Ultrastructural Characterization of Corticotropin-Releasing Factor and Neuropeptide Y in the Rat Locus Coeruleus: Anatomical Evidence for Putative Interactions.

As a neurochemical mediator of stress resilience, NPY has been shown to oppose excitatory effects of the pro-stress neuropeptide corticotropin-releasing factor (CRF). Previous studies have described the anatomical organization of NPY and CRF in the central nucleus of the amygdala, which sends CRF projections to the locus coeruleus (LC), activating LC norepinephrine release. However, the cellular substrates for interactions between NPY and CRF in the LC remain unknown. In this study, we investigated these anatomical substrates in the male rat LC, using immunocytochemistry, confocal microscopy, and immunoelectron microscopy to detect NPY and CRF, as well as CRF and Y1 or Y2 receptors (Y1R or Y2R). Immunofluorescence and electron microscopy revealed both co-localization of NPY and CRF in LC axon terminals, as well as separately labeled terminals, suggesting NPY and CRF may serve as co-transmitters in a subset of terminals. Semi-quantitative analysis showed that 32.4% of CRF-labeled terminals contained NPY, while 58.2% (152/261) of NPY-labeled terminals contained CRF. With respect to Y1R and CRF, dual immunoelectron microscopy showed that 23.3% (67/288) of CRF-labeled axon terminals directly contacted Y1R-labeled dendrites, while only 6.3% (18/288) of CRF-labeled axon terminals co-localized with Y1R. Dual immunoelectron microscopy also showed Y2R co-localized with 30.4% (103/339) CRF-labeled terminals, but only with 16.2% (55/339) of dendrites post-synaptic to CRF-labeled axon terminals in the LC. Taken together, these findings indicate multiple sites of interaction between CRF and NPY in the LC and suggest that conditions or drugs that modulate the NPY:CRF balance in the LC may promote stress resilience.

Intranasal neuropeptide Y reverses anxiety and depressive-like behavior impaired by single prolonged stress PTSD model.

PTSD is a debilitating neuropsychiatric disorder and many patients do not respond sufficiently to current treatments. Neuropeptide Y (NPY) is suggested to provide resilience to the development of PTSD and co-morbid depression. Injections of NPY to the rodent brain are anxiolytic. Recently we showed that intranasal delivery of NPY to rats before or immediately after exposure to single prolonged stress (SPS) animal model of PTSD prevented development of many biochemical and behavioral symptoms of PTSD, indicating its prophylactic potential. Here, we investigated whether intranasal NPY might provide benefits once symptoms have already developed. One week after exposure to SPS stressors, animals were given intranasal NPY or vehicle and tested on elevated plus maze 2h or 2 days later. The NPY treated rats had lower anxiety-like behavior than vehicle treated rats as indicated by more entries into open arms and fewer into closed arms, lower anxiety index, higher risk assessment and unprotected head dips and reduced grooming time. Their anxiety index was similar to that of unstressed controls. On most of these variables there was no effect of time interval and rats displayed similar overall changes 2h or 2 days after the infusion. Moreover, intranasal NPY led to reduced depressive-like behavior, assessed by forced swim test. Thus, intranasal NPY reversed several behavioral impairments triggered by the traumatic stress of SPS and has potential for non-invasive PTSD therapeutic intervention.

Single intranasal neuropeptide Y infusion attenuates development of PTSD-like symptoms to traumatic stress in rats.

Exposure to severe stress leads to development of neuropsychiatric disorders, including depression and Post-Traumatic Stress Disorder (PTSD) in at-risk individuals. Neuropeptide Y (NPY) is associated with resilience or improved recovery. Therefore exogenous administration to the brain has therapeutic potential although peripheral administration can trigger undesirable side effects. Here, we established conditions with intranasal (IN) NPY infusion to rats to obtain CSF concentrations in the proposed anxiolytic range without significant change in plasma NPY. Rats were pretreated with IN NPY or vehicle before exposure to single prolonged stress (SPS) animal model of PTSD and compared to untreated controls. The IN NPY appeared to lessen the perceived severity of stress, as these animals displayed less time immobile in forced swim part of the SPS. Thirty minutes after SPS the elevation of plasma adrenocorticotropic hormone (ACTH) and corticosterone was not as pronounced in NPY-infused rats and the induction of tyrosine hydroxylase (TH) in locus coeruleus (LC) was attenuated. Seven days after SPS, they displayed lower depressive-like behavior on Forced Swim Test and reduced anxiety-like behavior on Elevated Plus Maze. The prolonged effect of SPS on Acoustic Startle Response was also lower in NPY-infused rats. Plasma ACTH, corticosterone, and hippocampal glucocorticoid receptor levels were significantly above controls only in the vehicle - but not IN NPY-treated group 1week after SPS. Baseline TH mRNA levels in LC did not differ among groups, but increased with forced swim in the vehicle - but not NPY-pretreated animals. Administration of IN NPY after exposure to SPS led to similar, but not identical, reduction in development of anxiety, depressive-like behavior and hyperarousal. The results show that single IN NPY can alter stress-triggered dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis and activation of central noradrenergic activity. These findings provide proof of concept for potential of IN NPY for non-invasive prophylactic treatment or early intervention in response to traumatic stress.

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.

Role of N-terminus of tyrosine hydroxylase in the biosynthesis of catecholamines.

Tyrosine hydroxylase (TH) catalyzes the conversion of L: -tyrosine to L: -dopa, which is the initial and rate-limiting step in the biosynthesis of catecholamines [CA; dopamine (DA), noradrenaline, and adrenaline], and plays a central role in the neurotransmission and hormonal actions of CA. Thus, TH is related to various neuro-psychiatric diseases such as TH deficiency, Parkinson's disease (PD), and schizophrenia. Four isoforms of human TH (hTH1-hTH4) are produced from a single gene by alternative mRNA splicing in the N-terminal region, whereas two isoforms exist in monkeys and only a single protein exist in all non-primate mammals. A catalytic domain is located within the C-terminal two-thirds of molecule, whereas the part of the enzyme controlling enzyme activity is assigned to the N-terminal end as the regulatory domain. The catalytic activity of TH is end product inhibited by CA, and the phosphorylation of Ser residues (Ser(19), Ser(31), and especially Ser(40) of hTH1) in the N-terminus relieves the CA-mediated inhibition. Ota and Nakashima et al. have investigated the role of the N-terminus of TH enzyme in the regulation of both the catalytic activity and the intracellular stability by producing various mutants of the N-terminus of hTH1. The expression of the following three enzymes, TH, GTP cyclohydrolase I, which synthesizes the tetrahydrobiopterin cofactor of TH, and aromatic-L: -amino acid decarboxylase, which produces DA from L: -dopa, were induced in the monkey striatum using harmless adeno-associated virus vectors, resulting in a remarkable improvement in the symptoms affecting PD model monkeys Muramatsu (Hum Gene Ther 13:345-354, 2002). Increased knowledge concerning the amino acid sequences of the N-terminus of TH that control enzyme activity and stability will extend the spectrum of the gene-therapy approach for PD.

Adrenocorticotropic hormone elevates gene expression for catecholamine biosynthesis in rat superior cervical ganglia and locus coeruleus by an adrenal independent mechanism.

Classically, upon hypothalamic stimulation, adrenocorticotropic hormone (ACTH) is released from the pituitary and acts on melanocortin 2 receptors (MC2R) in the adrenal cortex, stimulating glucocorticoid synthesis and release. Our earlier studies suggested that ACTH might have a direct effect on sympathetic ganglia. To analyze further the involvement of ACTH in regulation of gene expression of norepinephrine (NE) biosynthetic enzymes, we examined the effect of bilateral adrenalectomy (ADX) of Sprague-Dawley male rats. Fourteen days post-ADX, as expected, plasma ACTH was elevated, and levels of tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH) and MC2R mRNAs in superior cervical ganglia (SCG), and TH mRNA in locus coeruleus (LC) were increased compared with sham-operated animals. To determine effect of pulsatile elevation of ACTH, corticosterone pellets were implanted to ADX rats. Similar to immobilization (IMO) stress ACTH injections to these animals caused a rise in ACTH in plasma and triggered elevation of TH and DBH mRNAs in SCG and in LC with single and repeated daily injections, and MC2R mRNA in SCG with single injections. To study the effect of ACTH in isolated cells, primary cultures of rat SCG were transfected with TH and DBH promoter constructs and treated with ACTH. In agreement with the in vivo data, ACTH elevated their promoter activities similar to levels triggered by cyclic AMP analog. ACTH in the human SK-N-SH neuroblastoma cells increased TH and DBH promoter activity and endogenous DBH mRNA levels. The results show that ACTH can have a direct effect on transcription and gene expression of NE biosynthetic enzymes even without contribution of adrenal hormones.

Gene expression of phenylethanolamine N-methyltransferase in corticotropin-releasing hormone knockout mice during stress exposure.

AIMS: Epinephrine (EPI) synthesizing enzyme phenylethanolamine N-methyltransferase (PNMT, EC 2.1.1.28) is primarily localized in the adrenal medulla (AM). We have recently described existence of the PNMT gene expression in cardiac atria and ventricles and in sympathetic ganglia of adult rats and mice. The aim of the present work was to study regulation of the PNMT gene expression in corticotropin-releasing hormone knockout mice (CRH KO) and matched control wild-type mice (WT) under normal and stress conditions. METHODS: Levels of the PNMT mRNA were determined by RT-PCR; PNMT immunoprotein and protein of transcription factor EGR-1 by Western Blot. Plasma EPI and corticosterone (CORT) levels were determined by radioenzymatic and RIA methods. Immobilization (IMMO) was used as a stressor. RESULTS: Stress-induced increases in the PNMT mRNA and protein levels observed in WT mice were almost completely absent in CRH KO mouse adrenal medulla, stellate ganglia, and cardiac atria, while ventricular PNMT mRNA elevation was not CRH-dependent. Plasma EPI and CORT levels were markedly reduced in CRH KO compared to WT mice both before and after the stress. Levels of EGR-1, crucial transcription factor for regulation of the PNMT were highly increased in stressed WT and CRH KO mice in cardiac areas, but not in the adrenal medulla. CONCLUSIONS: Data show that the CRH deficiency can markedly prevent immobilization-triggered induction of the PNMT mRNA and protein levels in the adrenal medulla and stellate ganglia. Reduced plasma epinephrine and corticosterone levels and adrenal medullary EGR-1 protein levels in CRH knockout versus WT mice during stress indicate that the HPA axis plays a crucial role in regulation of the PNMT gene expression in these organs. Cardiac atrial PNMT gene expression with stress is also dependent on intact HPA axis. However, in cardiac ventricles, especially after the single stress exposure, its expression is not impaired by CRH deficiency. Since cardiac EGR-1 protein levels in CRH KO mice are also not affected by the single stress exposure, we propose existence of different regulation of the PNMT gene expression, especially in the cardiac ventricles.Overall, our findings reveal that the PNMT gene expression is regulated through the HPA in both sympathoadrenal system and the heart and also via EGR-1 in the adrenal medulla, but apparently not in the heart. Regulation of the PNMT gene expression in various compartments of heart includes both corticosterone-dependent and independent mechanisms.

Estrogen-triggered activation of GTP cyclohydrolase 1 gene expression: role of estrogen receptor subtypes and interaction with cyclic AMP.

Guanosinetriphosphate cyclohydrolase I (GTPCH) catalyzes the initial step in the de novo biosynthesis of (6R)-5,6,7,8-tetrahydrobiopterin, an important determinant of the rate of catecholamine and nitric oxide biosynthesis. Administration of estrogen in vivo was found to elevate GTPCH mRNA levels in several catecholaminergic locations. To examine the mechanism, PC12 cells were co-transfected with a reporter construct containing 2988 bp of rat GTPCH promoter fused to luciferase gene, and expression vectors for estrogen receptors. Addition of 2.5-20 nM of 17 beta-estradiol increased GTPCH promoter-driven luciferase activity in the presence of either estrogen receptor alpha or estrogen receptor beta indicating, for the first time, that 17 beta-estradiol can regulate GTPCH gene expression via transcriptional mechanisms. However, there were differences in dose dependence and time course with estrogen receptor alpha or estrogen receptor beta. With estrogen receptor alpha, the effect was greater with lower doses of 17 beta-estradiol. At the same dose, the response with estrogen receptor beta was observed somewhat earlier than with estrogen receptor alpha and with 20 nM 17 beta-estradiol was effective even after 6 h. These responses to 17 beta-estradiol required estrogen receptors and specific agonists for estrogen receptor alpha and estrogen receptor beta, 4,4,4,-(4-propil-[1H-pyrazole-1,3,5-triyl)tris-phenol and 2,3-bis[4-hydroxyphenyl]propionitrile respectively, triggered increased GTPCH promoter activity. In addition, neither estradiol, nor the selective agonists activated GTPCH promoter without transfection of appropriate estrogen receptor expression vectors. Addition of 17 beta-estradiol, or the selective agonists, also elevated endogenous GTPCH mRNA levels. The results demonstrate that estrogen can have a direct effect on GTPCH gene expression. Although estradiol increased GTPCH promoter activity in the presence of estrogen receptors, it attenuated the response of the promoter and endogenous gene to cyclic AMP, suggesting the crosstalk between estrogen and cyclic AMP pathways in the regulation of GTPCH gene expression. These findings reveal the significance of estrogen in modulating regulation of rate limiting enzyme in the (6R)-5,6,7,8-tetrahydrobiopterin biosynthesis, which may have implications for sex-related differences in vulnerability in related disorders.

Identification of phenylethanolamine N-methyltransferase gene expression in stellate ganglia and its modulation by stress.

Phenylethanolamine N-methyltransferase (PNMT, EC 2.1.1.28) is the terminal enzyme of the catecholaminergic pathway converting noradrenaline to adrenaline. Although preferentially localized in adrenal medulla, evidence exists that PNMT activity and gene expression are also present in the rat heart, kidney, spleen, lung, skeletal muscle, thymus, retina and different parts of the brain. However, data concerning PNMT gene expression in sympathetic ganglia are still missing. In this study, our effort was focused on identification of PNMT mRNA and/or protein in stellate ganglia and, if present, testing the effect of stress on PNMT mRNA and protein levels in this type of ganglia. We identified both PNMT mRNA and protein in stellate ganglia of rats and mice, although in much smaller amounts compared with adrenal medulla. PNMT gene expression and protein levels were also increased after repeated stress exposure in stellate ganglia of rats and wild-type mice. Similarly to adrenal medulla, the immobilization-induced increase was probably regulated by glucocorticoids, as determined indirectly using corticotropin-releasing hormone knockout mice, where immobilization-induced increase of PNMT mRNA was suppressed. Thus, glucocorticoids might play an important role in regulation of PNMT gene expression in stellate ganglia under stress conditions.

Estrogen modifies stress response of catecholamine biosynthetic enzyme genes and cardiovascular system in ovariectomized female rats.

Estrogen is likely involved in the gender specific differences in coping with stress. Activation of catecholamine (CA) biosynthetic enzyme gene expression in central and peripheral CA systems plays a key role in response to stress and in regulation of the cardiovascular system. Here we examined whether estradiol can modulate response of hypothalamic-pituitary-adrenal axis (HPA), gene expression of enzymes related to CA biosynthesis in several noradrenergic locations, tetrahydrobiopterin (BH4) concentration and blood pressure (BP) in response to immobilization stress (IMO) of ovariectomized female rats. Rats were injected with 25 mug/kg estradiol benzoate (EB) or sesame oil once daily for 16 days and subsequently exposed to two hours of IMO. The IMO triggered elevation in plasma ACTH was lessened in EB-pretreated animals. However, estradiol did not alter the IMO-elicited rise of tyrosine hydroxylase mRNA levels in adrenal medulla (AM) and in the nucleus of solitary track (NTS) compared with controls. The response of GTP cyclohydrolase I (GTPCH) mRNA in AM to IMO was also similar in both groups. Several responses to IMO in EB-treated rats were reversed. Instead of IMO-elicited elevation in dopamine beta-hydroxylase mRNA levels in the locus coeruleus, GTPCH mRNA and BH4 levels in the NTS, they were reduced by IMO. In a parallel experiment, BP was monitored during restraint stress. The elevation of BP in response to single or repeated restraint stress was sustained during 2 h in controls and reduced after 70 min stress in EB treated rats. One month after withdrawal of EB treatment, the BP response to restraint was similar to that of rats which never received EB. The results demonstrate that estrogen can modulate responses to stress affecting HPA axis, CA biosynthesis, in central and peripheral noradrenergic systems, and BP.

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

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

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.

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.

Effect of novel stressors on tyrosine hydroxylase gene expression in the adrenal medulla of repeatedly immobilized rats.

The activity of the sympathetic-adrenomedullary system in rats submitted to novel stressors after prior repeated or chronic stress exposure is poorly understood. The purpose of the present work was to investigate changes in adrenomedullary (AM) tyrosine hydroxylase (TH) gene expression after a single or long-term repeated exposure of rats to immobilization stress (IMMO; 42 times), as well as in repeatedly immobilized rats (41 times) exposed once to various novel heterotypic stressors. Cold exposure for 5 h, administration of insulin (INS, 51U), or 2-deoxyglucose (2DG, 500 mg/kg) were used as novel stressors. A single exposure to cold, INS, or 2DG produced transient increases in TH mRNA levels in AM. Animals exposed to repeated homotypic IMMO stress showed permanently increased TH mRNA levels, TH activity, and protein levels; however, an exposure of such animals to heterotypic novel stressors did not induce any further changes. Thus the observed differences in TH mRNA levels in the AM of control rats and long-term repeatedly IMMO rats suggest that an adaptation to this stressor is displayed by a permanently increased TH gene expression, TH activity, and protein level. The exposure of repeatedly IMMO rats to a single episode of novel stressor does not induce exaggerated responses in TH gene expression, as some other stressors do. The mechanism of this finding could involve a central regulation and/or adrenomedullary signaling pathway(s), leading to additional modifications or accumulation of transcription factors. The precise mechanism(s) of this phenomenon remains to be elucidated.

Quantitation of changes in gene expression of norepinephrine biosynthetic enzymes in rat stellate ganglia induced by stress.

Enzymes involved in catecholamine synthesis are present in the highest concentration in the adrenal medulla, however they were found also in other, mainly nervous tissues. The aim of our study was to quantify the exact concentration of tyrosine hydroxylase (TH) and dopamine-ss-hydroxylase (DBH) mRNA in rat stellate ganglia under control conditions and at different intervals after exposure to immobilization stress (IMO). In rats immobilized once for 2h, we determined TH and DBH mRNA in different time intervals up to 22 h after the end of the stress stimulus. TH immunoreactive protein levels were also determined in stellate ganglia. TH and DBH mRNA levels were quantified by RT-competitive-PCR. In stellate ganglia, the concentration of TH mRNA was 17+/-1.6 amol/microg of total RNA, which is approximately 30-times lower than in the adrenal medulla. The concentration of DBH mRNA in the stellate ganglia was 2601+/-203 amol/microg of total RNA, which is the concentration similar to adrenal medulla, but is 150-times higher than concentration of TH mRNA in stellate ganglia. After a single 2-h immobilization the highest elevation of TH and DBH mRNA levels was measured 22 h after the termination of the stress stimulus. Repeated immobilization (7 days, 2h daily) did not produce further increase in TH and DBH mRNA levels compared to already elevated levels in adapted control group (immobilized for 6 days, 2h daily and decapitated 22 h later). Levels of TH protein were significantly changed only after the repeated immobilization. This study compared for the first time the precise amounts of TH and DBH mRNA in rat stellate ganglia under control conditions and after immobilization stress, and indicates large differences in their concentration. TH and DBH mRNA concentrations in stellate ganglia are markedly elevated for a prolonged period of time after termination of the stress stimuli.

Stress increases gene expression of phenylethanolamine N-methyltransferase in spleen of rats via pituitary-adrenocortical mechanism.

Gene expression of phenylethanolamine N-methyltransferase (PNMT), the enzyme catalyzing conversion of norepinephrine to epinephrine, has been detected in rat spleen using the reverse transcription polymerase chain reaction. PNMT identity was subsequently verified by Southern blots. Localization of the spleen cells responsible for the PNMT gene expression was investigated by the in situ hybridization and PNMT mRNA was found to be present in the white pulp. The hypothesis that stress may produce an increase in PNMT gene expression in rat spleen was tested and a robust rise in the relative abundance of PNMT mRNA levels was observed after a single or repeated immobilization (about 80%). Adrenalectomy or hypophysectomy completely prevented the immobilization-induced increase in spleen PNMT mRNA levels, suggesting that stress-induced PNMT gene expression in the spleen is regulated predominantly via pituitary-adrenocortical axis. In control animals, however, spleen PNMT was not significantly affected by the ectomies and therefore basal PNMT gene expression might be regulated by different mechanism(s).Thus, PNMT gene expression in the rat spleen is exaggerated by stress stimuli, suggesting its role in physiological regulations.

Identification of tyrosine hydroxylase gene expression in rat spleen.

This study was aimed to identify tyrosine hydroxylase (TH) gene expression in the rat spleen under basal and stress conditions. Using the reverse transcription polymerase chain reaction we did not detect TH mRNA in rat spleen either in control, or immobilized animals. Semi-nested PCR revealed a clear signal, demonstrating that TH mRNA is formed in the spleen, although in low abundance. We also detected both, TH immunoreactive protein and TH activity in the rat spleen that were in higher abundance than expected from the mRNA levels. This study identifies, for the first time, TH gene expression in rat spleen. Since TH protein and activity are present in the spleen in much higher abundance compared to corresponding mRNA, the majority of TH protein is most probably supplied by the sympathetic innervation of spleen.

Endogenous and exogenous ARC in serum withdrawal mediated PC12 cell apoptosis: a new pro-apoptotic role for ARC.

The role of ARC (Apoptosis Repressor with Caspase Recruitment Domain) in PC12 cell serum withdrawal driven apoptosis was studied. A progressive and massive increase in ARC protein occurs during serum withdrawal that correlates with declining survival and processing of caspase-2, previously shown to associate with ARC. This accumulation of ARC occurs in a transcriptional and translational independent manner. Additionally, ARC is localized exclusively in the nucleus of PC12 cells. Furthermore, transfection of PC12 cells with hARC-Flag promotes death and fails to protect the cells from apoptosis by serum withdrawal. Therefore, ARC functions in a pro-apoptotic manner in PC12 cell serum withdrawal induced apoptosis.

Gene expression of catecholamine biosynthetic enzymes following exercise: modulation by age.

Both age and exercise training are associated with tissue specific alterations in the catecholaminergic system. We examined the effect of short-term exercise training on tyrosine hydroxylase and dopamine beta-hydroxylase gene expression in adrenals and specific brain regions with aging. In addition, we examined activator protein-1 and cyclic AMP response element transcription factor binding activity in the adrenal medulla. Male, six- and 24-month-old F-344 rats were exercised by treadmill running for five consecutive days. One group was killed immediately and a second group was killed 2h after the last training session. Exercise significantly elevated tyrosine hydroxylase messenger RNA equally in adrenals of both young and old rats. Training had no effect on dopamine beta-hydroxylase messenger RNA in adrenals of young, but levels were elevated in old rats. Binding activities of both activator protein-1 and cyclic AMP response element binding protein were diminished with age in the adrenal medulla. Exercise training had no significant effect on the binding activity of cyclic AMP response element binding protein in either young or old animals, whereas activator protein-1 binding activity increased equally in young and old animals. Exercise training revealed divergent changes in tyrosine hydroxylase messenger RNA in brain catecholaminergic neurons. In the locus coeruleus and the ventral tegmental areas, training elevated tyrosine hydroxylase messenger RNA levels only in young rats. In the substantia nigra, there was no change in young, but a 45% increase in tyrosine hydroxylase messenger RNA in old rats. In the ventral tegmental area, training increased tyrosine hydroxylase gene expression 80% in young but not in old rats. These results indicate that short-term exercise training increases tyrosine hydroxylase messenger RNA levels in young animals in the adrenals, the locus coeruleus and the ventral tegmental area. The responses for exercise training of aged animals differed from the young in brain noradrenergic and dopaminergic nuclei, especially in the substantia nigra, and to some extent in the locus coeruleus and the ventral tegmental area.

Stress-triggered activation of gene expression in catecholaminergic systems: dynamics of transcriptional events.

Stress triggers important adaptive responses that enable an organism to cope with a changing environment. However, when prolonged or repeated, stress can be extremely harmful. The release of catecholamines is a key initial event in responses to stressors and is followed by an increase in the expression of genes that encode catecholamine-synthesizing enzymes. This process is mediated by transcriptional mechanisms in the adrenal medulla and the locus coeruleus. The persistence of transcriptional activation depends on the duration and repetition of the stress. Recent work has begun to identify the various transcription factors that are associated with brief or intermediate duration of a single or repeated stress. These studies suggest that dynamic interplay is involved in converting the transient increases in the rate of transcription into prolonged (potentially adaptive or maladaptive) changes in gene expression.