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.

Heightened transcription for enzymes involved in norepinephrine biosynthesis in the rat locus coeruleus by immobilization stress.

BACKGROUND: The locus coeruleus (LC), a target for CRH neurons, is critically involved in responses to stress. Various physiological stresses increase norepinephrine turnover, tyrosine hydroxylase (TH) enzymatic activity, protein and mRNA levels in LC cell bodies and terminals; however, the effect of stress on other enzymes involved in norepinephrine biosynthesis in the LC is unknown. METHODS: Rats were exposed to single (2 hour) or repeated (2 hour daily) immobilization stress (IMO). Recombinant rat dopamine b-hydroxylase (DBH) cDNA was expressed in E. coli and used to generate antisera for immunohistochemistry and immunoblots in LC. Northern blots were used to assess changes in mRNA levels for TH, DBH, and GTP cyclohydrolase I (GTPCH) in the LC in response to the stress. Conditions were found to isolate nuclei from LC and to use them for run-on assays of transcription. RESULTS: Repeated stress elevated the DBH immunoreactive protein levels in LC. Parallel increases in TH, DBH and GTPCH mRNA levels of about 300% to 400% over control levels were observed with single IMO, and remained at similar levels after repeated IMO. This effect was transcriptionally mediated, and even 30 min of a single IMO significantly increased the relative rate of transcription. CONCLUSIONS: This study is the first to reveal transcriptional activation of the genes encoding catecholamine biosynthetic enzymes in the LC by stress. In addition to TH, changes in DBH and GTPCH gene expression may also contribute to the development of stress-triggered affective disorders.

Transient or sustained transcriptional activation of the genes encoding rat adrenomedullary catecholamine biosynthetic enzymes by different durations of immobilization stress.

The impact of stress on the transcription of rat adrenal tyrosine hydroxylase and dopamine beta-hydroxylase genes was examined. Nuclear run-on assays revealed that repeated immobilization stress elicited marked (about threefold) increases in the relative rates of transcription, being sustained for at least one day. Parallel changes in the steady-state messenger RNA levels for tyrosine hydroxylase and dopamine beta-hydroxylase were also observed. A single episode of stress triggered similar enhancements in their relative transcription rates. Depending on the duration of the stress signal, the effect on gene transcription varied in its persistence. After very short (5 min) immobilization, there was a marked transient rise in the transcription of both genes, without an accumulation of the corresponding mRNAs. Longer episodes of stress (30 min) increased the relative rate of tyrosine hydroxylase transcription for hours, causing elevations in the steady-state messenger RNA levels. In contrast, although dopamine beta-hydroxylase transcription was elevated to a similar extent by 30-min immobilization stress, the effect was transient and not reflected in significant accumulation of its messenger RNA. The results of our studies emphasize that the stress-evoked increases in the expression of the genes encoding adrenomedullary catecholamine biosynthetic enzymes involve transcriptional activation. Depending on the duration and reiteration of the stress signal, different transcriptional mechanisms may be employed.

Promoter elements and second messenger pathways involved in transcriptional activation of tyrosine hydroxylase by ionomycin.

Membrane depolarization, or agents which increase intracellular calcium, elicit transcriptional activation of tyrosine hydroxylase (TH). In this study we analyze the factors involved in the regulation of the TH promoter by a calcium ionophore. PC12 cells were transiently transfected with plasmids containing wild type or mutated 5' flanking sequences of the rat TH gene, fused to bacterial chloramphenicol acetyl transferase (CAT). Point mutations introduced into the consensus cAMP-regulatory element (CRE) abolished the induction of CAT by ionomycin indicating that it is essential for mediating the calcium response. An intact and functional AP1 site did not confer calcium inducibility when the CRE/CaRE sequence was mutated. The extent and kinetics of the increase in intracellular calcium as well as the induction of CAT activity under the control of TH promoter by ionomycin were similar in PC12 cells and in the A123.7, protein kinase A (PKA) deficient cell line. In both cell lines addition of ionomycin rapidly increased the phosphorylation of transcription factor CREB at Ser-133. These results suggest that the activation of TH transcription by ionomycin does not require PKA. However, KN62 an inhibitor of Ca2+/calmodulin dependent (CaM) kinases prevented the induction indicating possible involvement of CAM kinases in the calcium response.

Glucocorticoids elevate GTP cyclohydrolase I mRNA levels in vivo and in PC12 cells.

GTP cyclohydrolase I (GTPCH) is the rate-limiting enzyme in the formation of tetrahydrobiopterin, the cofactor for catecholamine, indolamine and nitric oxide biosynthesis. The effect of glucocorticoids on GTPCH gene expression was examined by direct infusion of cortisol to rats and by incubation of PC12 cells with glucocorticoids. Northern blot analysis revealed that infusion of cortisol for 1 or 7 days elevated levels of the 3.6 kb GTPCH mRNA species in rat adrenal medulla, while the 1.2 kb mRNA species were only increased by 1 day cortisol. Cortisol administration to hypophysectomized animals elicited a 4-5-fold elevation in both forms of GTPCH mRNA. These results indicate that glucocorticoids may be directly involved in the regulation of adrenomedullary GTPCH mRNA levels by physiological stress. Incubation of PC12 cells with plasma from immobilized, but not control, animals increased the level of the 3.6 kb mRNA. Treatment of PC12 cells with dexamethasone for 12-48 h elicited a 4-6-fold elevation in both GTPCH mRNAs. Using the nuclear run-on assay, increased transcription of the GTPCH gene was observed in the rat adrenal medulla with immobilization stress, or in PC12 cells treated with dexamethasone. This is the first report that glucocorticoids can alter GTPCH expression.

Stress elicits trans-synaptic activation of adrenal neuropeptide Y gene expression.

Stress triggers responses important to maintain internal homeostasis, and yet when prolonged can cause medical consequences that are most likely mediated by changes in gene expression. In this study we examine the alterations in gene expression of neuropeptide Y (NPY), a potent vasoconstrictor, with stress. Stressors, such as immobilization and cold, were found to increase adrenal NPY gene expression in rats. Adrenal prepro-NPY mRNA levels were elevated by a relatively short period of stress. A single immobilization was sufficient for an increase in prepro-NPY mRNA, that remained elevated for as long as one day later. This rise in adrenal NPY mRNA was abolished by the transcriptional inhibitor actinomycin D. Repeated (2 and 7) daily immobilizations led to a further rise and sustained elevations of prepro-NPY mRNA levels. This increase persisted 2-3 days after the cessation of repeated stress. The stress-elicited increase in NPY gene expression is transsynaptic requiring splanchnic innervation and mediated by nicotinic receptors. Hypophysectomy did not prevent the stress elicited rise in adrenal prepro-NPY mRNA levels. These results suggest that long lasting changes in NPY gene expression might be an important component in the homeostatic mechanisms triggered by chronic stress.

Molecular biology of stress-elicited induction of catecholamine biosynthetic enzymes.

Immobilization stress elevates adrenal mRNA for several catecholamine biosynthetic enzymes. Phenylethanolamine N-methyltransferase (PNMT) mRNA is elevated in response to as little as 5 minutes of immobilization, which appears to be a direct consequence of the large rise of glucocorticoids. Tyrosine hydroxylase (TH) mRNA levels are not elevated under these conditions, but are maximally induced by 30 min of a single immobilization. Dopamine beta-hydroxylase (DBH) requires repeated stress for maximal induction. Transcriptional inhibitor actinomycin D prevented the elevation of TH and PNMT mRNA with a single immobilization. Oligonucleotides corresponding to the AP-1 like element in the TH promoter and an important regulatory element in the DBH promoter were used in binding assays with adrenomedullary extracts of rats exposed to stress. The complexes were UV cross-linked and analyzed by gel electrophoresis. The complex bound to the TH AP-1 site contained c-fos and other proteins (including cJun). The complex to the DBH-1 element contained several proteins, probably including fos related proteins but lacked c-fos. Immunoblots found that c-fos is induced in the adrenal medulla near maximally by one hour immobilization and cJun and JunD are present in higher constitutive levels and are only moderately regulated by immobilization stress. The results indicate that different mechanisms of transcriptional activation are used by several members of the catecholamine biosynthetic pathway.

Sympathoadrenal system in stress. Interaction with the hypothalamic-pituitary-adrenocortical system.

Exposure of an organism to any of a variety of stressors markedly activates the sympathoadrenal and hypothalamic-pituitary-adrenocortical systems. Interactions of these major stress systems occur at several levels in the periphery and the brain. In the present study, we used sham-operated or adrenalectomized cortisol-treated conscious rats to examine glucocorticoid effects on indices of CA release, metabolism, and synthesis, and on CA biosynthetic enzyme activities and gene expression at baseline and during immobilization stress (IMO). Adrenalectomy (ADX) stimulated basal and stress-induced increments in norepinephrine release, reuptake, metabolism, turnover, and biosynthesis. Loss of adrenomedullary hormones after ADX did not appear to contribute to these increments. Cortisol treatment reversed the ADX effects on CA indices and suppressed catecholaminergic responses to IMO in intact rats. These results suggest that endogenous glucocorticoids restrain responses of catecholamine turnover, synthesis, release, reuptake, and metabolism during stress. In contrast, in intact rats, continuous administration of cortisol lasting for 7 days exaggerated the IMO-induced increases in plasma CA levels. Inhibition of DOPA conversion to dopamine elevated plasma DOPA levels in chronically cortisol-treated stressed rats compared to saline-treated ones, suggesting a cortisol-induced increase in tyrosine hydroxylation. Stress increases TH and PNMT activities and mRNA levels in the adrenal medulla. Hypophysectomy reduced adrenal PNMT but not TH mRNA levels in control and IMO rats. Pretreatment of hypophysectomized animals with ACTH fully restored the control and IMO-induced adrenal PNMT mRNA levels and augmented PNMT but not TH mRNA responses in intact rats. Long-term cortisol administration to intact rats also elevated adrenal PNMT but not TH mRNA levels. The results indicate a suppressive effect of endogenous glucocorticoids and a stimulatory effect of chronically elevated glucocorticoid levels on sympathoadrenal activity during stress. The results also suggest that a nonneuronal, nonpituitary factor contributes to TH gene expression during some forms of stress, whereas pituitary-adrenocortical factors play the essential role in the regulation of PNMT gene expression.

Immobilization Stress Elevates GTP Cyclohydrolase I mRNA Levels in Rat Adrenals Predominantly by Hormonally Mediated Mechanisms.

GTP cyclohydrolase I (GTPCH) is the rate-limiting enzyme in the biosynthesis of tetrahydrobiopterin, the cofactor for catecholamine, indolamine and nitric oxide biosynthesis. In this study we examined the effect of immobilization stress on GTPCH mRNA levels and the mechanism(s) of stress-induced changes in adrenomedullary GTPCH mRNA levels. We used reverse-polymerase chain reaction to isolate and clone a cDNA corresponding to nucleotides 269 to 570 of rat GTPCH. Northern blot analysis with a cRNA probe revealed two species of GTPCH mRNA (about 3.6 and 1.2 kb) in rat adrenal medulla and cortex, and in PC12 cells. The levels of both forms of GTPCH mRNA were significantly increased 3-5 fold in adrenal medulla by a single 2 hour immobilization and by repeated immobilizations (2 hours a day for 2 days). Hypophysectomy had little effect on their basal levels but prevented the stress elicited rise in both GTPCH mRNAs. In contrast, unilateral transection of the splanchnic nerve did not affect induction of the 3.6 kb GTPCH mRNA by stress. Combined denervation with hypophysectomy completely blocked the induction of both GTPCH mRNA species by immobilization stress. Thus, stress elicits elevation of both forms of GTPCH mRNA by a mechanism requiring an intact pituitary-adrenocortical axis.

Adrenocorticotropic hormone (MC-2) receptor mRNA is expressed in rat sympathetic ganglia and up-regulated by stress.

Stress triggered cardiovascular disorders are associated with elevated activity of the sympathetic nervous system, the major source of elevated plasma norepinephrine levels. Our previous studies revealed that administration of adrenocorticotropic hormone (ACTH) increases the gene expression of norepinephrine biosynthetic enzymes and several neuropeptides in rat sympathetic ganglia as much as stress. Here, we examine whether an ACTH-responsive receptor is expressed in rat superior cervical (SCG) and stellate ganglia (StG). Using reverse transcriptase-polymerase chain reaction (RT-PCR) we found expression of MC-2 receptor mRNA in these ganglia. Identical DNA fragments were amplified with mRNA from SCG, StG or from adrenal cortex. Sequencing revealed extensive homology to published sequences of mouse and human MC-2 receptor. Real time PCR was used to quantitate MC-2 receptor mRNA levels in the SCG under basal conditions and following immobilization stress. Immobilization stress triggered a large increase in MC-2 receptor mRNA in SCG. The results provide the first evidence that rat sympathetic ganglia express MC-2 receptor gene and are a target tissue for the peripheral actions of ACTH in response to stress.

Nicotinic receptor partial agonists alter catecholamine homeostasis and response to nicotine in PC12 cells.

Repeated stress is a major public health concern where many stress responses are mediated by neuronal nicotinic acetylcholine receptors. In the present study we evaluated the effects of the nicotinic receptor partial agonists, cytisine and its derivative 3-(pyridin-3'-yl)-cytisine (3-pyr-Cyt) on two main biological outputs associated with activation of nAChR-release of neurotransmitters and increase in catecholamine biosynthesis to replenish the releasable pool. We compared these substances to the maximal response triggered by nicotine (full agonist) in PC12 cells. Cytisine, 3-pyr-Cyt or nicotine induced time-, dose- and Ca(2+)-dependent significant release of norepinephrine (NE) into the culture media. These effects were completely inhibited by mecamylamine but not by α-bungarotoxin, and only partially affected by α-conotoxin AulB, consistent with the involvement of α3ß4 receptors. Co-application of cytisine (or 3-pyr-Cyt) and nicotine resulted in attenuated nicotine-induced NE release. Cytisine or 3-pyr-Cyt alone induced a modest rise in tyrosine hydroxylase (TH) mRNA levels (index of the cell's catecholamine biosynthetic capacity). We conclude that both, cytisine and 3-pyr-Cyt (i) display typical partial agonist properties at naturally existing ganglionic nAChR (α3ß4 and α7 nAChR) with regard to catecholamine homeostasis (i.e. NE release and re-synthesis) and (ii) modulated the effect of nicotine during combined treatment.

Multiple signalling pathways exist in the stress-triggered regulation of gene expression for catecholamine biosynthetic enzymes and several neuropeptides in the rat adrenal medulla.

A critical component of the response to stress is the coincident activation of the hypothalamic-pituitary-adrenal axis and the sympathoadrenal system - comprised of sympathetic ganglia and the adrenal medullae. The sympathoadrenal system produces the catecholamines - noradrenaline and adrenaline, and several neuropeptides, involved in the homeostatic mechanisms that govern the adaptation to stress. This brief survey aims to provide a general overview of the present knowledge about the impact of stress on neurotransmitter gene expression in the adrenal medulla, with particular attention paid to the apparent heterogeneity in stress-evoked signals and regulatory pathways.

Activated ribosomal RNA synthesis in regenerated rat liver upon inhibition of protein synthesis.

Cycloheximide (Cyh), administered at a dose of 5 mg/kg body wt blocks protein synthesis in normal rat liver (NRL) and regenerating rat liver (RRL). The rate of synthesis of 45S pre-rRNA in RRL, studied after RNA labelling in vivo is activated 2.8 times. Pre-r RNA synthesis in RRL is more sensitive to the stopped translation, but never falls down to the level in NRL. The major contribution to the rDNA transcription activation in RRL comes from the 20-fold increase in the number of pol I molecules engaged in the transcription, the elongation rate being 1.4-fold accelerated. Cyh quenches partially the enhanced rDNA transcription in RRL: the number of pol I molecules and their elongation rate are about 1.7-fold and 1.5-fold higher, respectively, than the corresponding values in NRL after Cyh treatment. The results show that two different mechanisms control the number and the rate of initiation and elongation of RNA polymerase I in rat liver; one of them depends on continuous protein synthesis and can be inactivated by Cyh, the other is Cyh resistant.

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.

Fos-related antigen 2: potential mediator of the transcriptional activation in rat adrenal medulla evoked by repeated immobilization stress.

The precise mechanisms by which beneficial responses to acute stress are transformed into long-term pathological effects of chronic stress are largely unknown. Western blot analyses revealed that members of the AP1 transcription factor family are differentially regulated by single and repeated stress in the rat adrenal medulla, suggesting distinct roles in establishing stress-induced patterns of gene expression in this tissue. The induction of c-fos was transient, whereas marked elevation of long-lasting Fos-related antigens, including Fra2, was observed after repeated immobilization. We investigated DNA protein interactions at the AP1-like promoter elements of two stress-responsive genes, tyrosine hydroxylase and dopamine beta-hydroxylase. Increased DNA-binding activity was displayed in adrenomedullary extract from repeatedly stressed rats, which was predominantly composed of c-Jun- and Fra2-containing dimers. The induction of Fra2 and increased AP1-like binding activity was reflected in sustained transcriptional activation of tyrosine hydroxylase and dopamine beta-hydroxylase genes after repeated episodes of stress. The functional link between Fra2 and regulation of tyrosine hydroxylase and dopamine beta-hydroxylase transcription was confirmed in PC12 cells coexpressing this factor and the corresponding promoter-reporter gene constructs. These studies emphasize the potential importance of stress-evoked increases in the expression of the Fra2 gene for in vivo adaptations of the adrenal catecholamine producing system.

Repeated immobilization stress increases the binding of c-Fos-like proteins to a rat dopamine beta-hydroxylase promoter enhancer sequence.

Repeated immobilization stress elicits a large elevation in adrenal dopamine beta-hydroxylase (DBH) mRNA levels. This study attempts to analyze the molecular mechanism of increased DBH gene expression in stress. Adrenomedullary nuclear proteins were prepared from controls and rats exposed to various intervals of immobilization stress. Electrophoretic mobility shift assays showed that repeated stress led to increased binding of adrenomedullary nuclear factors to a cis-acting regulatory element in the rat DBH promoter (DBH-1). One of the partners in the DNA-protein complex is c-Fos or a Fos-related protein. There was a correlation between promoter binding activity and elevated steady-state levels of DBH mRNA. Our data indicate that this cis regulatory element in the rat DBH promoter is functional in vivo, and increased binding of AP1-like transcription factors to this motif is induced by immobilization stress.

Selective in vivo stimulation of stress-activated protein kinase in different rat tissues by immobilization stress.

Stress activated protein kinases (SAPK) are key enzymes mediating the cellular response to stressful stimuli. While they are intensively studied in cultured cells, little is known about their physiological role in vivo, or relevance to pathological conditions. Therefore we examined the effect of various times of immobilization on c-Jun N-terminal protein kinase (JNK) activity in several rat stress responsive tissues and in a number of other locations. The abundance and relative distribution of JNK isoforms, the basal levels, time course and relative magnitude of stress induced JNK activity differed among tissues and regions of the brain of the same animal. JNK immunoreactive proteins were most abundant in the brain, especially in the hippocampus, hypothalamus and frontal cortex. Marked activation in response to immobilization stress was observed in adrenal medulla, adrenal cortex, aorta and hippocampus, less pronounced in locus coeruleus. JNK was not affected in superior cervical ganglia, pituitary, hypothalamus, frontal cortex and cerebellum. In adrenal medulla, the activation of JNK by single immobilization stress is correlated with increased transcription of stress-responsive genes, tyrosine hydroxylase and dopamine beta-hydroxylase. These data suggest a potential role of JNK signal transduction pathway in mediating the long term adaptation to stressful stimuli in vivo.

Induction of adrenal tyrosine hydroxylase mRNA by single immobilization stress occurs even after splanchnic transection and in the presence of cholinergic antagonists.

Immobilization (IMO) stress elevates plasma catecholamines and increases tyrosine hydroxylase (TH) gene expression in rat adrenals. This study examined the mechanism(s) of IMO-induced changes in adrenal TH mRNA levels. Innervation of the adrenal medulla is predominantly cholinergic and splanchnicotomy as well as nicotinic receptor antagonists prevent the cold-induced rise in TH mRNA levels. In this study, the IMO-induced rise in plasma catecholamines, but not TH mRNA levels, was reduced by the antagonist chlorisondamine. Muscarinic antagonist atropine also did not prevent the IMO stress-elicited rise in TH mRNA. Furthermore, denervation of the adrenals by unilateral splanchnicotomy did not block the IMO-induced rise in TH mRNA but completely prevented the induction of neuropeptide Y mRNA. These results suggest that (1) the large increase in adrenal TH gene expression elicited by a single IMO stress is not regulated via cholinergic receptors or splanchnic innervation, and (2) there is a dissociation between regulatory mechanisms of catecholamine secretion and elevation of TH gene expression in the adrenal medulla of rats during IMO stress.