Expression Of Dopamine Beta-hydroxylase Research Articles

Kinin B1 receptor modulates glucose homeostasis and physical exercise capacity by altering adrenal catecholamine synthesis and secretion

Our group has shown in several papers that kinin B1 receptor (B1R) is involved in metabolic adaptations, mediating glucose homeostasis and interfering in leptin and insulin signaling. Since catecholamines are involved with metabolism management, we sought to evaluate B1R role in catecholamine synthesis/secretion. Using B1R global knockout mice, we observed increased basal epinephrine content, accompanied by decreased hepatic glycogen content and increased glucosuria. When these mice were challenged with maximal intensity exercise, they showed decreased epinephrine and norepinephrine response, accompanied by disturbed glycemic responses to effort and poor performance. This phenotype was related to alterations in adrenal catecholamine synthesis: increased basal epinephrine concentration and reduced norepinephrine content in response to exercise, as well decreased gene expression and protein content of tyrosine hydroxylase and decreased gene expression of dopamine beta hydroxylase and kinin B2 receptor. We conclude that the global absence of B1R impairs catecholamine synthesis, interfering with glucose metabolism at rest and during maximal exercise.

Candidate gene association study suggests potential role of dopamine beta-hydroxylase in pain heterogeneity in sickle cell disease.

Introduction: Pain is a lifelong companion of individuals with sickle cell disease (SCD) and has a severe impact on their quality of life. Both acute crisis pain and chronic non-crisis pain exhibit high variability between individuals, making it difficult to effectively manage sickle cell-related pain. We investigated the role of dopamine beta-hydroxylase (DBH) gene polymorphisms on pain variability in SCD. DBH is a key enzyme in the catecholamine biosynthesis pathway that catalyzes the conversion of dopamine to norepinephrine, both of which are known mediators of pain and pain-related behaviors. Methods: Acute crisis pain-related utilization and non-crisis chronic pain scores of 131 African Americans with SCD were obtained. Results and discussion: Association analyses revealed that the T allele of upstream variant rs1611115 and downstream variant rs129882 correlated with higher severity of chronic pain in an additive model. On the other hand, the A allele of missense variant rs5324 associated with lower risk of both acute crisis pain and chronic pain. Similarly, the C allele of intronic variant rs2797849 was associated with lower incidence of acute crisis pain in the additive model. In addition, tissue-specific eQTL revealed that the T allele of rs1611115 correlated with decreased expression of DBH in the frontal cortex and anterior cingulate cortex (GTEx), and decreased expression of DBH-AS1 in blood (eQTLGen). Bioinformatic approaches predicted that rs1611115 may be altering a transcription factor binding site, thereby, contributing to its potential effect. Taken together, findings from this study suggest that potential functional polymorphisms of DBH may modulate pain perception in SCD.

Weight-gain propensity and morphine withdrawal alters locomotor behavior and regional norepinephrine-related gene expression in male and female mice

Interactions between obesity and opioid use are poorly understood. The objective of this study was to determine whether phenotypic differences in diet-induced weight gain altered morphine withdrawal responses. Male and female C57BL/6J mice were characterized as obese prone (OP) or obese resistant (OR) based on median split in body weights following exposure to high-fat diet (45% fat). After classification into OP or OR, all mice were fed a low-fat diet (10% fat) for the remainder of the study (≥5 weeks) to remain weight matched. Mice were treated with a 7-day escalating dosing scheme of morphine (20–100 mg/kg; IP) or saline and underwent a spontaneous withdrawal. Morphine-induced weight loss was restored by withdrawal day 7. On withdrawal day 8, male OP demonstrated less total time mobile in the open field test (OFT). In females, OR-morphine traveled less distance than OR-saline, and OR-morphine spent less time mobile compared with all other groups in the OFT. Female OP also increased time spent in the center of the apparatus, regardless of treatment. On withdrawal day 8, relative gene expression was measured by qPCR. For males, expression of dopamine beta-hydroxylase (dbh), alpha-adrenergic receptor 2 a (adra2a), and orexin receptor 1 (orx1) were increased in the locus coeruleus (LC) region of OP mice, regardless of treatment. In comparison, in females, dbh and adra2a were decreased in the LC region of OP mice, regardless of treatment. Also, in the LC region of females, OP-morphine had lower expression of alpha-adrenergic receptor 1 a (adra1a) than OR-morphine and OP-saline. In the hypothalamic paraventricular nucleus (PVN) of females, adra2a was increased in OP-morphine compared with OP-saline and OR-morphine. Our findings suggest morphine withdrawal responses and regional expression of noradrenergic-related genes are differentially influenced by weight gain propensity.

Immunohistochemical Expression of Choline Acetyltransferase and Catecholamine-Synthesizing Enzymes in Head-and-Neck and Thoracoabdominal Paragangliomas and Pheochromocytomas.

Paragangliomas (PGLs) are neural-crest-derived, non-epithelial neuroendocrine tumors distributed along the parasympathetic and sympathetic nerves. Head-and-neck PGLs (HNPGLs) have been recognized as nonchromaffin, nonfunctional, parasympathetic tumors. By contrast, thoracoabdominal paragangliomas and pheochromocytomas (PPGLs) are chromaffin, functional, sympathetic tumors. Although HNPGLs and PPGLs have the same histological structure, the zellballen pattern, composed of chief and sustentacular cells surrounded by abundant capillaries, the pathobiological differences between these types of PGLs remain unclarified. To determine the phenotypic features of these PGLs, we performed an immunohistochemical study using specific antibodies against choline acetyltransferase (ChAT), an enzyme involved in acetylcholine synthesis, and enzymes for the catecholamine-synthesis, tyrosine hydroxylase (TH), and dopamine beta-hydroxylase (DBH), in 34 HNPGLs from 31 patients, 12 thoracoabdominal PGLs from 12 patients, and 26 pheochromocytomas from 22 patients. The expression of ChAT, TH, and DBH was 100%, 23%, and 10% in the HNPGLs; 12%, 100%, and 100% in the pheochromocytomas; and 25%, 67%, and 100% in the thoracoabdominal PGLs, respectively. These results designate HNPGLs as acetylcholine-producing parasympathetic tumors, in contrast to PPGLs being catecholamine-producing tumors. The other most frequently used neuroendocrine markers are synaptophysin and chromogranin A expressed 100% and 80%, respectively, and synaptophysin was superior to chromogranin A in HNPGLs. This is the first report of HNPGLs being acetylcholine-producing tumors. Immunohistochemistry of ChAT could be greatly useful for pathologic diagnosis of HNPGL. Whether measurement of acetylcholine levels in the blood or urine could be a tumor marker of HNPGLs should be investigated soon.

Thioacetamide-Induced Norepinephrine Production by Hepatocytes is Associated with Hepatic Stellate Cell Activation and Liver Fibrosis.

Collagen production by activated hepatic stellate cells (HSCs) to encapsulate injury is part of the natural wound-healing response in injured liver. However, persistent activation of HSCs can lead to pathological fibrogenesis. Such persistent HSC activation could be mediated by norepinephrine (NE), a reaction product of dopamine beta-hydroxylase (DBH). To investigate the potential paracrine role of NE in hepatotoxin thioacetamide (TAA)-induced liver fibrosis. In TAA-treated mice, fibrotic liver tissue showed significant increases in the mRNA expression of DBH up to 14-fold and collagen up to 7-fold. Immunohistochemical staining showed increased DBH protein expression in fibrotic liver tissue. Parenchymal hepatocyte cell line HepG2 expressed DBH and secreted NE, and the conditioned medium of HepG2 cells promoted collagenesis in nonparenchymal HSC cell line LX-2. TAA treatment increased DBH expression by 170% in HepG2 cells, as well as increased NE by 120% in the conditioned medium of HepG2 cells. The conditioned medium of TAA-treated HepG2 cells was used to culture LX-2 cells, and was found to increase collagen expression by 80% in LX-2 cells. Collagen expression was reduced by pre-treating HepG2 cells with siRNA targeting DBH or by adding NE antagonists to the conditioned medium. Finally, TAA-induced oxidative stress in HepG2 cells was associated with induction of DBH expression. Collectively, our results suggest a potential role for DBH/NE-mediated crosstalk between hepatocytes and HSCs in fibrogenesis. From a therapeutic standpoint, antagonism of DBH/NE induction in hepatocytes might be a useful strategy to suppress pathological fibrogenesis.

The role of the brain in the regulation of peripheral noradrenaline-producing organs in rats during morphogenesis

This work represents one part of our research project, in which we try to prove, that in the perinatal period exist a humoral regulation between noradrenaline producing organs. In this study we used a rat model that allowed blocking synthesis of noradrenalin in the brain and evaluated gene expression and protein levels of noradrenaline key synthesis enzymes such as tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH) in peripheral noradrenaline producing organs. As a result we showed increased gene expression of TH and DBH in adrenal glands. This data indicate that if neonatal rat brain lacks an ability to produce noradrenaline, then as a compensatory process synthesis of noradrenaline increased in adrenal glands, so that the concentration levels in blood are kept at normal levels. This indicates that there is a humoral regulation between brain and adrenal glands which is not fully understood yet.

The Role of the Brain in the Regulation of Peripheral Noradrenaline-producing Organs in Rats During Morphogenesis.

This work represents one part of our research project, in which we attempted to prove that a humoral regulation between noradrenaline-producing organs exist in the perinatal period. In this study, we used a rat model that allowed blocking the synthesis of noradrenalin in the brain and evaluated gene expression and protein levels of noradrenaline key synthesis enzymes such as tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH) in peripheral noradrenaline-producing organs. As a result, we showed an increased gene expression of TH and DBH in adrenal glands. These data indicate that, if neonatal rat brain lacks the ability to produce noradrenaline, then the synthesis of noradrenaline in adrenal glands increased as a compensatory process, so that the concentration levels in blood are maintained at normal levels. This indicates that there is a humoral regulation between brain and adrenal glands, which is not fully understood yet.

Melatonin inhibits attention-deficit/hyperactivity disorder caused by atopic dermatitis-induced psychological stress in an NC/Nga atopic-like mouse model

Atopic dermatitis (AD) is a chronic inflammatory skin disease with the hallmark characteristics of pruritus, psychological stress, and sleep disturbance, all possibly associated with an increased risk of attention-deficit/hyperactivity disorder (ADHD). However, the etiology of the possible association between AD and ADHD is still not well understood. 2,4-dinitrochlorobenzene or corticosterone was used to evaluate the atopic symptom and its psychologic stress in the atopic mice model. Melatonin, corticotropin-releasing hormone, corticotropin-releasing hormone receptor, urocortin, proopiomelanocortin, adrenocorticotropic hormone, corticosterone, cAMP, cAMP response element-binding protein, dopamine and noradrenaline were analyzed spectrophotometrically, and the expression of dopamine beta-hydroxylase and tyrosine hydroxylase were measured by Western blotting or immunohistochemistry. AD-related psychological stress caused an increase in the levels of dopamine beta-hydroxylase and tyrosine hydroxylase, degradation of melatonin, hyper-activity of the hypothalamic-pituitary-adrenal axis, and dysregulation of dopamine and noradrenaline levels (ADHD phenomena) in the locus coeruleus, prefrontal cortex, and striatum of the AD mouse brain. Notably, melatonin administration inhibited the development of ADHD phenomena and their-related response in the mouse model. This study demonstrated that AD-related psychological stress increased catecholamine dysfunction and accelerated the development of psychiatric comorbidities, such as ADHD.

Role of the area postrema in the hypophagic effects of oleoylethanolamide

The satiety-promoting action of oleoylethanolamide (OEA) has been associated to the indirect activation of selected brain areas, such as the nucleus of the solitary tract (NST) in the brainstem and the tuberomammillary (TMN) and paraventricular (PVN) nuclei in the hypothalamus, where noradrenergic, histaminergic and oxytocinergic neurons play a necessary role. Visceral ascending fibers were hypothesized to mediate such effects. However, our previous findings demonstrated that the hypophagic action of peripherally administered OEA does not require intact vagal afferents and is associated to a strong activation of the area postrema (AP). Therefore, we hypothesized that OEA may exert its central effects through the direct activation of this circumventricular organ. To test this hypothesis, we subjected rats to the surgical ablation of the AP (APX rats) and evaluated the effects of OEA (10mgkg−1 i.p.) on food intake, Fos expression, hypothalamic oxytocin (OXY) immunoreactivity and on the expression of dopamine beta hydroxylase (DBH) in the brainstem and hypothalamus. We found that the AP lesion completely prevented OEA’s behavioral and neurochemical effects in the brainstem and the hypothalamus. Moreover OEA increased DBH expression in AP and NST neurons of SHAM rats while the effect in the NST was absent in APX rats, thus suggesting the possible involvement of noradrenergic AP neurons. These results support the hypothesis of a necessary role of the AP in mediating OEA’s central effects that sustain its pro-satiety action.

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.

Identification and functional characterisation of a novel dopamine beta hydroxylase gene variant associated with attention deficit hyperactivity disorder

Objectives. Dysregulation in neurotransmitter signalling has been implicated in the aetiology of attention deficit hyperactivity disorder (ADHD). Polymorphisms of the gene encoding dopamine beta hydroxylase (DBH) have been reported to be associated with ADHD; however, small sample sizes have led to inconsistency. Methods. We conducted transmission disequilibrium test analysis in 794 nuclear families to examine the relationship between DBH and ADHD. The effects of the ADHD-associated polymorphisms on gene expression were assessed by luciferase reporter assays in a human neuroblastoma cell line, SH-SY5Y. Results. A SNP within the 3′ untranslated region of DBH rs129882 showed a significant association with ADHD (χ2 = 9.71, p = 0.0018, OR = 1.37). This association remained significant after Bonferroni correction for multiple testing (p = 0.02). Further, allelic variation in rs129882 significantly impacted luciferase expression. Specifically, the C allele of the ADHD-associated rs129882 SNP produced a 2-fold decrease (p < 0.001) in luciferase activity. Conclusions. These data demonstrate for the first time that a DBH gene variant, rs129882, which confers risk to ADHD is also associated with reduced in vitro gene expression. Reduced DBH expression would be consistent with decreased conversion of dopamine to noradrenaline and thus with a relative hypo-noradrenergic state in ADHD.

The catecholamine biosynthetic enzyme dopamine β-hydroxylase (DBH): first genome-wide search positions trait-determining variants acting additively in the proximal promoter.

Dopamine beta-hydroxylase (DBH) is the biosynthetic enzyme catalyzing formation of norepinephrine. Changes in DBH expression or activity have been implicated in the pathogenesis of cardiovascular and neuropsychiatric disorders. Genetic determination of DBH enzymatic activity and its secretion are only incompletely understood. We began with a genome-wide association search for loci contributing to DBH activity in human plasma. Initially, in a population sample of European ancestry, we identified the proximal DBH promoter as a region harboring three common trait-determining variants (top hit rs1611115, P = 7.2 × 10(-51)). We confirmed their effects on transcription and showed that the three variants each acted additively on gene expression. Results were replicated in a population sample of Native American descent (top hit rs1611115, P = 4.1 × 10(-15)). Jointly, DBH variants accounted for 57% of DBH trait variation. We further identified a genome-wide significant SNP at the LOC338797 locus on chromosome 12 as trans-quantitative trait locus (QTL) (rs4255618, P = 4.62 × 10(-8)). Conditional analyses on DBH identified a third genomic region contributing to DBH variation: a likely cis-QTL adjacent to DBH in SARDH (rs7040170, P = 1.31 × 10(-14)) on chromosome 9q. We conclude that three common SNPs in the DBH promoter act additively to control phenotypic variation in DBH levels, and that two additional novel loci (SARDH and LOC338797) may also contribute to the expression of this catecholamine biosynthetic trait. Identification of DBH variants with strong effects makes it possible to take advantage of Mendelian randomization approaches to test causal effects of this intermediate trait on disease.

Loss of Hand2 in a population of Periostin lineage cells results in pronounced bradycardia and neonatal death

The Periostin Cre (Postn-Cre) lineage includes endocardial and neural crest derived mesenchymal cells of the cardiac cushions, neural crest-derived components of the sympathetic and enteric nervous systems, and cardiac fibroblasts. In this study, we use the Postn-Cre transgenic allele to conditionally ablate Hand2 (H2CKO). We find that Postn-Cre H2CKOs die shortly after birth despite a lack of obvious cardiac structural defects. To ascertain the cause of death, we performed a detailed comparison of the Postn-Cre lineage and Hand2 expression at mid and late stages of embryonic development. Gene expression analyses demonstrate that Postn-Cre ablates Hand2 from the adrenal medulla as well as the sphenopalatine ganglia of the head. In both cases, Hand2 loss-of-function dramatically reduces expression of Dopamine Beta Hydroxylase (Dbh), a gene encoding a crucial catecholaminergic biosynthetic enzyme. Expression of the genes Tyrosine Hydroxylase (Th) and Phenylethanolamine N-methyltransferase (Pnmt), which also encode essential catecholaminergic enzymes, were severely reduced in postnatal adrenal glands. Electrocardiograms demonstrate that 3-day postnatal Postn-Cre H2CKO pups exhibit sinus bradycardia. In conjunction with the aforementioned gene expression analyses, these results strongly suggest that the observed postnatal lethality occurs due to a catecholamine deficiency and subsequent heart failure.

Electroacupuncture Analgesia Is Improved by Adenoviral Gene Transfer of Dopamine Beta-hydroxylase into the Hypothalamus of Rats

Electroacupuncture (EA) is a modified form of acupuncture that utilizes electrical stimulation. We previously showed that EA stimulated rats were divided into responders that were sensitive to EA and non-responders that were insensitive to EA based on the tail flick latency (TFL) test. The dopamine beta-hydroxylase (DBH) gene was more abundantly expressed in the hypothalamus of responder rats than non-responder rats. To determine whether overexpression of DBH gene expression in the hypothalamus modulate EA analgesia, we constructed a DBH encoding adenovirus and which was then injected into the hypothalamus of SD rats. Microinjection of DBH or control GFP virus into the hypothalamus had no changes on the basal pain threshold measured by a TFL test without EA treatment. However, the analgesic effect of EA was significantly enhanced from seven days after microinjection of the DBH virus, but not after injection of the control GFP virus. DBH expression was significantly higher in the hypothalamus of DBH virus injected rat than control GFP virus or PBS injected rats. Moreover, expression of the DBH gene did not affect the body core temperature, body weight, motor function or learning and memory ability. Although the functional role of DBH in the hypothalamus in the analgesic effect of EA remains unclear, our findings suggest that expression of the DBH gene in the hypothalamus promotes EA analgesia without obvious side-effects.

Effects of treadmill training on noradrenaline fibers and the recovery of hindlimb motor function in rats after spinal cord contusion

Objective To observe the effects of treadmill training on hindlimb motor recovery and noradrenaline (NA) fibers in rats with spinal cord contusion.Methods Spinal cord contusions at the T10 level were induced in 3-month old female SD rats by dropping a 10 g rod from a height of 25 mm.The injured rats were then randomly divided into a control group and a treadmill training group ( n =15 in each group).Treadmill training began 1 week post-injury.No treatment was administered to the control group rats.Hindlimb motor function was evaluated weekly from the 1 st to the 9th week post-injury using Basso Beattie Bresnahan (BBB) scores and average combined scores (ACOs).The expression of dopamine beta hydroxylase (DBH) in the spinal cord caudal to the injured area was detected by immunohistochemical staining.Results The treadmill training group exhibited significantly greater improvement in BBB scores from the 3rd week to the 7th week and in ACOs from the 2nd to the 9th week post-injury compared with the control group.The number of DBH positive fibers in the injured spinal cords and the number of DBH positive neurons in the injured frontal horns were also significantly higher in the training group.Conclusions Treadmill training can promote the recovery of hindlimb motor function in rats after spinal cord contusion,perhaps by increasing the number of DBH positive fibers and DBH positive neurons. Key words: Spinal cord contusion; Treadmill training; Noradrenaline

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 48 h (standard deviation (SD) = 5%, 25% and 50% of mean cycle length) at a constant rate of 5 Hz. 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 5 Hz 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.

Expression of the paralogous tyrosine hydroxylase encoding genes th1 and th2 reveals the full complement of dopaminergic and noradrenergic neurons in zebrafish larval and juvenile brain

The development of dopaminergic and noradrenergic neurons has received much attention based on their modulatory effect on many behavioral circuits and their involvement in neurodegenerative diseases. The zebrafish (Danio rerio) has emerged as a new model organism with which to study development and function of catecholaminergic systems. Tyrosine hydroxylase is the entry enzyme into catecholamine biosynthesis and is frequently used as a marker for catecholaminergic neurons. A genome duplication at the base of teleost evolution resulted in two paralogous zebrafish tyrosine hydroxylase-encoding genes, th1 and th2, the expression of which has been described previously only for th1. Here we investigate the expression of th2 in the brain of embryonic and juvenile zebrafish. We optimized whole-mount in situ hybridization protocols to detect gene expression in the anatomical three-dimensional context of whole juvenile brains. To confirm whether th2-expressing cells may indeed use dopamine as a neurotransmitter, we also included expression of dopamine beta hydroxylase, dopa decarboxylase, and dopamine transporter in our analysis. Our data provide the first complete account of catecholaminergic neurons in the zebrafish embryonic and juvenile brain. We identified four major th2-expressing neuronal groups that likely use dopamine as transmitter in the zebrafish diencephalon, including neurons of the posterior preoptic nucleus, the paraventricular organ, and the nuclei of the lateral and posterior recesses in the caudal hypothalamus. th2 Expression in the latter two groups resolves a previously reported discrepancy, in which strong dopamine but little tyrosine hydroxylase immunoreactivity had been detected in the caudal hypothalamus. Our data also confirm that there are no mesencephalic DA neurons in zebrafish. J. Comp. Neurol. 518:423–438, 2010. © 2009 Wiley-Liss, Inc.

Effects of transcription factors Phox2 on expression of norepinephrine transporter and dopamine β‐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.

Linkage studies of catechol-O-methyltransferase (COMT) and dopamine-beta-hydroxylase (DBH) cDNA expression levels.

The COMT and DBH genes are physically located at chromosomes 22q11 and 9q34, respectively, and both COMT and DBH are involved in catecholamine metabolism and are strong candidates for certain psychiatric and neurological disorders. Although the genetic determinants for both enzymes' activities have been widely studied, their genetic involvement on gene mRNA expression levels remains unclear. In this study we performed quantitative linkage analysis of COMT and DBH cDNA expression levels, identifying transcriptional regulatory regions for both genes. Multiple Haseman-Elston regression was used to detect both additive and interactive effects between two loci. We found that the master transcriptional regulatory region 20q13 had an additive effect on the COMT expression level. We also found that chromosome 19p13 showed both additive and interactive effects with 9q34 on DBH expression level. Furthermore, a potential interaction between COMT and DBH was indicated.

Dopamine beta‐hydroxylase (DBH) activity and ‐1021C/T polymorphism of DBH gene in combat‐related post‐traumatic stress disorder

The roles of dopamine (DA) and norepinephrine (NE) in posttraumatic stress disorder (PTSD) are unclear. The aim of the study was to determine plasma dopamine beta-hydroxylase (DBH) activity and DBH-1021C/T gene polymorphism in combat veterans with (N = 133) or without (N = 34) chronic PTSD. Similar frequencies in genotype or allele distribution were found between veterans with or without PTSD. War veterans with PTSD had lower DBH activity, associated with the DBH-1021C/T variant in DBH genes, than veterans without PTSD. A significantly lower plasma DBH activity was found in combat veterans with PTSD carrying the CC genotype as compared to veterans without PTSD carrying the corresponding genotype. Since both groups were exposed to the same trauma, it is possible that a pre-existing trait difference in regulation of NE function contributed to a differential vulnerability to develop PTSD, or that the regulation of DBH expression was different in response to trauma. The results suggest that that genotype-controlled measurement of plasma DBH activity might be used as a potential biological marker of the response to trauma, and that further studies of DBH and other loci related to DA and NA in PTSD are warranted.