Richard Kvetňanský

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.

Effects of Stress on Neurotrophic Factor Expression in the Rat Brain

Changes in neurotrophic factor expression in the brain are part of the stress response. Decreased BDNF may contribute to hippocampal damage that occurs during chronic stress or aging. Stress-induced increases in NT-3 may be important for neural plasticity and adaptation or sensitization to repeated stress. Stress-induced changes in neurotrophic factors may be particularly relevant to the cognitive changes that occur in recurrent depression, aging, and posttraumatic stress disorder.

Sympathoadrenal System in Stress

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.

Chronic Stress, Combined with a High‐Fat/High‐Sugar Diet, Shifts Sympathetic Signaling toward Neuropeptide Y and Leads to Obesity and the Metabolic Syndrome

In response to stress, some people lose while others gain weight. This is believed to be due to either increased β‐adrenergic activation, the bodys main fat‐burning mechanism, or increased intake of sugar‐ and fat‐rich “comfort foods.” A high‐fat, high‐sugar (HFS) diet alone, however, cannot account for the epidemic of obesity, and chronic stress alone tends to lower adiposity in mice. Here we discuss how chronic stress, when combined with an HFS diet, leads to abdominal obesity by releasing a sympathetic neurotransmitter, neuropeptide Y (NPY), directly into the adipose tissue. In vitro, when “stressed” with dexamethasone, sympathetic neurons shift toward expressing more NPY, which stimulates endothelial cell (angiogenesis) and preadipocyte proliferation, differentiation, and lipid‐filling (adipogenesis) by activating the same NPY‐Y2 receptors (Y2Rs). In vivo, chronic stress, consisting of cold water or aggression in HFS‐fed mice, stimulates the release of NPY and the expression of Y2Rs in visceral fat, increasing its growth by 50% in 2 weeks. After 3 months, this results in metabolic syndrome‐like symptoms with abdominal obesity, inflammation, hyperlipidemia, hyperinsulinemia, glucose intolerance, hepatic steatosis, and hypertension. Remarkably, local intra‐fat Y2R inhibition pharmacologically or via adenoviral Y2R knock‐down reverses or prevents fat accumulation and metabolic complications. These studies demonstrated for the first time that chronic stress, via the NPY‐Y2R pathway, amplifies and accelerates diet‐induced obesity and the metabolic syndrome. Our findings also suggest the use of local administration of Y2R antagonists for treatment of obesity and NPY‐Y2 agonists for fat augmentation in other clinical applications.

Catecholamines in Individual Hypothalamic Nuclei of Acutely and Repeatedly Stressed Rats

Norepinephrine (NE) and dopamine (DA) concentrations in 17 individual hypothalamic nuclei and 3 other brain regions were measured in rats, acutely or repeatedly stressed by immobilization, using a microdissection technique and a radioisotopic-enzymatic assay. Following the first 20 min immobilization (IMO) a significant NE decrease in the ventromedial (NVM) and supraoptic (NSO) nuclei and a DA decrease in the arcuate nucleus (NA) as well as NE and DA increase in the dorsomedial nucleus (NDM) were seen. Repeated IMO (40 times) produced a NE increase in the NVM, NDM, NSO paraventricular nucleus (NPV) and median eminence (ME), and a DA increase in the NDM and NPV. Changes of NE and DA concentration found in some individual hypothalamic nuclei under the influence of stress indicate that catecholamines (CAs), particularly in the medial basal hypothalamus, could be involved in the regulation of some neuroendocrine processes which are being activated during stress, especially ACTH release.

ADRENERGIC RESPONSES TO STRESS: TRANSCRIPTIONAL AND POST-TRANSCRIPTIONAL CHANGES

Stress effects on adrenergic responses in rats were examined in adrenal medulla, the primary source of circulating epinephrine (Epi). Irrespective of duration, immobilization (IMMO) increased adrenal corticosterone to the same extent. In contrast, Epi changed little, suggesting that Epi synthesis replenishes adrenal pools and sustains circulating levels for the heightened alertness and physiological changes required of the “flight or fight” response. IMMO also induced the Epi‐synthesizing enzyme, phenylethanolamine N‐methyltransferase (PNMT). The rise in its mRNA and protein was preceded by increases in Egr‐1 and Sp1 mRNA, protein, and protein‐DNA binding complex formation. With repeated and prolonged stress, PNMT protein did not reflect the magnitude of change in mRNA. The latter suggests that post‐transcriptional, in addition to transcriptional mechanisms, regulate PNMT responses to stress. To further reveal molecular mechanisms underlying stress‐induced changes in adrenergic function, the effects of hypoxia on PNMT promoter‐driven gene expression are being examined in adrenal medulla‐derived PC12 cells. Hypoxia activates the PNMT promoter to increase PNMT promoter‐driven luciferase reporter gene expression and endogenous PNMT in PC12 cells. Induction of both appear mediated via activation of multiple signaling pathways and downstream activation of hypoxia inducible factor and PNMT transcriptional activators, Egr‐1 and Sp1. Hypoxia generates both partially and fully processed forms of PNMT mRNA. The former reportedly is translated into a truncated, nonfunctional protein, and the latter into enzymatically active PNMT. Together, findings suggest that stress increases PNMT gene transcriptional activity but post‐transcriptional regulatory mechanisms limit the biological end‐point of functional PNMT enzyme and, thereby, Epi.

Stress-Induced Norepinephrine Release in the Paraventricular Nucleus of Rats with Brainstem Hemisections: A Microdialysis Study

Immobilization (IMMO) of conscious rats evokes marked increases in release of norepinephrine (NE) in the paraventricular nucleus (PVN) of the hypothalamus, consistent with a role of NE in the PVN release of corticotropin-releasing hormone and therefore in pituitary-adrenocortical activation during stress. The present study examined the effects of surgical hemisection of the brainstem between the locus ceruleus and rostral portion of the medulla on release of NE in the PVN of the hypothalamus in vivo in conscious rats, at baseline and during IMMO. Concentrations of NE, the intraneuronal NE metabolite dihydroxyphenylglycol (DHPG), and the dopamine metabolite dihydroxyphenylacetic acid (DOPAC) were measured in microdialysate samples obtained beginning 24 h after implantation of a microdialysis probe in the PVN either ipsilateral or contralateral to the hemisection. On the lesioned side, baseline levels of NE, DHPG, and DOPAC were significantly lower and IMMO-induced increases were smaller than in sham-operated rats. Contralateral to the hemisection, DOPAC levels were significantly reduced. Neither baseline levels nor IMMO-induced increases in plasma corticosterone levels differed between lesioned and sham-operated animals. The present results indicate that: (1) NE release in the PVN at baseline and during IMMO depends mainly on ascending medullary tracts from ipsilateral brainstem A1 and A2 catecholaminergic areas, with small contributions from the locus ceruleus and from contralateral medullary cells, and (2) brainstem hemisection does not influence IMMO-induced activation of the hypothalamic-pituitary-adrenocortical axis as indicated by plasma corticosterone levels in conscious rats.

Tyrosine hydroxylase mRNA levels in locus ceruleus of rats during adaptation to long-term immobilization stress exposure

The major central norepinephrinergic nucleus, locus ceruleus (LC), is thought to participate in modulation of such brain areas as cerebral cortex, septum, hippocampus, thalamus, hypothalamus, and cerebellum in animals facing various physiological challenges, including stress. Exposure of experimental animals to different stressors causes an increase in LC activity and gene expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis. The aim of this work was to investigate the effect of a single and repeated (7 times) or long-term repeated (42 times) daily immobilization stress (IMMO) on TH mRNA levels in LC of laboratory rats by in situ hybridization method. A single IMMO caused significant elevation of LC TH mRNA levels in comparison to unstressed controls. This was found immediately and at 3 and 6 h after IMMO, and progressively increased up to 24 h after the first IMMO terminated. Further exposure to IMMO did not cause additional increases in LC TH mRNA levels, which stayed significantly elevated in comparison to unstressed rats. In animals that underwent IMMO for 42 times, the LC TH gene expression, 24 h after the last stress exposure, was significantly lower when compared to that of singly or seven times stressed rats. Thus, our results indicate a possible adaptation of catecholamine-synthesizing system at the level of TH gene expression in LC of rats exposed to long-term repeated IMMO.

Changes of plasma and adrenal catecholamines and corticosterone in stressed rats with septal lesions

The effect of septal lesions on plasma catecholamine and corticosterone (B) levels has been studied in rats during single and 7 times repeated immobilization stress (IMO). Blood samples were obtained via a catheter in the tail artery or by decapitation. The increased circulating epinephrine (EPI) and norepinephrine (NE) levels observed in the initial phase of acute stress as well as the elevated baseline EPI level after six times repeated IMO are indicative of an enhanced response of the sympathetic adrenomedullary system after lesions of the septum. After decapitation of rats with septal lesions there was a significant increase in plasma NE one day after the sixth IMO and a block of EPI increase after the seventh IMO compared to sham-operated rats. The adrenocortical system was similarly found to be activated after septal lesions, exhibiting increased baseline plasma B levels. It has been suggested that the septal region affects the studied systems by exerting an inhibitory tonus. The removal of this inhibitory system results in an increase of adrenocortical and sympathetic-adrenomedullary activities.

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

Abstract: 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.