Repeated Stress Exaggerates Lipopolysaccharide-Induced Inflammatory Response in the Rat Spleen.

Spleen is an immune organ innervated with sympathetic nerves which together with adrenomedullary system control splenic immune functions. However, the mechanism by which prior stress exposure modulates the immune response induced by immunogenic challenge is not sufficiently clarified. Thus, the aim of this study was to investigate the effect of a single (2 h) and repeated (2 h daily for 7 days) immobilization stress (IMO) on the innate immune response in the spleen induced by lipopolysaccharide (LPS, 100 µg/kg). LPS elevated splenic levels of norepinephrine and epinephrine, while prior IMO prevented this response. LPS did not alter de novo production of catecholamines, however, prior IMO attenuated phenylethanolamine N-methyltransferase gene expression. Particularly repeated IMO exacerbated LPS-induced down-regulation of α1B- and ß1-adrenergic receptors (ARs), while enhanced α2A- and ß2-AR mRNAs. Elevated expression of inflammatory mediators (iNOS2, IL-1ß, IL-6, TNF-α, IL-10) was observed following LPS and repeated IMO again potentiated this effect. These changes were associated with enhanced Ly6C gene expression, a monocyte marker, and elevated MCP-1, GM-CSF, and CXCL1 mRNAs suggesting an increased recruitment of monocytes and neutrophils into the spleen. Additionally, we observed increased Bax/Bcl-1 mRNA ratio together with reduced B cell numbers in rats exposed to repeated IMO and treated with LPS but not in acutely stressed rats. Altogether, these data indicate that repeated stress via changes in CA levels and specific α- and ß-AR subtypes exaggerates the inflammatory response likely by recruiting peripheral monocytes and neutrophils to the spleen, resulting in the induction of apoptosis within this tissue, particularly in B cells. These changes may alter the splenic immune functions with potentially pathological consequences.

Fos expression in tyrosine hydroxylase containing hypothalamic neurons in CRH-KO mice: effect of immobilization stress.

OBJECTIVE: Little is known about the response of tyrosine hydroxylase (TH) containing hypothalamic neurons to stress in corticoliberine deficient (CRH-KO) mice. This study was aimed to extend this issue and reveal the data leading to a better understanding of physiological/anatomical plasticity of hypothalamic TH cells in response to acute immobilization stress (IMO) as well as of possible of CRH body deficiency contribution in the regulation of TH cells during stress. We examined the topographic distribution of TH protein immunolabeled perikarya in selected hypothalamic structures including the paraventricular (PVN), supraoptic (SON), periventricular (PeVN), arcuate (ArcN), dorsomedial (DMN), and ventromedial (VMN) nuclei and extrahypothalamic zona incerta (ZI) in CRH-KO and wild type (WT) mice. METHODS: The animals were perfused with fixative 120 min after a single IMO stress. The brains were removed, cryo-sectioned throughout the hypothalamus and Fos-TH co-localizations were processed immunohistochemically. Fos protein was visualized by diaminobenzidine (DAB) intensified with nickel ammonium sulphate, while TH cells were labeled only with DAB chromogen. The evaluation of Fos-TH co-labeled perikarya was performed with the use of computerized Leica light microscope and expressed as the percentage of total amount of TH labeled cells. RESULTS: From the qualitative point of view, the present data indicate similar anatomical distribution of TH immunoreactive perikarya in all brain structures investigated in both WT and CRH-KO mice, while from the quantitative point of view only TH cells in the DMN of CRH-KO mice showed a trend for increased activation by IMO. CONCLUSIONS: In several hypothalamic structures the basic population of TH neurons was not affected by the absence of endogenous CRH. Based on the data of this study it can also be assumed that despite of the presence of direct reciprocal connections between PVN and DMN neurons, PVN CRH neurons possibly are not participating in the regulation of TH neurons in the DMN during IMO stress. KEYWORDS: Hypothalamic nuclei - Fos-immunohistochemistry - Tyrosine hydroxylase - Immobilization stress - CRH knockout mice.

Animal model of metastatic pheochromocytoma: evaluation by MRI and PET.

OBJECTIVE: The development of metastatic pheochromocytoma animal model provides a unique opportunity to study the physiology of these rare tumors and to evaluate experimental treatments. Here, we describe the use of small animal imaging techniques to detect, localize and characterize metastatic lesions in nude mice. METHODS: Small animal positron emission tomography (PET) imaging and magnetic resonance imaging (MRI) were used to detect metastatic lesions in nude mice following intravenous injection of mouse pheochromocytoma cells. [18F]-6-fluoro-dopamine ([18F]-DA) and [18F]-L-6-fluoro-3,4-dihydroxyphenylalanine, which are commonly used for localization of pheochromocytoma lesions in clinical practice, were selected as radiotracers to monitor metastatic lesions by PET. RESULTS: MRI was able to detect liver lesions as small as 0.5mm in diameter. Small animal PET imaging using [18F]-DA and [18F]-DOPA detected liver, adrenal gland, and ovarian lesions. CONCLUSION: We conclude that MRI is a valuable technique for tumor growth monitoring from very early to late stages of tumor progression and that animal PET confirmed localization of metastatic pheochromocytoma in liver with both radiotracers.

Response of hypothalamic oxytocinergic neurons to immobilization stress is not dependent on the presence of corticotrophin releasing hormone (CRH): a CRH knock-out mouse study.

This study explores the quantitative patterns of immunolabeled Fos protein incidence in the hypothalamic paraventricular (PVN) and supraoptic nuclei (SON) oxytocinergic (OXY) neurons in response to immobilization (IMO) stress in corticotrophin releasing hormone deficient (CRH-KO) mice. Adult male mice, taken directly from cages or 120 min after a single IMO, were sacrificed by intracardial perfusion with fixative. Coronal brain sections of 30 mum thickness were processed for dual Fos/OXY immunohistochemistry. In control wild type (WT) and CRH-KO mice, scattered Fos immunoreactivity was observed in hypothalamus, including the PVN where scanty Fos signal occurred in both parvocellular and magnocellular PVN subdivisions. Dual Fos/OXY immunostainings revealed higher basal Fos expression in the PVN of control CRH-KO mice. IMO evoked a marked rise in Fos expression in OXY neurons of the PVN and SON in both WT and CRH-KO groups of mice. The present data demonstrate that 1/ CRH deficiency upregulates the basal activity of hypothalamic PVN OXY cells in CRH-KO mice and 2/ IMO stress in both WT and CRH-KO mice affects distinctly the activity of OXY cells in both SON and PVN. Our data indicate that CRH deficiency does not alter the responsiveness of PVN and SON OXY cells to IMO stress.

Hormone response to stress in rat strains of different susceptibility to immunologic challenge.

OBJECTIVE: The aim of this study was to compare the changes in plasma levels of hormones involved in modulation of the immune system function after exposure to stress in two rat strains with different susceptibility to immunoantigens. METHODS: Adult rat males of Lewis (LEW) and Fischer 344 (FIS) strains were exposed to restrain stress for 2 hours and blood samples were collected during stress exposure. Other groups of animals were exposed to restrain stress for 2 hours and sacrificed 3 hours later for blood and organ collection. Corticosterone, testosterone, dehydroepiandrosterone, 17beta-estradiol and progesterone were estimated by radioimmunoassay, epinephrine and norepinephrine levels were determined by radioenzymatic method. RESULTS: The levels of plasma corticosterone and catecholamines were significantly higher during stress exposure in FIS as compared to LEW rats. Greater decrease of testosterone levels and higher levels of estradiol were noted after exposure to stress in LEW rats. Higher values of progesterone plasma levels were noted in FIS rats after stress. CONCLUSIONS: These results demonstrated the differences in the response of catecholamines, adrenal and gonadal steroids after exposure to stress in LEW and FIS rats with lower levels of hormones with anti-inflammatory action in LEW rats.

Developmental changes of some G-protein coupled receptors affected by c-fos knock-out.

The role of c-fos gene disruption on binding characteristics of selected G protein-coupled receptors has been investigated. The following receptors were studied: muscarinic receptors (MR), alpha1-adrenoceptors (AAR), beta-adrenoceptors (BAR), D1-like dopamine receptors (D1R), D2-like dopamine receptors (D2R). We have tested the hypothesis that c-fos gene disruption would not influence the receptor density in central nervous system (CNS: brain cortex and cerebellum) and in the periphery (lung, heart). We found that both in the periphery and in the CNS there were important changes in receptor density. Both MR and BAR were increased in the lung and heart. The effects of c-fos gene disruption in CNS were more selective. In general, the receptors that activate Gq-phospholipase C-proteinkinase C pathway (AAR, MR) were affected, while the others (that activate/or inhibit adenylyl cyclase: BAR, D1R, D2R) were not. These results show that disruption of c-fos gene could significantly change the expression of G protein-coupled receptors. Moreover, these changes could be comprehended as one of the adaptive mechanisms that help the organism to cope with c-fos gene disruption.

Hormone concentrations in synovial fluid of patients with rheumatoid arthritis.

OBJECTIVE: Alterations in local concentrations of hormones, affecting directly synovial cells, could be involved in the modulation of the rheumatic inflammatory processes. The aim of present study was to investigate the levels of selected hormones (steroids, peptide and thyroid hormones) in synovial fluid of knee joint of patients with rheumatoid arthritis (RA) and control individuals with non-rheumatic exudate (with osteoarthrosis, OA). METHODS: Thirty-eight patients, 22 female and 16 males, with rheumatoid arthritis (RA) and 12 subjects with osteoarthrosis (OA, control group, 6 females and 6 males) participated in the study. Concentrations of cortisol (CS), 17-beta-estradiol (ES), dehydroepiandrosterone (DHEA), progesterone (PRG), aldosterone ALD), prolactin (PRL), insulin (INS), and C-peptide were determined by radioimmunoassay in synovial fluid. Insulin binding to isolated cell membrane of cells from synovial sediment was estimated by using radioiodine labeled insulin. In a group of patients (10 with RA and 4 with OS), the levels of free threeiodothyronine (FT3), TSH and growth hormone (GH) were also determined in synovial fluid. RESULTS: Increased levels of ES in synovial fluid of RA patients were observed, and higher differences were noted in men. TE concentrations were moderately elevated in synovial fluid of RA patients, however the ratio of ES/TE was significantly higher in male RA compared to OA patients. Higher levels of PRG, ALD and growth hormone were noted in synovial fluid of RA patients. Besides the steroid hormones the presence of insulin and C-peptide was noted in synovial fluid and the correlation between the levels of these two peptides was highly significant. The concentrations of INS and C-peptide in synovial fluid of patients from RA and OA group were not significantly different, however, highly significant increase of insulin binding to isolated membrane of synovial cells was found. Concentrations of cortisol, dehydroepiandosterone, prolactin, TSH and FT3 in synovial fluid were not significantly different in RA and OA groups. CONCLUSIONS: Besides the steroids also insulin, c-peptide, GH and FT3 were found in synovial fluid. The elevated ALD and GH levels in synovial fluid of RA patients and the presence of INS in synovial fluid with increase of INS binding to plasma membranes of cells from synovial fluid of RA patients suggest that besides the gonadal steroids also these hormones may affect the local inflammatory processes.

Low molecular weight proteomic information distinguishes metastatic from benign pheochromocytoma.

Metastatic lesions occur in up to 36% of patients with pheochromocytoma. Currently there is no way to reliably detect or predict which patients are at risk for metastatic pheochromocytoma. Thus, the discovery of biomarkers that could distinguish patients with benign disease from those with metastatic disease would be of great clinical value. Using surface-enhanced laser desorption ionization protein chips combined with high-resolution mass spectrometry, we tested the hypothesis that pheochromocytoma pathologic states can be reflected as biomarker information within the low molecular weight (LMW) region of the serum proteome. LMW protein profiles were generated from the serum of 67 pheochromocytoma patients from four institutions and analyzed by two different bioinformatics approaches employing pattern recognition algorithms to determine if the LMW component of the circulatory proteome contains potentially useful discriminatory information. Both approaches were able to identify combinations of LMW molecules which could distinguish all metastatic from all benign pheochromocytomas in a separate blinded validation set. In conclusion, for this study set low molecular mass biomarker information correlated with pheochromocytoma pathologic state using blinded validation. If confirmed in larger validation studies, efforts to identify the underlying diagnostic molecules by sequencing would be warranted. In the future, measurement of these biomarkers could be potentially used to improve the ability to identify patients with metastatic disease.

Phenylethanolamine N-methyltransferase gene expression in transplanted human heart.

BACKGROUND: Phenylethanolamine N-methyltransferase (PNMT) is an enzyme involved in the epinephrine synthesis. The aim of this study was to investigate PNMT gene expression in the transplanted human heart in relation to the time elapsed from heart transplantation (HTx) and selected clinical characteristics. PATIENTS AND METHODS: The messenger RNA (mRNA) levels of PNMT in myocardial tissue were determined in 22 (21 males) patients at 0-12 years after HTx. Relative quantification of mRNA levels was performed using reverse transcription and polymerase chain reaction (RT-PCR) for correlation with heart rate (HR), blood pressure (BP), parameters of heart rate variability, and graft systolic and diastolic functions. RESULTS: During the first 3 years after HTx, PNMT mRNA levels were higher (mean +/- SEM) (0.75 +/- 0.06; n = 12) compared with later years (0.33 +/- 0.06; n = 10); (P < .01). HR variability in the low frequency band of the power spectrum (LF) was lower among patients during the first 3 years after HTx (2.73 +/- 0.31 vs 5.67 +/- 0.69 ms; P < .01). A significant negative linear correlation was observed between PNMT mRNA and LF (P = .05; r = -0.47). No association was noted between HR, BP, parameters of systolic or diastolic function, standard deviation of all RR intervals, or high frequency (0.15-0.40 Hz) bands of the 24-hour RR interval power spectrum. CONCLUSION: This work provides the first evidence of the presence of local PNMT transcription in human heart after HTx. Appearance of PNMT transcription in the myocardium after HTx may reflect autonomous "sympathicotrophy." Decreased PNMT expression with time elapsed after HTx suggests graft reinervation.

Low levels of dehydroepiandrosterone sulphate in plasma, and reduced sympathoadrenal response to hypoglycaemia in premenopausal women with rheumatoid arthritis.

OBJECTIVES: To evaluate the function of the hypothalamic-pituitary-adrenal axis and sympathoadrenal system in premenopausal women with rheumatoid arthritis (RA). METHODS: Insulin-induced hypoglycaemia (0.1 IU/kg) was produced in 15 glucocorticoid-naive patients with long term RA with low disease activity and in 14 healthy women matched for age and body mass index. Concentrations of glucose, adrenocorticotropic hormone (ACTH), cortisol, Delta4-androstenedione (ASD), dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulphate (DHEAS), 17alpha-hydroxyprogesterone (17OHP), epinephrine (EPI), norepinephrine (NE), interleukin 6 (IL6), and tumour necrosis factor alpha (TNFalpha) were analysed in plasma. RESULTS: Patients had comparable responses of glucose, cortisol, ACTH, ASD, and 17OHP to hypoglycaemia, without any signs of hypothalamic insufficiency. Patients had lower basal DHEAS than controls (3.03 (0.37) micromol/l v 5.1 (0.9) micromol/l, respectively; p<0.05); borderline lower basal DHEA levels (p = 0.067); while the response of DHEA to hypoglycaemia was comparable to that of controls. Patients with RA had lower EPI (p = 0.005) and NE (p<0.001) responses to hypoglycaemia. TNFalpha and IL6 were higher (p<0.05) in patients with RA (TNFalpha 8 (2.8) pg/ml in RA v 1.1 (0.5) pg/ml in controls and IL6 15.1 (6.7) pg/ml v 1.4 (0.7) pg/ml). CONCLUSIONS: Lower basal DHEAS levels, without concomitant differences or changes in DHEA, ASD, 17OHP, and cortisol responses to hypoglycaemia in patients with RA, indicate an isolated decrease in adrenal androgen production. Significantly lower responses of EPI and NE to hypoglycaemia may suggest sympathoadrenal hyporeactivity in patients with RA.

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.

Stress- as well as suckling-induced prolactin release is blocked by a structural analogue of the putative hypophysiotrophic prolactin-releasing factor, salsolinol.

Prolactin is secreted from the anterior lobe of the pituitary gland in response both to suckling and to stress. We recently observed that 1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (salsolinol), produced in the neurointermediate lobe of the pituitary gland, as well as in the medial basal hypothalamus, can selectively release prolactin from the anterior pituitary. Therefore, it has been proposed that salsolinol is a putative endogenous prolactin-releasing factor (PRF). Here, we report that one structural analogue of salsolinol, 1-methyl-3,4-dihydroisoquinoline (1MeDIQ), can block salsolinol-induced release of prolactin, but does not affect prolactin release in response to thyrotropin releasing hormone (TRH), alpha-methyl-p-tyrosine (alpha MpT) (an inhibitor of tyrosine hydroxylase), domperidone (a D(2) dopamine receptor antagonist), or 5-hydroxytryptophan (5-HTP), a precursor of serotonin). 1MeDIQ profoundly inhibited suckling-, immobilization-, as well as formalin-stress induced prolactin release without any influence on corticosterone secretion. The 1MeDIQ-induced reduction in prolactin response to immobilization stress was dose-dependent. These results suggest that salsolinol can play a pivotal role in the regulation of prolactin release induced by either physiological (suckling) or environmental (stress) stimuli.

[Use of knock-out mice in studies of stress reactions]. / Vyuzitie "knock-out" mysí pri stúdiu stresovej reakcie organizmu.

Modern human population intimately recognizes the stress reaction of the organism, which has developed as a response to variety physical and/or psychical stressors. This reaction represents not only a complex of adaptive mechanisms enabling the successful overcoming of the conflicting situation, but in its pathological form it can significantly contribute to the development of serious diseases, from carcinogenic, cardiovascular, gastrointestinal, up to the psychical. New, developing scientific disciplines permit to widen knowledge of the basis of this undesirable side of the stress. Transgenic animals, which either has integrated the gene of interest, or studied gene is not functioning (i.e. knockout animals), permit at least partially to study the physiological importance of the product of this gene in the organism. In our review we described in detail three lines of knockout mice--with the knockout gene for the dopamine-beta-hydroxylase, corticoliberin and c-fos--that significantly contribute to the regulation of the stress response.

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.

Altered function of peripheral organ systems in rats exposed to chronic mild stress model of depression.

1. In depression, psychiatric symptoms are frequently associated with impaired cardiovascular function and perhaps also increased risk for cancer diseases. Pathophysiological basis of this comorbidity is not clearly understood. Molecular events involved, particularly factors modified by chronic stress exposure, may only be evaluated in animal models of depression. 2. Present experiments were aimed to study parameters related to cardiovascular system (tyrosine hydroxylase (TH) gene expression in adrenal glands) and carcinogenesis (retinoic acid receptors in the liver) in the chronic mild stress model of depression. 3. Chronic mild stress induced a rise in adrenal TH gene expression in both male and female rats. Gender dependent changes were found in retinoic acid receptor binding with stress-induced activation in females but not males. Ovariectomized animals exhibited higher retinoic acid receptor binding. slightly elevated TH mRNA levels and failed to respond to chronic mild stress exposure with further increase in TH mRNA levels. Similarly, chronic mild stress induced an anhedonic state manifested by decreased sucrose preference in control but not ovariectomized rats. 4. Presented data document that central neurochemical and behavioral changes in animals exposed to chronic mild stress model of depression are associated with changes in adrenal TH gene expression and with gender dependent changes in retinoic acid receptor status in the liver. Such alterations may participate in the development of pathological changes and could participate on increased risk for cardiovascular and oncologic comorbidity in depressive patients.

Changes in plasma catecholamine and corticosterone levels and gene expression of key enzymes of catecholamine biosynthesis in partially hepatectomized rats.

OBJECTIVE: To reexamine the possible role of catecholamines and corticosterone in the early period of liver regeneration after partial hepatectomy (PH)in conscious cannulated rats under carefully controlled conditions which would allow to obtain reliable information about sympathetic-adrenomedullary function after PH in the rat in vivo. METHODS: Plasma levels catecholamines (epinephrine - EPI, norepinephrine - NE) were estimated by radioenzymatic assay and these of corticosterone by competitive protein binding assay. The total RNA was isolated from the adrenals and tyrosine hydroxylase (TH) mRNA expression was estimated by hybridisation with cDNA after Northern blot. The level of immunoreactive protein was measured by using a monoclonal antibody to rat TH, visualized by Western light chemiluminescent detection system and analyzed by densitometry. The level of TH in adrenals was estimated with the aid of 3H-tyrosine and TH cofactor DL-6-methyl-5,6,7,8-tetrahydropterine and the formed radioactive water was measured by scintillation spectometry. RESULTS: The plasma level of norepinephrine (NE), epinephrine (EPI) and corticosterone rapidly increased 20 min. after PH or sham operation (laparotomy). Although the increase of plasma NE was about the same after both PH and laparotomy, that of EPI and corticosterone in PH rats was significantly higher as compared to the laparotomy. One hour after the surgery plasma NE levels in both groups decreased to the basal value and remained still unchanged 4 and 24 h later. At the interval of 4h the plasma level of EPI was higher than in laparotomized controls, but after 24 h the EPI levels returned to basal values. Adrenal tyrosine hydroxylase (TH) mRNA level was significantly elevated in both PH and laparotomized rats, however 24 h after the surgery they returned to the baseline. Adrenal TH immunoprotein levels and TH activity were significantly elevated in both groups 4 h after the surgery, while 24 h later they returned to the baseline in laparotomized rats bur remained elevated in PH rats. Adrenal phenylethanolamine N-methyl-transferase (PNMT) mRNA levels were increased 4 h after both the PH and laparotomy and declined within 24 h. CONCLUSIONS: The first peak of catecholamine and corticosterone levels might result from unspecific stressor associated with the surgery. These levels could be accompanied with the mechanism of the rat liver regeneration. Prolonged elevations of EPI found after PH seems to be specific for liver regeneration indicating that the rise in the adrenal TH mRNA appears to be translated into immunoreactive protein which further leads to the elevation of TH activity. These results contrast markedly with previous studies indicating that the regeneration is modulated predominantly by norepinephrine.

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 increases brain-derived neurotropic factor messenger ribonucleic acid in the hypothalamus and pituitary.

Brain-derived neurotropic factor (BDNF) is a member of the nerve growth factor family that is important for neuronal survival and plasticity. We recently demonstrated that stress decreases BDNF messenger RNA (mRNA) levels in the hippocampus, which raises the possibility that BDNF may play a role in regulation of the hypothalamic-pituitary-adrenal axis. The purpose of this study was to determine whether BDNF expression is present and influenced by stress in other brain areas relevant to control of the hypothalamic-pituitary-adrenal axis. Using in situ hybridization, we found that BDNF mRNA is present in the parvocellular portion of the hypothalamic paraventricular nucleus (PVN), the lateral hypothalamus, and the anterior and neurointermediate lobes of the pituitary in rats. Acute (2-h) or repeated immobilization stress increased BDNF mRNA in all of these areas. This was in distinct contrast to stress-induced decreases in extrahypothalamic areas, including the basolateral amygdala, claustrum, and cingulate cortex as well as the hippocampus. BDNF was expressed in both CRF and TRH neurons in the PVN. Reducing glucocorticoid or thyroid negative feedback increased BDNF mRNA in the PVN and anterior pituitary, but not in the neurointermediate lobe. These results suggest that BDNF is a stress-responsive intercellular messenger that may be an important component of the stress response.