Restoration of Noradrenergic Function in Parkinson's Disease Model Mice.

Dysfunction of the central noradrenergic and dopaminergic systems is the primary neurobiological characteristic of Parkinson's disease (PD). Importantly, neuronal loss in the locus coeruleus (LC) that occurs in early stages of PD may accelerate progressive loss of dopaminergic neurons. Therefore, restoring the activity and function of the deficient noradrenergic system may be an important therapeutic strategy for early PD. In the present study, the lentiviral constructions of transcription factors Phox2a/2b, Hand2 and Gata3, either alone or in combination, were microinjected into the LC region of the PD model VMAT2 Lo mice at 12 and 18 month age. Biochemical analysis showed that microinjection of lentiviral expression cassettes into the LC significantly increased mRNA levels of Phox2a, and Phox2b, which were accompanied by parallel increases of mRNA and proteins of dopamine ß-hydroxylase (DBH) and tyrosine hydroxylase (TH) in the LC. Furthermore, there was considerable enhancement of DBH protein levels in the frontal cortex and hippocampus, as well as enhanced TH protein levels in the striatum and substantia nigra. Moreover, these manipulations profoundly increased norepinephrine and dopamine concentrations in the striatum, which was followed by a remarkable improvement of the spatial memory and locomotor behavior. These results reveal that over-expression of these transcription factors in the LC improves noradrenergic and dopaminergic activities and functions in this rodent model of PD. It provides the necessary groundwork for the development of gene therapies of PD, and expands our understanding of the link between the LC-norepinephrine and dopamine systems during the progression of PD.

Hindbrain metabolic deficiency regulates ventromedial hypothalamic nucleus glycogen metabolism and glucose­regulatory signaling.

The catecholamine norepinephrine (NE) links hindbrain metabolic­sensory neurons with downstream gluco­regulatory loci, including the ventromedial hypothalamic nucleus (VMN). Exogenous NE up­regulates VMN expression of glutamate decarboxylase (GAD), biomarker for the gluco­inhibitory transmitter γ­aminobutryic acid (GABA). Brain glycogen phosphorylase (GP)­muscle (GPmm) and ­brain (GPbb) variants are stimulated in vitro by NE or energy deficiency, respectively. Current research investigated whether lactoprivic­driven VMN NE signaling regulates GABA and if VMN GPmm and GPbb profiles react differently to that deficit cue. Male rats were pretreated by caudal fourth ventricle delivery of the selective catecholamine neurotoxin 6­hydroxydopamine (6OHDA) ahead of the monocarboxylate transporter inhibitor alpha­cyano­4­hydroxycinnamic acid (4CIN). Micropunch­dissected VMN tissue was analyzed by Western blot and ELISA to assess NE­dependent 4CIN regulation of GAD and GP variant protein expression and NE activity. 4CIN caused 6OHDA­reversible augmentation of VMN NE content and plasma glucose and counter­regulatory hormone levels. 6OHDA stimulated basal VMN GAD expression, but prevented 4CIN stimulation of this profile. Neurotoxin inhibited or increased baseline VMN GPmm and GPbb levels, respectively, in non­4CIN­injected rats. 6OHDA deterred 4CIN inhibition of GPmm, but did not prevent drug stimulation of GPbb. Results affirm hindbrain lactoprivic regulation of glucostasis. Hindbrain NE exerts opposite effects on VMN GABA transmission during hindbrain lactostasis vs. ­privation. VMN norepinephrine­ vs. energy­sensitive GP variants are subject to dissimilar NE regulation during energy homeostasis, and respond differently to hindbrain lactoprivation.

Effects of transgenic expression of dopamine beta hydroxylase (Dbh) gene on blood pressure in spontaneously hypertensive rats.

The spontaneously hypertensive rat (SHR) is the most widely used animal model of essential hypertension and left ventricular hypertrophy. Catecholamines play an important role in the pathogenesis of both essential hypertension in humans and in the SHR. Recently, we obtained evidence that the SHR harbors a variant in the gene for dopamine beta hydroxylase (Dbh) that is associated with reduced adrenal expression of Dbh mRNA and reduced DBH enzymatic activity which correlated negatively with blood pressure. In the current study, we used a transgenic experiment to test the hypothesis that reduced Dbh expression predisposes the SHR to hypertension and that augmentation of Dbh expression would reduce blood pressure. We derived 2 new transgenic SHR-Dbh lines expressing Dbh cDNA under control of the Brown Norway (BN) wild type promoter. We found modestly increased adrenal expression of Dbh in transgenic rats versus SHR non-transgenic controls that was associated with reduced adrenal levels of dopamine and increased plasma levels of norepinephrine and epinephrine. The observed changes in catecholamine metabolism were associated with increased blood pressure and left ventricular mass in both transgenic lines. We did not observe any consistent changes in brainstem levels of catecholamines or of mRNA levels of Dbh in the transgenic strains. Contrary to our initial expections, these findings are consistent with the possibility that genetically determined decreases in adrenal expression and activity of DBH do not represent primary determinants of increased blood pressure in the SHR model.

Melatonin mediated antidepressant-like effect in the hippocampus of chronic stress-induced depression rats: Regulating vesicular monoamine transporter 2 and monoamine oxidase A levels.

The hippocampus is sensitive to stress which activates norepinephrine terminals deriving from the locus coeruleus. Melatonin exerts positive effects on the hippocampal neurogenic process and on depressive-like behaviour. Thus, in the present study, an examination was made of the effect of chronic melatonin treatment on norepinephrine content, synthesis, uptake, vesicular transport and degradation in the hippocampus of rats exposed to CUMS. This entailed quantifying the norephinephrine, mRNA and protein levels of DBH, NET, VMAT 2, MAO-A and COMT. The results show that CUMS evoked prolonged immobility. Melatonin treatment decreased immobility in comparison with the placebo group, reflecting an antidepressant-like effect. Compared with the placebo group, a dramatic decrease in norepinephrine content, decreased VMAT2 mRNA and protein and increased MAO-A protein levels in the hippocampus of the CUMS rats were observed. However, no significant differences in the levels of DBH, NET, COMT mRNA and protein and MAO-A mRNA levels between the placebo and the stressed groups were found. The results showed the restorative effects of melatonin on the stress-induced decline in the norepinephrine content of the hippocampus. It was observed that melatonin treatment in the CUMS rats prevented the stress-induced decrease in VMAT2 mRNA and protein levels, whereas it reduced the increase of the mRNA of COMT and protein levels of MAO-A. Chronic treatment with melatonin failed to alter the gene expression of DBH or NET in the hippocampus of the CUMS rats. Additionally, the results show that melatonin enhances VMAT2 expression and norepinephrine storage, whilst it reduces norepinephrine degrading enzymes.

Catecholamine metabolism in paraganglioma and pheochromocytoma: similar tumors in different sites?

Pheochromocytoma (PHEO) and paraganglioma (PGL) are catecholamine-producing neuroendocrine tumors that arise respectively inside or outside the adrenal medulla. Several reports have shown that adrenal glucocorticoids (GC) play an important regulatory role on the genes encoding the main enzymes involved in catecholamine (CAT) synthesis i.e. tyrosine hydroxylase (TH), dopamine ß-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT). To assess the influence of tumor location on CAT metabolism, 66 tissue samples (53 PHEO, 13 PGL) and 73 plasma samples (50 PHEO, 23 PGL) were studied. Western blot and qPCR were performed for TH, DBH and PNMT expression. We found a significantly lower intra-tumoral concentration of CAT and metanephrines (MNs) in PGL along with a downregulation of TH and PNMT at both mRNA and protein level compared with PHEO. However, when PHEO were partitioned into noradrenergic (NorAd) and mixed tumors based on an intra-tumoral CAT ratio (NE/E >90%), PGL and NorAd PHEO sustained similar TH, DBH and PNMT gene and protein expression. CAT concentration and composition were also similar between NorAd PHEO and PGL, excluding the use of CAT or MNs to discriminate between PGL and PHEO on the basis of biochemical tests. We observed an increase of TH mRNA concentration without correlation with TH protein expression in primary cell culture of PHEO and PGL incubated with dexamethasone during 24 hours; no changes were monitored for PNMT and DBH at both mRNA and protein level in PHEO and PGL. Altogether, these results indicate that long term CAT synthesis is not driven by the close environment where the tumor develops and suggest that GC alone is not sufficient to regulate CAT synthesis pathway in PHEO/PGL.

Corticosterone administration up-regulated expression of norepinephrine transporter and dopamine ß-hydroxylase in rat locus coeruleus and its terminal regions.

Stress has been reported to activate the locus coeruleus (LC)-noradrenergic system. In this study, corticosterone (CORT) was orally administrated to rats for 21 days to mimic stress status. In situ hybridization measurements showed that CORT ingestion significantly increased mRNA levels of norepinephrine transporter (NET) and dopamine ß-hydroxylase (DBH) in the LC region. Immunofluorescence staining and western blotting revealed that CORT treatment also increased protein levels of NET and DBH in the LC, as well as NET protein levels in the hippocampus, the frontal cortex and the amygdala. However, CORT-induced increase in DBH protein levels only appeared in the hippocampus and the amygdala. Elevated NET and DBH expression in most of these areas (except for NET protein levels in the LC) was abolished by simultaneous treatment with combination of corticosteroid receptor antagonist mifepristone and spironolactone (s.c. for 21 days). Also, treatment with mifepristone alone prevented CORT-induced increases of NET expression and DBH protein levels in the LC. In addition, behavioral tasks showed that CORT ingestion facilitated escape in avoidance trials using an elevated T-maze, but interestingly, there was no significant effect on the escape trial. Corticosteroid receptor antagonists failed to counteract this response in CORT-treated rats. In the open-field task, CORT treatment resulted in less activity in a defined central zone compared to controls and corticosteroid receptor antagonist treatment alleviated this increase. In conclusion, this study demonstrates that chronic exposure to CORT results in a phenotype that mimics stress-induced alteration of noradrenergic phenotypes, but the effects on behavior are task dependent. As the sucrose consumption test strongly suggests CORT ingestion-induced depression-like behavior, further elucidation of underlying mechanisms may improve our understanding of the correlation between stress and the development of depression.

Co-expression pattern of dopamine beta-hydroxylase (DßH) and neuropeptide Y (NPY) within sympathetic innervation of ovary and umbilical cord of the European bison (Bison bonasus L.).

Co-expression of dopamine ß-hydroxylase (DßH) and neuropeptide Y (NPY) has never been examined in ovary (OV) and umbilical cord (UC) of the European bison (Eb), the endangered wild species. The OV and UC samples were harvested from seasonally eliminated Eb females (45-120 days post coitum). Frozen histological sections were examined by double fluorescent immunohistochemistry (dF-IHC), using the primary mouse anti-DßH monoclonals and rabbit anti-NPY polyclonals and then the immunocomplexes were visualized with FITC and CY3 fluorophores, respectively. Numerous DßH immunoreactive nerve fibers (DßH-IRs) and a little less frequent NPY-IRs were found in the bundle-like structures, innervating mainly perivascular regions of the OV. The NPY-IRs constantly co-expressed DßH, while some DßH-IRs did not express NPY. This specific pattern of innervation was observed both in the stromal and cortical regions of the OV. The simultaneous co-expression of DßH and NPY were also detected in the UC, in which specific single or bundle-like structures ran along the smooth muscles of blood vessels. The spatial-specific co-expression of DßH and NPY in OV and UC, may suggest that these markers are involved in the control of vascularization that regulates nourishing blood circulation required for proper pregnancy maintenance and efficient embryo/fetus development in the Eb.

Subchronic exposure to arsenic disturbed the biogenic amine neurotransmitter level and the mRNA expression of synthetase in mice brains.

Little is known about the influence of arsenic (As) exposure on monoamine neurotransmitters and the underlying mechanisms, although arsenic toxicity on the central nervous system has been well documented. In the present study, the levels of norepinephrine (NE), dopamine (DA), and 5-HT were determined by high performance liquid chromatography in the cerebrum and cerebellum of mice exposed to 1, 2 and 4 ppm As2O3 through drinking water for 60 days. The ultra-structural change of vesicles in the synapses of mice brains was observed by transmission electron microscopy; the mRNA expressions of dopamine beta hydroxylase (DBH), tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH) as NE, DA and 5-HT synthetases were quantitatively assessed by real time reverse transcription-polymerase chain reaction. It was shown that the concentrations of NE, DA and 5-HT in the cerebrum or cerebellum of mice exposed to As were significantly lower than those in the control group. The number of synaptic vesicles significantly decreased in the brain of mice exposed to As. Moreover, the expressions of TH, TPH and DBH genes were significantly lower in the brains of mice exposed to As than those in the controls. These results suggested that subchronic exposure to As might decrease the concentrations of the three monoamine neurotransmitters in the mouse brain and downregulate TH, TPH and DBH gene expressions. It was also indicated that the decreased concentrations of the three monoamine neurotransmitters in the brain might be related to the down-regulated gene expressions of these synthetases by As.

Forced exercise changes catecholamine synthesis in the spleen of adult rats.

Treadmill training produces modulation of neuro-endocrine and immune functions. This study examined the effects of chronic forced running (CFR) on the plasma concentration of catecholamines and the expression of splenic catecholamine biosynthetic enzymes in rats by using real-time RT-PCR and Western blot analyses. We found that CFR increases the plasma catecholamine levels, decreases splenic tyrosine hydroxylase (TH), dopamine-ß-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT) mRNA levels and increases splenic PNMT protein levels. This shows that CFR is a very strong stressor which activates the sympatho-adrenomedullary system and increases synthesis of splenic PNMT by 20%, which both can modulate the immune function.

Irregular electrical activation of intrinsic cardiac adrenergic cells increases catecholamine-synthesizing enzymes.

BACKGROUND: Recently, increased cardiac norepinephrine levels were observed in patients who were exposed to irregular stimulation during electrophysiological testing. The molecular mechanisms remain unclear. Intrinsic cardiac adrenergic (ICA) cells are present in mammalian hearts and contain catecholamine-synthesizing enzymes sufficient to produce biologically active norepinephrine levels. Thus, we aimed to investigate the expression of catecholamine-synthesizing enzymes by ICA cells exposed to irregular pacing. METHODS: Co-cultures of cardiomyocytes and ICA cells were exposed to irregular pacing for 48h (standard deviation (SD)=5%, 25% and 50% of mean cycle length) at a constant rate of 5Hz. The expression of catecholamine-synthesizing enzymes including tyrosine hydroxylase (TH) and dopamine beta hydroxylase (DBH) were analyzed on mRNA and protein levels. RESULTS: First, immunolabeling identified ICA cells presenting TH and DBH staining around the cell nucleus. Irregular pacing with 25% SD at a constant rate of 5Hz significantly increased the expression of TH and DBH enzyme synthesis. Pharmacological approaches have shown that both metoprolol and losartan reversed the irregular pacing induced DBH increase, whereas the expression of TH was only blocked by metoprolol in a significant manner. Blockade of the endothelin-A receptor by BQ123 or the calcineurin-NFAT pathway by cyclosporine-A, 11R-VIVIT or FK506 revealed a potential role of both cascades in irregular pacing induced catecholamine-synthesizing enzyme expression. CONCLUSIONS: ICA cells respond to irregular electrical activation with an increase in catecholamine-synthesizing enzymes. Drugs commonly used in clinical routine significantly influence the expression of TH and DBH by ICA cells via different signaling routes.

Behavioral coping strategies in response to social stress are associated with distinct neuroendocrine, monoaminergic and immune response profiles in mice.

Individual variation in behavioral coping strategies to stress implies that animals may have a distinct physiological adaptation to stress; these differences may underlie differences in vulnerability to stress-related diseases. This study was designed to test the hypothesis that different behavioral coping strategies (active vs. passive) are stable over time and that they would be associated with differences in hypothalamic-pituitary-adrenal (HPA) and sympathetic-adreno-medular (SAM) axes, and monoaminergic and immune activity. Male mice were subjected to social stress. Twelve days after the first social interaction, mice were subjected to a second identical social stress interaction. Behavior was videotaped and assessed during both sessions. One hour after the final social interaction, serum was collected for corticosterone and adrenaline concentrations and brains were collected for hypothalamic corticotrophin-releasing hormone (CRH) mRNA expression. Monoaminergic system activity was determined by mRNA expression of serotonin, dopamine and noradrenaline synthetic enzymes in the brain stem. Immune system activity was determined by mRNA expression of hypothalamic interleukin-1ß (IL-1ß) and splenic IL-1ß and interleukin-2 (IL-2). Mice engaging in a passive strategy had higher serum corticosterone and lower serum adrenaline concentrations than the active group. The passive group showed lower hypothalamic mRNA expression of IL-1ß and CRH and lower splenic mRNA expression of IL-2 and IL-1ß relative to mice in the active group. An active strategy was associated with higher expression of the dopaminergic synthetic enzyme, while a passive strategy was associated with decreased expression of the serotonergic synthetic enzyme. These findings indicate that individual coping strategies are stable over time and are related to differences in the physiological stress response and immune activity.

Transcription factor Phox2 upregulates expression of norepinephrine transporter and dopamine ß-hydroxylase in adult rat brains.

Degeneration of the noradrenergic locus coeruleus (LC) in aging and neurodegenerative diseases is well documented. Slowing or reversing this effect may have therapeutic implications. Phox2a and Phox2b are homeodomain transcriptional factors that function as determinants of the noradrenergic phenotype during embryogenesis. In the present study, recombinant lentiviral eGFP-Phox2a and -Phox2b (vPhox2a and vPhox2b) were constructed to study the effects of Phox2a/2b over-expression on dopamine ß-hydroxylase (DBH) and norepinephrine transporter (NET) levels in central noradrenergic neurons. Microinjection of vPhox2 into the LC of adult rats significantly increased Phox2 mRNA levels in the LC region. Over-expression of either Phox2a or Phox2b in the LC was paralleled by significant increases in mRNA and protein levels of DBH and NET in the LC. Similar increases in DBH and NET protein levels were observed in the hippocampus following vPhox2 microinjection. In the frontal cortex, only NET protein levels were significantly increased by vPhox2 microinjection. Over-expression of Phox2 genes resulted in a significant increase in BrdU-positive cells in the hippocampal dentate gyrus. The present study demonstrates an upregulatory effect of Phox2a and Phox2b on the expression of DBH and NET in noradrenergic neurons of rat brains, an effect not previously shown in adult animals. Phox2 genes may play an important role in maintaining the function of the noradrenergic neurons after birth, and regulation of Phox2 gene expression may have therapeutic utility in aging or disorders involving degeneration of noradrenergic neurons.

Norepinephrine deficiency is caused by combined abnormal mRNA processing and defective protein trafficking of dopamine beta-hydroxylase.

Human norepinephrine (NE) deficiency (or dopamine ß-hydroxylase (DBH) deficiency) is a rare congenital disorder of primary autonomic failure, in which neurotransmitters NE and epinephrine are undetectable. Although potential pathogenic mutations, such as a common splice donor site mutation (IVS1+2T→C) and various missense mutations, in NE deficiency patients were identified, molecular mechanisms underlying this disease remain unknown. Here, we show that the IVS1+2T→C mutation results in a non-detectable level of DBH protein production and that all three missense mutations tested lead to the DBH protein being trapped in the endoplasmic reticulum (ER). Supporting the view that mutant DBH induces an ER stress response, exogenous expression of mutant DBH dramatically induced expression of BiP, a master ER chaperone. Furthermore, we found that a pharmacological chaperone, glycerol, significantly rescued defective trafficking of mutant DBH proteins. Taken together, we propose that NE deficiency is caused by the combined abnormal processing of DBH mRNA and defective protein trafficking and that this disease could be treated by a pharmacological chaperone(s).

Varied mechanisms of oestradiol-mediated regulation of dopamine ß-hydroxylase transcription.

Experiments performed in vivo and in cell culture have demonstrated that oestradiol induces dopamine ß-hydroxylase (DBH) gene transcription. In the present study, we examined oestrogen-responsive elements of the rat DBH gene promoter aiming to characterise the mechanisms of oestradiol-induced DBH transcription. Various mutations and deletions of DBH promoter reporter constructs were tested for responsiveness to 17ß-oestradiol (E(2) ). Mutation of the half palindromic oestrogen response element (ERE) at position -759 reduced the response to E(2) in PC12 cells co-transfected with oestrogen receptor (ER) α, indicating a functional role for this motif. In cells co-transfected with ERß, mutations at the -759 site were unresponsive to E(2) . To characterise the additional E(2) responsive elements, mediated by ERα, the DBH promoter was truncated to the proximal 249 or 200 nucleotides upstream of the transcription start site. Despite either truncation, 10 nm E(2) still elicited an approximately two-fold induction of DBH promoter activity. Mutation of a possible ERE-like sequence at -59 had no effect. The lack of a functional ERE in the proximal region of the rat DBH promoter despite E(2) -mediated DBH promoter activity, suggests regulation by a nonclassical mechanism, such as a membrane-initiated signalling pathway. Moreover, the induction of DBH promoter activity and the rise in DBH mRNA levels were observed within hours. To determine whether membrane-initiated E(2) signalling is involved in rat DBH gene transcription, a membrane impermeable E(2) conjugate, ß-oestradiol-6-(O-carboxy-methyl) oxime-bovine serum albumin (E(2) BSA), was used. Incubation with E(2) -BSA induced luciferase activity and elicited a significant rise in DBH mRNA levels in the ERα transfected cells. The findings indicate two different mechanisms whereby DBH transcription is regulated by E(2) in the presence of ERα. The results implicate both genomic and membrane-initiated mechanisms, mediated by ERα, in E(2) -induced DBH gene transcription.

Agonist-induced internalization of κ-opioid receptors in noradrenergic neurons of the rat locus coeruleus.

Kappa-opioid receptors (κOR) are positioned to modulate pre- and post-synaptic responses of norepinephrine-containing neurons in the rat locus coeruleus (LC). The ability of an acute systemic injection of a long acting κOR agonist, U50,488, to induce trafficking of κOR was assessed in the LC using immunogold-silver detection in male Sprague-Dawley rats. U50,488 administration shifted immunogold-silver labeling indicative of κOR from primarily plasmalemmal sites to intracellular sites when compared to vehicle-treated subjects. This translocation from the plasma membrane to the cytoplasmic compartment was prevented by pre-treatment with the κOR antagonist, norbinaltorphimine (norBNI). To determine whether agonist stimulation could induce adaptations in the expression of the noradrenergic synthesizing enzyme, dopamine beta hydroxylase (DßH), and κOR expression, Western blot analysis was used to compare expression levels of DßH and κOR following U50,488 administration. Expression levels for DßH and κOR were significantly increased following U50,488 administration when compared to controls. These data indicate that a systemic injection of a κOR agonist stimulates internalization of κORs in noradrenergic neurons and can impact κOR and DßH expression levels in this stress-sensitive brain region.

Effects of transcription factors Phox2 on expression of norepinephrine transporter and dopamine beta-hydroxylase in SK-N-BE(2)C cells.

Phox2a and Phox2b are two homeodomain proteins that control the differentiation of noradrenergic neurons during embryogenesis. In the present study, we examined the possible effect of Phox2a/2b on the in vitro expression of the norepinephrine transporter (NET) and dopamine beta-hydroxylase (DBH), two important markers of the noradrenergic system. SK-N-BE(2)C cells were transfected with cDNAs or short hairpin RNAs specific to the human Phox2a and Phox2b genes. Transfection of 0.1 to 5 mug of cDNAs of Phox2a or Phox2b significantly increased mRNA and protein levels of NET and DBH in a concentration-dependent manner. As a consequence of the enhanced expression of NET after transfection, there was a parallel increase in the uptake of [(3)H]norepinephrine. Co-transfection of Phox2a and Phox2b did not further increase the expression of noradrenergic markers when compared with transfection of either Phox2a or Phox2b alone. Transfection of shRNAs specific to Phox2a or Phox2b genes significantly reduced mRNA and protein levels of NET and DBH after shutdown of endogenous Phox2, which was accompanied by a decreased [(3)H]norepinephrine uptake. Furthermore, there was an additive effect after cotransfection with both shRNAs specific to Phox2a or Phox2b genes on NET mRNA levels. Finally, the reduced DBH expression caused by the shRNA specific to Phox2a could be reversed by transfection with Phox2b cDNA and vice versa. The present findings verify the determinant role of Phox2a and Phox2b on the expression and function of NET and DBH in vitro. Further clarifying the regulatory role of these two transcription factors on key proteins of the noradrenergic system may open a new avenue for therapeutics of aging-caused dysfunction of the noradrenergic system.

Effect of age on angiotensin II-mediated downregulation of adrenomedullary catecholamine biosynthetic enzymes.

Expression of catecholamine biosynthesizing enzymes, tyrosine hydroxylase (TH) and dopamine beta hydroxylase (DbetaH) increase with age in the adrenal medulla, however, the underlying mechanisms are unclear. In the present study, we examined the effect of peripheral angiotensin II (AngII) on the expression of TH and DbetaH, in the adrenal medulla of young (6 mo) and old (23 mo) Fischer-344 rats. Saline or AngII (230 ng/kg/min sc) was infused for 3 days using osmotic minipumps. Adrenomedullary TH and DbetaH mRNA levels increased significantly with age, and while AngII reduced the expression of these enzymes in young animals, it had no such effect in the old animals. Neuropeptide Y (NPY), which is co-released with catecholamines in the adrenal medulla and stimulates the synthesis of TH and DbetaH, was also upregulated with age and downregulated in response to AngII in young rats. However, in the old animals, the already elevated NPY expression remained unchanged following AngII treatment. This data indicate that the hypertensive effect of peripheral AngII is compensated by an inhibition of adrenomedullary catecholamine biosynthesis in young animals, but this mechanism is impaired in senescence, potentially contributing to the age-related increase in catecholamine biosynthesis.

Long-term hypoxia modulates expression of key genes regulating adrenomedullary function in the late gestation ovine fetus.

We previously communicated that long-term hypoxia (LTH) resulted in a selective reduction in plasma epinephrine following acute stress in fetal sheep. The present study tested the hypothesis that LTH selectively reduces adrenomedullary expression of phenylethanolamine-N-methyltransferase (PNMT), the rate-limiting enzyme for epinephrine synthesis. We also examined the effect of LTH on adrenomedullary nicotinic, muscarinic, and glucocorticoid receptor (GR) expression. Ewes were maintained at high altitude (3,820 m) from 30 to 138 days gestation (dGA); adrenomedullary tissue was collected from LTH and age-matched, normoxic control fetuses at 139-141 dGA. Contrary to our hypothesis, in addition to PNMT, adrenomedullary expression (mRNA, protein) of tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH) were reduced in the LTH fetus. Immunocytochemistry indicated that TH and DBH expression was lower throughout the medulla, while PNMT appeared to reflect a reduction in PNMT-expressing cells. Nicotinic receptor alpha 1, 2, 3, 5, 6, 7, beta 1, 2, and 4 subunits were expressed in the medulla of LTH and control fetuses. Messenger RNA for alpha 1 and 7 and beta 1 and 2 subunits was lower in LTH fetuses. Muscarinic receptors M1, M2, and M3 as well as the GR were also expressed, and no differences were noted between groups. In summary, LTH in fetal sheep has a profound effect on expression of key enzymes mediating adrenomedullary catecholamine synthesis. Further, LTH impacts nicotinic receptor subunit expression potentially altering cholinergic neurotransmission within the medulla. These findings have important implications regarding fetal cardiovascular and metabolic responses to stress in the LTH fetus.

Pitfalls in detection of contaminating neuroblastoma cells by tyrosine hydroxylase RT-PCR due to catecholamine-producing hematopoietic cells.

BACKGROUND: RT-PCR analysis of compounds of catecholamine metabolism (in particular tyrosine hydroxylase, TH) is widely used for the detection of contaminating neuroblastoma cells in hematopoietic stem cell preparations. Due to reports in the literature showing that hematopoietic cells are also able to produce catecholamines, we investigated whether TH-RT-PCR is really suitable for this purpose. MATERIALS AND METHODS: Besides neuroblastoma cells, mononuclear blood cells, apheresis preparations and hematopoietic stem cells were used for single and nested RT-PCR. In addition to TH, the expressions of dopamine-beta-hydroxylase and noradrenaline transporter were analyzed. RESULTS: Using single RT-PCR, a clear discrimination between neuroblastoma and hematopoietic cells was possible. However, by using nested RT-PCR, the "neuroblastoma markers" were also detected in a significant percentage of non-mobilized mononuclear blood cells, in mononuclear blood cells of healthy donors mobilized with G-CSF, and in highly purified CD34+ and CD133+ stem cells from healthy mobilized donors. CONCLUSION: Our results raise the question of whether the RT-PCR analysis of compounds of catecholamine metabolism is suitable and selective enough to detect the contamination of hematopoietic stem cells by a low number of neuroblastoma cells.