Dopamine Beta-hydroxylase mRNA Levels Research Articles

Changes in Noradrenergic Synthesis and Dopamine Beta-Hydroxylase Activity in Response to Oxidative Stress after Iron-induced Brain Injury.

Noradrenaline (NA) levels are altered during the first hours and several days after cortical injury. NA modulates motor functional recovery. The present study investigated whether iron-induced cortical injury modulated noradrenergic synthesis and dopamine beta-hydroxylase (DBH) activity in response to oxidative stress in the brain cortex, pons and cerebellum of the rat. Seventy-eight rats were divided into two groups: (a) the sham group, which received an intracortical injection of a vehicle solution; and (b) the injured group, which received an intracortical injection of ferrous chloride. Motor deficits were evaluated for 20 days post-injury. On the 3rd and 20th days, the rats were euthanized to measure oxidative stress indicators (reactive oxygen species (ROS), reduced glutathione (GSH) and oxidized glutathione (GSSG)) and catecholamines (NA, dopamine (DA)), plus DBH mRNA and protein levels. Our results showed that iron-induced brain cortex injury increased noradrenergic synthesis and DBH activity in the brain cortex, pons and cerebellum at 3 days post-injury, predominantly on the ipsilateral side to the injury, in response to oxidative stress. A compensatory increase in contralateral noradrenergic activity was observed, but without changes in the DBH mRNA and protein levels in the cerebellum and pons. In conclusion, iron-induced cortical injury increased the noradrenergic response in the brain cortex, pons and cerebellum, particularly on the ipsilateral side, accompanied by a compensatory response on the contralateral side. The oxidative stress was countered by antioxidant activity, which favored functional recovery following motor deficits.

Effect of immobilization stress on gene expression of catecholamine biosynthetic enzymes in heart auricles of socially isolated rats

Chronic stress is associated with the development of cardiovascular diseases. The sympathoneural system plays an important role in the regulation of cardiac function both in health and disease. In the present study, the changes in gene expression of the catecholamine biosynthetic enzymes tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT) and protein levels in the right and left heart auricles of naive control and long-term (12 weeks) socially isolated rats were investigated by Taqman RT-PCR and Western blot analysis. The response of these animals to additional immobilization stress (2 h) was also examined. Long-term social isolation produced a decrease in TH mRNA level in left auricles (about 70%) compared to the corresponding control. Expression of the DBH gene was markedly decreased both in the right (about 62%) and left (about 81%) auricles compared to the corresponding control, group-maintained rats, whereas PNMT mRNA levels remained unchanged. Exposure of group-housed rats to acute immobilization for 2 h led to a significant increase of mRNA levels of TH (about 267%), DBH (about 37%) and PNMT (about 60%) only in the right auricles. Additional 2-h immobilization of individually housed rats did not affect gene expression of these enzymes in either the right or left auricle. Protein levels of TH, DBH and PNMT in left and right heart auricles were unchanged either in both individually housed and immobilized rats. The unchanged mRNA levels of the enzymes examined after short-term immobilization suggest that the catecholaminergic system of the heart auricles of animals previously exposed to chronic psychosocial stress was adapted to maintain appropriate cardiovascular homeostasis.

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.

Ethanol and Stress Activate Catecholamine Synthesis in the Adrenal

Ethanol consumption and mental stress activate the sympathetic nervous system, which can adversely affect bone. We compared six groups of 10 young adult rats, three with and three without 2 h daily restraint stress. Two groups consumed food and water ad libitum, two received food and 6% (w/v) ethanol as drinking water, and two received the amount of food consumed by ethanol rats the previous day plus water ad libitum (pairfed). After 6 weeks, rats were killed. Plasma, femurs, lumbar vertebrae, and adrenals were harvested. Femoral dimensions were measured and biomechanical properties were tested by three-point bending. Plasma osteocalcin, vertebral osteocalcin mRNA levels, and adrenomedullary tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), and phenylethanolamine N-methyl transferase (PNMT) mRNA levels were quantified. Daily restraint decreased weight gain and femoral length compared to dietary controls, and appeared to partially preserve bone strength, especially in calorie-restricted pairfed rats. Femoral strength was significantly affected by treatment in that bones of pairfed controls were weakest, ethanol drinkers were intermediate, and ad libitum restrained were strongest. Femoral yield load, displacement, and work at yield load were negatively correlated with TH and DBH mRNA levels, but not PNMT, suggesting a negative influence of norepinephrine. Plasma osteocalcin and dry weight of lumbar 3-5 vertebrae were unaffected; however, osteocalcin mRNA in second lumbar vertebrae was positively correlated with TH, DBH, and PNMT levels. Ethanol consumption at this level had little effect on femur morphology or strength. In contrast, the data suggested possible stimulation rather than inhibition of vertebral bone formation.

Age‐associated changes in mRNA levels of Phox2, norepinephrine transporter and dopamine β‐hydroxylase in the locus coeruleus and adrenal glands of rats

Age-related changes in the gene expression of the transcription factors, Phox2a and 2b, and two marker proteins, norepinephrine transporter (NET) and dopamine beta-hydroxylase (DBH), of noradrenergic neurons were characterized in the locus coeruleus (LC) and adrenal glands using in situ hybridization. Analysis of changes was performed in rats that were 1-23 months of age. Compared to 1-month-old rats, there was a 62% increase of Phox2a messenger RNA (mRNA) in the LC of 3-month-old rats, and a decline of 37% in 23-month-old rats. In contrast, levels of Phox2b mRNA in the LC remained unchanged in 3-month-old rats, but declined to a 30% reduction in 23-month-old rats. Interestingly, mRNA levels of NET in the LC decreased with increasing age to a reduction of 29%, 30% and 43% in 3-, 8- and 23-month-old rats, respectively. Similarly, DBH mRNA in the LC declined with increasing age to a 56% reduction in 23-month-old rats. mRNA levels of Phox2a, Phox2b, NET and DBH in the adrenal medulla of 23-month-old rats were significantly lower than those of 1-month-old rats. Semi-quantitative reverse transcription assays of the same genes yielded data similar to in situ hybridization experiments, with beta-actin mRNA levels being unchanged across the ages. Taken together, these data reveal that reduced Phox2 mRNAs in the LC and adrenal medulla of aging rats are accompanied by a coincidental decline in mRNA levels of NET and DBH and suggest a possible relationship between Phox2 genes and the marker genes in noradrenergic neurons after birth.

Catecholamine synthesizing enzymes and their modulation by immobilization stress in knockout mice.

The c-fos knockout mice (c-fos KO) and corticotropin-releasing hormone knockout mice (CRH KO) can serve as interesting models for studying mechanisms involved in response of the organism to stress, focused mainly on the hypothalamic-pituitary-adrenal (HPA) axis and sympathoadrenal system (SAS). The present study focused on the investigation of changes in gene expression of catecholamine biosynthesizing enzymes tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH), and phenylethanolamine N-methyltransferase (PNMT) in adrenal medulla of c-fos KO and CRH KO mice stressed by immobilization. Levels of TH, DBH, and PNMT mRNA were determined by reverse transcription-polymerase chain reaction (RT-PCR). Single immobilization for 2 h significantly increased adrenomedullary TH, DBH, and PNMT mRNA levels in both c-fos KO and wild-type (WT) mice compared to unstressed controls. In CRH KO mice, PNMT gene expression was not increased to the same extent after single, but especially after repeated immobilization as in WT mice, in contrast to TH and DBH mRNA levels. Thus, our data indicate that CRH deficiency can influence the PNMT mRNA level in adrenal medulla during stress, confirming the idea that the HPA axis plays the crucial role in PNMT gene regulation in mice. On the other hand, c-Fos protein probably does not play a crucial role in TH, DBH, and PNMT gene expression in adrenal medulla under stress conditions.

Expression of estrogen receptor-alpha and cFos in norepinephrine and epinephrine neurons of young and middle-aged rats during the steroid-induced luteinizing hormone surge.

Norepinephrine (NE) and epinephrine are important stimulators of GnRH release during the preovulatory surge in female rats. Previous studies have shown that the catecholaminergic neurons are sensitive to estradiol and that NE release in the hypothalamus is decreased in middle-aged rats at the time when the estrous cycles become irregular and later cease to exist. The aims of the present study were to determine whether the NE and epinephrine neurons continue to express estrogen receptor (ER)-alpha in middle-aged rats; temporal expression of ER-alpha and cFos changes with age during the steroid-induced surge; and tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH), and phenylethanol-N-methyltransferase mRNA content in catecholaminergic neurons of the brain stem changes during the surge with age. The results show that there was no difference in TH mRNA content; however, DBH mRNA levels in areas A1, A2, and C1 of the middle-aged animals did not rise during the surge as was observed in the young animals. Although the percentage of NE and epinephrine neurons that express ER-alpha was unchanged during the surge in both young and middle-aged animals, cFos expression was enhanced in areas A1 and A2 of the middle-aged animals but not in the young animals. Together the results suggest that NE and epinephrine neurons in the middle-aged rat continue to express appropriate basal levels of TH, DBH, and phenylethanol-N-methyltransferase mRNAs as well as ER-alpha and cFos; however, the enhancement of DBH expression, as seen in the young animals during the steroid-induced surge, was not detected in middle-aged animals. On the other hand, cFos expression in the middle-aged rat was higher in areas A1 and A2 during the surge. It is concluded that the reduced catecholamine release during the surge in middle-aged rats is caused, in part, by an altered sensitivity of the NE neurons to estradiol, which results in an aberrant cFos expression and probably not by major deficits in the expression of transmitter synthesizing enzymes or steroid receptors.

Involvement of alpha7 nicotinic acetylcholine receptors in activation of tyrosine hydroxylase and dopamine beta-hydroxylase gene expression in PC12 cells.

Nicotine treatment increases intracellular free Ca(2+) concentration [Ca(2+)](i), stimulates catecholamine release, and elevates gene expression for the catecholamine biosynthetic enzymes tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH). However, the type of nicotinic acetylcholine receptors (nAChRs) mediating these events is unclear. The nAChR receptor antagonists alpha-bungarotoxin (alphaBTX) and methyllycaconitine greatly reduced the nicotine-triggered initial transient rise in [Ca(2+)](i) and prevented the second prolonged elevation of [Ca(2+)](i), suggesting the involvement of alpha7 nAChRs. Two specific alpha7 nicotinic agonists, 3-(2,4-dimethoxybenzilidene)anabaseine (DMXB) and E, E-3-(cinnamylidene)anabaseine (3-CA), were found to elicit a small, delayed increase in [Ca(2+)](i) with kinetics and magnitude similar to the second elevation observed with nicotine. This increase was inhibited by the inositol trisphosphate receptor antagonist xestospongin C. Exposure to 3-CA or DMXB for 6 or 24 h elevated TH and DBH mRNA levels two- to fourfold over control levels. These agonists were more effective than nicotine alone in increasing TH and DBH gene expression and significantly elevated [Ca(2+)](i) for up to 6 h. The increase in [Ca(2+)](i) or the elevation in TH mRNA by 3-CA was completely inhibited by alphaBTX. This study, for the first time, implicates stimulation of alpha7 nAChRs in the activation of TH and DBH gene expression.

Stimulation of human DBH gene expression by prostaglandin E2 in human neuroblastoma SK-N-BE(2)C cells

Prostaglandin E2 (PGE2) enhances transcription of the human dopamine beta-hydroxylase (DBH) gene in human neuroblastoma SK-N-BE(2)C cells. To identify a PGE2-responsive cis-acting element in the human DBH gene, serial deletion constructs of the human DBH 5'-upstream region fused to the chloramphenicol acetyltransferase (CAT) reporter gene were transiently transfected into SK-N-BE(2)C cells. Treatment of the transformed cells with PGE2 increased CAT expression two- to threefold in all constructs except where the promoter region was shortened beyond position -114 bp. There are several cis-regulatory elements in the region between -262 and -114 bp from the transcription initiation site that include a cyclic AMP response element (CRE) and a putative AP1 sequence. We presupposed that the CRE and AP1 might be candidates for PGE2 stimulation, and therefore, used site-directed mutagenesis to change the CRE and AP1 motives and test which of the two elements mediated the transcriptional enhancement. Only a specific mutation within the CRE sequence abolished the PGE2 effect. In addition, cotransfection with an expression vector expressing PKA inhibitor resulted in the specific blockage of the PGE2 effect on DBH gene expression. Northern blot analysis revealed that the increase in DBH gene transcription caused by PGE2 results in elevated DBH mRNA levels. Gel-retardation and competition assays confirmed that the binding of nuclear factors to the CRE site is sequence specific. Our data, therefore, indicate that PGE2 enhances the transcription of the human DBH gene. The effect is mediated by the CRE motif through activation of PKA.

Pituitary adenylate cyclase activating polypeptide (PACAP) regulates expression of catecholamine biosynthetic enzyme genes in bovine adrenal chromaffin cells.

Pituitary adenylate cyclase activating polypeptide (PACAP) elevates levels of the mRNAs encoding the catecholamine synthesizing enzymes tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), and phenylethanolamine N-methyltransferase (PNMT) in primary cultures of bovine adrenal chromaffin cells. PACAP potently (in nanomolar concentrations) increases the amount of mRNA for each of the three catecholamine biosynthetic enzymes. At 10 nM PACAP, TH and DBH mRNA levels increase approx 10-fold; 1 nM PACAP produces an approx 2.5-fold elevation of PNMT mRNA. In contrast to depolarizing or cholinergic stimuli, PACAP does not enhance expression of 5' upstream regions of the PNMT gene transiently transfected into chromaffin cells. Nor does PACAP stimulate the rate of PNMT gene transcription, thereby indicating that the effects of this neuropeptide do not involve enhanced transcription of this gene. However, after 16 h in the presence of transcriptional inhibitors, more PNMT mRNA is present in cultures treated with PACAP relative to control cultures, whereas amounts of TH and DBH mRNAs are not changed. PACAP likely elevates PNMT mRNA levels posttranscriptionally, possibly by stabilizing this message against degradation. Thus, although PACAP is an effective regulator for expression of all three catecholamine enzyme genes, its mechanism of action on PNMT mRNA appears to be distinctive from its effects on TH and DBH gene transcription.

Protein kinase A coordinately regulates both basal expression and cyclic AMP-mediated induction of three catecholamine-synthesizing enzyme genes.

Studies have shown that the cyclic AMP-regulated pathway is involved in the activation of tyrosine hydroxylase (TH) and in the induction of gene expression of the three catecholamine-synthesizing enzymes, TH, dopamine beta-hydroxylase (DBH), and phenylethanolamine N-methyltransferase (PNMT). In the present study we investigated further the role of protein kinase A (PKA) in the regulation of both basal and cyclic AMP-inducible transcription of the three catecholamine-synthesizing enzymes in primary cultured bovine chromaffin cells by using the PKA-specific inhibitor N-[2-(p-bromocinnamylamine)ethyl]-5-isoquinolinesulfonamide (H-89). In the presence of 40 microM H-89, mRNA levels of TH, DBH, and PNMT were reduced to 17 +/- 8, 19 +/- 8, and 14 +/- 2% of the untreated control, respectively, in 24 h, and intracellular norepinephrine and epinephrine levels were decreased to 20 and 34%, respectively, in 72 h. At 20 microM, although the basal enzyme gene expression levels were little affected, their induction by forskolin was abolished and norepinephrine and epinephrine levels fell to 55 and 74%. This reduction in catecholamines at 20 microM was probably due to changes in the phosphorylation state of TH, as its enzymatic activity was found to be decreased to 66 and 69% in 48 and 72 h, respectively. Thus, PKA activity in bovine adrenal medullary cells coordinately regulates both basal and cyclic AMP-inducible gene expression of specific catecholamine-synthesizing enzymes, resulting in changes in intracellular catecholamine levels available for consequent neurohormonal activities.

Expression of catecholamine-synthesizing enzymes, peptidylglycine alpha-amidating monooxygenase, and neuropeptide Y mRNA in the rat adrenal medulla after acute systemic nicotine.

The expression of catecholamine-synthesizing enzymes in the adrenal medulla is upregulated in parallel by stress and pharmacological treatments. In this study we examined whether a neuropeptide and its processing enzyme are regulated in parallel with catecholamine enzyme genes after drug treatment. Because the main effect of stress on the adrenal medulla is via splanchnic nerve stimulation of nicotinic receptors, we used nicotine to stimulate the medulla and visualized expression of catecholamine enzyme genes, the medullary peptide neuropeptide Y (NPY), and the neuropeptide-processing enzyme peptidylglycine alpha-amidating monooxygenase (PAM) by in situ hybridization quantified by image analysis of autoradiographic images. Rats received a single injection of nicotine (0, 1, or 5 mg/kg sc). Six hours later, rats were transcardially perfused. Free-floating adrenal gland sections were hybridized with 35S-labeled cDNA probes for tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), phenylethanolamine N-methyltransferase (PNMT), PAM, and NPY. Nicotine treatment upregulated the expression of TH, PNMT, and NPY genes in a dose-dependent fashion. Small but nonsignificant increases were observed in DBH and PAM mRNA levels. These results suggest that common transcriptional activation mechanisms may upregulate both catecholamine and neuropeptide synthesis in the adrenal medulla after nicotinic stimulation.

Immobilization stress elevates gene expression for catecholamine biosynthetic enzymes and some neuropeptides in rat sympathetic ganglia: effects of adrenocorticotropin and glucocorticoids.

Sympathetic ganglia are the major contributors to the stress-elicited rise in circulating norepinephrine, enkephalins, and neuropeptide Y. Here we examined the effect of immobilization stress and treatment with ACTH and glucocorticoids on messenger RNA (mRNA) levels for tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), preproneuropeptide Y (pre-NPY), and proenkephalin in rat superior cervical ganglia (SCG) and in stellate ganglia. Our results show a severalfold increase in the relative abundance of TH and NPY mRNAs in response to a single immobilization. Repeated stress elevated expression of all the genes studied and increased TH immunoreactivity in both ganglia. The effect of stress was more pronounced in SCG. Prolonged cortisol administration failed to alter the mRNA levels of TH, DBH, and NPY in control animals but attenuated the response to stress. In contrast, TH and DBH mRNA levels in the SCG, but not in adrenal medulla, were elevated by ACTH administration, similar to the levels attained after immobilization. The results revealed that the regulation of gene expression in response to immobilization stress in sympathetic neurons differs from the regulation in adrenal medulla. The study implicates hormonal involvement in the stress-induced changes in TH, DBH, NPY, and proenkephalin gene expression in sympathetic ganglia.

Induction of gene expression of the catecholamine-synthesizing enzymes by insulin-like growth factor-I.

The effects of insulin-like growth factor-I (IGF-I) on gene expression and the activities of the three enzymes specific for catecholamine biosynthesis, tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), and phenylethanolamine N-methyltransferase (PNMT), were determined in bovine adrenomedullary chromaffin cells primary cultured in serum-free medium. The mRNA level of TH was maximally elevated in the presence of IGF-I by 3.1 +/- 0.4-fold after 48 h, DBH by 5.1 +/- 0.3-fold in 24 h, and PNMT by 2.8 +/- 0.5-fold in 72 h. In addition, the activity of TH was increased by 77%, DBH by 70%, and PNMT by 23% in IGF-I-exposed cultures. In the absence of the growth factor, the mRNA levels of TH and DBH were decreased to 45 +/- 10% and 35 +/- 12% of the time-zero control within 48 h while PNMT mRNA was decreased to 82 +/- 5% only after 72 h. When the cells were cotreated with the protein tyrosine kinase inhibitor genistein, DBH induction by IGF-I was suppressed, confirming that the effect is mediated by tyrosine kinase. Cotreatment with the protein kinase A (PKA) inhibitor H89 caused complete reversal of the IGF-I-induced DBH increase and the effects of IGF-I treatment and PKA activation by forskolin were not additive, suggesting that PKA is involved in the signaling initiated by IGF-I in these cells.

Nicotine increases expression of tyrosine hydroxylase gene. Involvement of protein kinase A-mediated pathway.

Nicotine, a major component of tobacco smoke, stimulates catecholamine secretion and activates catecholamine biosynthetic enzymes such as tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH) in adrenal medullary cells. We investigated the effect of long term treatment with nicotine on TH and DBH gene expression in rat PC12 pheochromocytoma cells. Nicotine treatment for 1-2 days increased both the TH and DBH mRNA levels. The effect of nicotine on TH mRNA seems to be transcriptionally mediated. Deletion analysis of the 5' promoter region of the TH gene showed that the region containing a cyclic AMP/calcium regulatory element is sufficient for the nicotinic induction of TH. Nicotine did not induce TH mRNA or chloramphenicol acetyltransferase reporter activity in mutant PC12 cells deficient in protein kinase A activity. However, the deficiency in protein kinase A activity did not affect the elevation in intracellular calcium concentration caused by nicotine, indicating normal receptor function. These results suggest that a cAMP-mediated pathway plays a crucial role in the long term nicotine-induced activation of the TH gene.

Regulation of expression of dopamine beta-hydroxylase in PC12 cells by glucocorticoids and cyclic AMP analogues.

Regulation of catecholamine biosynthesis is crucial in the adaptation to various physiological conditions, such as stress, and in several disorders, including hypertension and depression. In this study we have found that in PC12 cells, the mRNA levels of dopamine beta-hydroxylase (DBH), the enzyme that catalyzes the formation of norepinephrine from dopamine, can be regulated by glucocorticoids and cyclic AMP (cAMP) analogues. Treatment with dexamethasone increased DBH mRNA levels by 6 h. with maximal elevation (four- to fivefold) obtained after 1 day of exposure, and these levels were maintained for up to 4 days. DBH mRNA levels were also elevated on treatment of PC12 cells with 8-bromo cAMP for 8 h to 1 day. The response to 8-bromo cAMP, however, was bimodal, because DBH mRNA levels declined below control values on treatment for > 1 day. In combined treatments with 8-bromo cAMP and dexamethasone, the cAMP effect was dominant. To begin to characterize the regulation of DBH mRNA, genomic clones for rat DBH were isolated, and 1 kb of the 5' flanking region was sequenced. Several putative regulatory elements, which may be involved in cAMP and glucocorticoid regulation, were identified, including two adjacent cAMP response elements, another element that can also bind members of the ATF/CREB family of transcription factors, a NF-kappa B-like sequence, several AP-2 sites, and three core glucocorticoid receptor binding sequences.

Regulation of tyrosine hydroxylase and dopamine beta-hydroxylase mRNA levels in rat adrenals by a single and repeated immobilization stress.

Adrenal catecholamines are known to mediate many of the physiological consequences of the "fight or flight" response to stress. However, the mechanisms by which the long-term responses to repeated stress are mediated are less well understood and possibly involve alterations in gene expression. In this study the effects of a single and repeated immobilization stress on mRNA levels of the adrenal catecholamine biosynthetic enzymes, tyrosine hydroxylase and dopamine beta-hydroxylase, were examined. A repeated 2-hr daily immobilization for 7 consecutive days markedly elevated both tyrosine hydroxylase and dopamine beta-hydroxylase mRNA levels (about six- and fourfold, respectively). In contrast, tyrosine hydroxylase but not dopamine beta-hydroxylase mRNA levels were elevated immediately following a single immobilization. The elevation in tyrosine hydroxylase mRNA with a single immobilization was as high as with seven daily repeated immobilizations. This elevation was not sustained and returned toward control values 24 hr later. Both tyrosine hydroxylase and dopamine beta-hydroxylase mRNA levels were elevated immediately following two daily immobilizations to levels similar to those observed after seven immobilizations and were maintained 24 hr later. The results indicate that both tyrosine hydroxylase and dopamine beta-hydroxylase mRNA levels are elevated by stress; however, the mechanism and/or timing of their regulation are not identical.

Rat dopamine β‐hydroxylase: Molecular cloning and characterization of the cDNA and regulation of the mRNA by reserpine

A number of cDNA clones for rat dopamine beta-hydroxylase (DBH) were isolated from a rat pheochromocytoma tumor cDNA library. The 2445 nucleotide sequence revealed a single open reading frame of 1860 nucleotides and a 3' untranslated region containing two polyadenylation addition signals. The cDNA coded for a 620 amino acid protein of 69,883 daltons. Six potential glycosylation sites and one potential phosphorylation site were identified. Amino acid residues likely to be involved in the active site of DBH and in copper ligand binding were identified. The N-terminal 42 amino acids appeared to constitute a typical but unusually long signal sequence. Hydropathy analysis indicated that this N-terminal region contained the only extensive hydrophobic domain and thus constituted the only obvious potential membrane attachment site. Northern analysis detected two mRNA species of 2.5 and 2.7 kb. The relative abundance of the 2.7 vs. 2.5 kb mRNAs was differentially regulated in PC12 cells and adrenals. DBH mRNA levels were induced in vivo in rat adrenals upon treatment with reserpine.