terminal deletion analysis of bacterially expressed enzyme

Tyrosine hydroxylase (TH) catalyzes the rate-limiting step in catecholamine biosynthesis. This enzyme is hypothesized to consist of an amino-terminal regulatory domain and a carboxy-terminal catalytic domain. In the present studies, we have utilized recombinant DNA techniques to map the boundaries of the regulatory and catalytic domains of TH. We have isolated a full-length cDNA clone for rat pheochromocytoma TH and have expressed the enzyme in bacteria. Utilizing this bacterial expression system and polymerase chain reaction technology, we have constructed and subcloned genes for five amino-terminal deletion mutants (NΔ40, NΔ155, NΔ165, NΔ175 and NΔ200; NΔ denotes amino-terminal deletion and the numerical value denotes the number of amino acids deleted) and two carboxy-terminal deletion mutants (CΔ19 and CΔ50). The catalytic core of rat tyrosine hydroxylase has been identified to include the region from amino acid #165 to amino acid #479. The amino-terminal deletion mutants, NΔ40, NΔ155 and NΔ165 are from 1.85 to 2.5-fold more active than unmodified recombinant TH, while the removal of 19 amino acids from the C-terminus (CΔ19) results in a 70% reduction in enzyme activity. Removal of additional sequences (ten more residues from the N-terminus [NΔ175]; or an additional 31 amino acids from the C-terminus [CΔ50]) results in protein that is totally without enzyme activity. As expected, removal of 40 (or more) N-terminal amino acids abolishes the ability of the catalytic subunit of the cAMP-dependent protein kinase to phosphorylate the recombinant enzyme; serine-40 is the phosphorylation site on TH for PKA. We conclude that the N-terminal boundary for the TH catalytic domain resides between residues 165 and 175 and that removal of this N-terminal domain (totally or partially) increases the activity of the enzyme. These findings confirm previous reports that proteolytic cleavage at amino acid #158 produces an active (and activated) catalytic fragment.

Linkage analysis between manic depressive illness and the dopamine beta-hydroxylase gene.

The dopamine beta-hydroxylase (DBH) gene is a candidate gene in manic depressive illness. DBH is required for conversion of dopamine to norepinephrine, the third step in catecholamine biosynthesis. A few earlier linkage studies have found low to moderately positive lod scores in manic depressive families for ABO which is closely linked to DBH. Based on several studies an association between manic depressive illness and ABO blood type has been suggested. Mutations at the DBH locus might thus be involved in the etiology of manic depressive illness in some families. The DBH gene is reported here as unlikely to be a major gene causing manic depressive illness in a large family. Linkage was excluded assuming a dominant mode of transmission. Several methods were used to minimize misclassification.