Immobilization Research Articles

Acute Stress Differently Modulates Beta 1, Beta 2 and Beta 3 Adrenoceptors in T Cells, but Not in B Cells, from the Rat Spleen

Objectives: Stress-induced rise in circulating catecholamines (CAs), followed by modulation of β-adrenergic receptors (adrenoceptors, ARs), is one of the pathways involved in the stress-mediated effects of immune functions. The spleen is an organ with a high number of lymphocytes and provides a unique microenvironment in which they reside. Thus, lymphocytes may respond differently to CAs in the spleen than in the circulation. No reports exist concerning the involvement of β-ARs in stress-mediated effects on T and B cells isolated from the spleen. Therefore, our aim was to investigate the effect of single stress exposure on gene expression and cellular localization of β-adrenoceptor subtypes in splenic T and B cells. We tried to correlate changes in adrenoceptors with the expression of apoptotic proteins. Methods: Immobilization (IMMO) was used as a stress model. T and B cells were isolated from rat spleen using magnetically labeled antibodies. The gene expression of individual adrenoceptors and apoptotic proteins was evaluated by real-time PCR. Immunofluorescence was used to evaluate localization and adrenoceptor expression. Results: We have found T cells to be more vulnerable to stress compared to B cells, because of increased β₁-, β₂- and β₃-ARs after a single IMMO. Moreover, β₂-ARs translocated from the nucleus to the plasma membrane in T cells after IMMO. The rise in β-ARs most probably led to the rise of Bax mRNA and Bax to Bcl-2 mRNA ratio. This might suggest the induction of an apoptotic process in T cells. Conclusion: Higher susceptibility of T cells to stress via modulation of β-ARs and apoptotic proteins might shift the immune responsiveness in the spleen.

The characteristics of supramammillary cells projecting to the hippocampus in stress response in the rat.

The hypothalamus-pituitary-adrenocortex (HPA) axis is the central mediator of the stress response. The supramammillary (SuM) region is relatively unique among the hypothalamic structures in that it sends a large, direct projection to the hippocampal formation. It has been shown that mild stress could activate the SuM cells that project to the hippocampus. However, the role of these cell populations in modulating the stress response is not known. The present study examined the effect of stress on different populations of SuM cells that project to the hippocampus by injecting the fluorescent retrograde tracer, fluorogold (FG), into the hippocampus and utilizing the immunohistochemistry of choline acetyltransferase (ChAT), corticotrophin releasing factor (CRF), serotonin (5-HT), glutamate decarboxylase (GAD), tyrosine hydroxylase (TH) and NADPH-d reactivity. Immobilization (IMO) stress (2 hr) produced an increase in the expression of ChAT-immunoreactivity, and tended to increase in CRF, 5-HT, GAD, TH-immunoreactivity and nitric oxide (NO)-reactivity in the SuM cells. Fifty-three percent of 5-HT, 31% of ChAT and 56% of CRF cells were double stained with retrograde cells from the hippocampus. By contrast, a few retrogradely labeled cells projecting to the hippocampus were immunoreactive for dopamine, γ-aminobutyric acid (GABA) and NO. These results suggest that the SuM region contains distinct cell populations that differentially respond to stress. In addition, the findings suggest that serotonergic, cholinergic and corticotropin releasing cells projecting to the hippocampus within the SuM nucleus may play an important role in modulating stress-related behaviors.

Epinephrine: a short- and long-term regulator of stress and development of illness : a potential new role for epinephrine in stress.

Epinephrine (Epi), which initiates short-term responses to cope with stress, is, in part, stress-regulated via genetic control of its biosynthetic enzyme, phenylethanolamine N-methyltransferase (PNMT). In rats, immobilization (IMMO) stress activates the PNMT gene in the adrenal medulla via Egr-1 and Sp1 induction. Yet, elevated Epi induced by acute and chronic stress is associated with stress induced, chronic illnesses of cardiovascular, immune, cancerous, and behavioral etiologies. Major sources of Epi include the adrenal medulla and brainstem. Although catecholamines do not cross the blood-brain barrier, circulating Epi from the adrenal medulla may communicate with the central nervous system and stress circuitry by activating vagal nerve β-adrenergic receptors to release norepinephrine, which could then stimulate release of the same from the nucleus tractus solitarius and locus coeruleus. In turn, the basal lateral amygdala (BLA) may activate to stimulate afferents to the hypothalamus, neocortex, hippocampus, caudate nucleus, and other brain regions sequentially. Recently, we have shown that repeated IMMO or force swim stress may evoke stress resiliency, as suggested by changes in expression and extinction of fear memory in the fear-potentiated startle paradigm. However, concomitant adrenergic changes seem stressor dependent. Present studies aim to identify stressful conditions that elicit stress resiliency versus stress sensitivity, with the goal of developing a model to investigate the potential role of Epi in stress-associated illness. If chronic Epi over expression does elicit illness, possibilities for alternative therapeutics exist through regulating stress-induced Epi expression, adrenergic receptor function and/or corticosteroid effects on Epi, adrenergic receptors and the stress axis.

Active coping with stress suppresses glucose metabolism in the rat hypothalamus

We used 18F-fluorodeoxyglucose small-animal positron-emission tomography to determine whether different styles of coping with stress are associated with different patterns of neuronal activity in the hypothalamus. Adult rats were subjected to immobilization (IMO)-stress or to a non-immobilized condition for 30 min, in random order on separate days, each of which was followed by brain-scanning. Some rats in the immobilized condition were allowed to actively cope with the stress by chewing a wooden stick during IMO, while the other immobilized rats were given nothing to chew on. Voxel-based statistical analysis of the brain imaging data shows that chewing counteracted the stress-induced increased glucose uptake in the hypothalamus to the level of the non-immobilized condition. Region-of-interest analysis of the glucose uptake values further showed that chewing significantly suppressed stress-induced increased glucose uptake in the paraventricular hypothalamic nucleus and the anterior hypothalamic area but not in the lateral hypothalamus. Together with the finding that the mean plasma corticosterone concentration at the termination of the IMO was also significantly suppressed when rats had an opportunity to chew a wooden stick, our results showed that active coping by chewing inhibited the activation of the hypothalamic–pituitary–adrenal axis to reduce the endocrine stress response.

Stress-induced alternations in CuZnSOD and MnSOD activity in cellular compartments of rat liver

Exposure to different stressors initiates generation of reactive oxygen species (ROS), which create harmful environment for cellular macromolecules. Superoxide dismutases (SODs) represent the first line of antioxidant defense. Hence, any alternation in their function might be potentially damaging. To better define the role of SODs, we investigated the CuZnSOD activity in cytosolic and the nuclear fraction as well as mitochondrial MnSOD activity in the liver of Wistar male rats after exposure to 2 h of acute immobilization (IM) or cold (4°C) stress, 21 days of chronic social isolation (IS) or their combination (chronic stress followed by acute stress). Serum corticosterone (CORT) was monitored as an indicator of the stress response. Acute IM stress, with elevated CORT level, led to increased hepatic CuZnSOD activity in the nuclear fraction. Chronic isolation stress, where CORT was close to control value, did not change the CuZnSOD activity either in nuclei or in cytosolic fraction, while combined stress IS+Cold led to increased cytosolic CuZnSOD activity. MnSOD activity in mitochondrial fraction was decreased in all treated groups. Data have shown that different stressors have diverse effect on hepatic CuZnSOD and MnSOD activity as well as on serum CORT level. Increased nuclear CuZnSOD activity after acute stress represents physiological response since the named activity protects cells against oxidative stress, while chronic IS stress compromises CuZnSOD function, suggesting an inefficient defense against ROS. Observed decrease of MnSOD activities indicate inadequate elimination of ROS after acute or chronic stress, which is characteristic of the oxidative stress.

Skeletal muscle deficits following spinal cord injury in a new rat model

Spontaneous recovery in contusion spinal cord injury (SCI) animal models is a limitation in preclinical rehabilitation studies. The objective of this study was to test a new cast immobilization (IMM) rat model of SCI, in which IMM minimizes muscle activation and loading of hindlimbs. Sprague Dawley female rats (16-week old) were randomly assigned into three groups: 1) controls, 2) SCI and 3) SCI plus cast immobilization (SCI+IMM). Severe contusions were performed at the T8 level by a NYU impactor (10g, 50mm weight drop). For SCI+IMM, joints of the lower body were immobilized at resting angles with casting tape after 1 week of SCI. Following 3 weeks of intervention, muscle mass and contractile properties were assessed. Muscle mass was reduced by 34% in soleus, 22% in gastrocnemius, 16% tibialis anterior and 13% in extensor digitorum longus in SCI. IMM induced an additional atrophy of 15% to 20% in both anterior and posterior compartments of hindlimb muscles. Maximal tetanic force was reduced by 30% in SCI and 50% in SCI+IMM. However, specific force was not different. Muscles in SCI+IMM showed shorter time to peak tension and half-relaxation time compared to SCI and controls. These preliminary results indicate that unloading of the muscles after SCI+IMM leads to profound muscle atrophy and loss of force capabilities of the hindlimb muscles without spontaneous recovery. NICHD PO1 HD059751-01A1 supports this work.

Repeated exposure to immobilization or two different footshock intensities reveals differential adaptation of the hypothalamic–pituitary–adrenal axis

Factors involved in adaptation to repeated stress are not well-characterized. For instance, acute footshock (FS) of high intensity appears to be less severe than immobilization (IMO) in light of the speed of post-stress recovery of the hypothalamic-pituitary-adrenal (HPA) axis and other physiological variables. However, repeated exposure to IMO consistently resulted in reduction of the HPA response to the same stressor (adaptation), whereas failure to adapt has been usually reported after FS. Thus, in the present work we directly compared the activation of HPA axis and other physiological changes in response to both acute and repeated exposure to IMO and two intensities of FS (medium and high) in adult male rats. Control rats were exposed to the FS boxes but they did not receive shocks. Daily repeated exposure to IMO resulted in significant adaptation of the overall ACTH and corticosterone responses to the stressor. Such a reduction was also observed with repeated exposure to FS boxes and FS-medium, whereas repeated exposure to FS-high only resulted in a small reduction of the corticosterone response during the post-stress period. This suggests that some properties of FS-high make adaptation to it difficult. Interestingly, overall changes in food intake and body weight gain throughout the week of exposure to the stressors reveal a greater impact of IMO than FS-high, indicating that factors other than the intensity of a stressor, at least when evaluated in function of the above physiological variables, can influence HPA adaptation. Since FS exposure is likely to cause more pain than IMO, activation of nociceptive signals above a certain level may negatively affect HPA adaptation to repeated stressors.

Immobilization of poligalacturonase on pm-dvb beads for oenological applications

Immobilization of poligalacturonase on pm-dvb beads for oenological applications

Stress and adrenergic function: HIF1α, a potential regulatory switch.

Stress elicits adrenal epinephrine and cortisol release into the bloodstream to initiate physiological and behavioral responses to counter and overcome stress, the classic "fight or flight" response (Cannon and De La Paz, Am J Physiol 28:64-70, 1911). Stress and the stress hormone epinephrine also contribute to the pathophysiology of illness, e.g., behavioral disorders, cardiovascular disease, and immune dysfunction. Epinephrine itself is regulated by stress through its biosynthesis by phenylethanolamine N-methyltransferase (PNMT, EC 2.1.1.28). Single and repeated immobilization (IMMO) stress in rats stimulates adrenal PNMT mRNA and protein expression via the transcription factors, Egr-1 and Sp1. Moderate hypoxic stress increases PNMT promoter-driven gene expression and endogenous PNMT mRNA and protein in PC12 cells. Induction is initiated through cAMP and PLC signaling, with PKA, PKC, PI3K, ERK1/2 MAPK, and p38 MAPK continuing downstream signal transduction, followed by activation of HIF1α, Egr-1, and Sp1. While functional Egr-1 and Sp1 binding sites exist within the proximal PNMT promoter, a putative hypoxia response element is a weak HIF binding site. Yet, HIF1α overexpression increases PNMT promoter-driven luciferase activity and endogenous PNMT. When the Egr-1 or Sp1 sites are mutated, HIF1α does not stimulate the PNMT promoter. siRNA knock down of Egr-1 or Sp1 prevents promoter activation while siRNA knock down of HIF1α inhibits Egr-1 and Sp1 induction. Findings suggest that hypoxia activates the PNMT gene indirectly via HIF1α stimulation of Egr-1 and Sp1. Thus, for stress-induced illnesses where adrenergic dysfunction is implicated, HIF1α may be an "on-off" switch regulating adrenergic responses to stress and a potential target for therapeutic intervention.

Noninvasive approach to the assessment of activity of the hypothalamic-pituitary adrenal system of the Amur tigers

The state of animals in natural populations can be often assessed only by noninvasive methods. The use of EIA antibodies against corticosterone makes it pos� sible to reveal an increase in the concentration of immunoreactive compounds that bind to antibodies against corticosterone in faeces of tigers both after artificial stimulation of adrenal glands by injection of adrenocorticotropic hormone and after stress (trans� portation of animals). This confirms the possibility of using antibodies against corticosterone for noninva� sive monitoring of the state of the hypothalamicpitu� itaryadrenal system (HPAS) in Amur tigers. The Amur tiger ( Panthera tigris altaica) is a subspe� cies inhabiting the periphery of the species range under conditions that are suboptimal for this species. In this case, exposure to adverse factors with a higher probability may cause an increase in the activity of the hypothalamicpituitaryadrenal system (HPAS), which may suppress breeding and even result in ani� mal's death. Rehabilitation of tigers withdrawn from nature and their subsequent return to natural environ� ment becomes a relevant method to conserve this spe� cies. However, these operations are associated with transportation of animals, which is a strong stressor. With allowance for the fact that the abundance of the Amur tiger is very low (approximately 500 animals (1)), establishing the factors that endanger this species and determination of the effect of transportation on the stress level of animals are very important for con� servation of Amur tigers and development of rehabili� tation programs for these animals. The welfare of animals is estimated primarily by the HPAS activity, because adverse factors stimulate it and cause an increase in the level of glucocorticoids in blood plasma. Changes in the concentration of gluco� corticoids (first of all, cortisol and corticosterone) in blood plasma compared to their baseline level can serve as a reliable indicator of animal's state. At the same time, this method has serious limitations: stress procedures, such as capture, handling, and immobili� zation of animals as such often drastically increase the level of glucocorticoids in blood plasma of animals and leads to a bias in results, because in this case only the response to manipulations is recorded (2).

Restraint Stress Induces Connexin-43 Translocation via α-Adrenoceptors in Rat Heart

Immobilization (IMO) confers emotional stress in animals and humans. It was recently reported that IMO in rats induced translocation of connexin-43 (Cx43) to gap junctions (GJs) and attenuated arrhythmogenesis with GJ inhibition, and Cx43 translocation in the ischemic heart was also shown. Few reports show the contribution of adrenoceptors to Cx43 upregulation in cardiomyocytes, but the involvement of adrenoceptors and ischemia in Cx43 translocation in IMO remains elusive. Male Sprague-Dawley rats underwent IMO and the ventricular distribution of Cx43 was examined by western blotting. IMO induced translocation of Cx43 to the GJ-enriched membrane fraction, with a peak at 60min. The IMO-induced Cx43 translocation was inhibited by pretreatment with the α(1)-adrenoceptor blockers, prazosin (1mg/kg, PO) and bunazosin (4mg/kg, PO), but not with either the β(1)-blocker, metoprolol (10mg/kg, IP), or the β(1+2)-blocker, propranolol (1mg/kg, PO). The translocation was inhibited by the nitric oxide, donor isosorbide dinitrate (100µg·kg(-1)·min(-1), IV), possibly through sympathetic inhibition. Hypoxia inducible factor-1α was not redistributed by IMO. The β-blockers, but not the α-blockers, inhibited the premature ventricular contractions (PVCs) induced by IMO. Translocation of Cx43 to the GJ-enriched fraction occurs via the α(1)-adrenoceptor pathway, independently of ischemia. The β-adrenoceptor pathway contributes to the inducing of PVCs in IMO.

Connexin-43 Redistribution and Gap Junction Activation During Forced Restraint Protects Against Sudden Arrhythmic Death in Rats

Connexin-43 (Cx43) expression is reduced or redistributed in heart disease. Restraint or other emotional stressors might cause sudden death in persons with such diseases, but the mechanism of death and its connection to Cx43 during restraint remain unknown. Whether Cx43 distribution or gap junction (GJ) function during restraint is involved in sudden arrhythmic death in rats is addressed in this study. Male Sprague-Dawley rats underwent immobilization (IMO), and individual electrocardiographic responses were monitored by telemetry. Heart sections were used to examine ventricular Cx43 distribution, and GJ intercellular communication (GJIC) activity was assessed using a dye-transfer assay. IMO induced the translocation of Cx43 into to the GJ-rich fraction, with a peak at 60 min. During IMO, Cx43 immunofluorescence was enhanced at intercalated discs, in association with GJIC activation, and premature ventricular contractions (PVCs) increased. In the presence of the GJ inhibitor, carbenoxolone (0.25 mg.kg(-1).h(-1)), IMO induced lethal ventricular tachycardia or fibrillation in 21.7% of rats, in association with QRS prolongation and increased PVCs. IMO causes Cx43 translocation to intercalated discs, thereby reducing vulnerability to lethal arrhythmias via enhancing GJ coupling.

Acute and/or chronic stress models modulate CuZnSOD and MnSOD protein expression in rat liver

Cellular protection against oxidative stress is afforded by the enzyme superoxide dismutase (SOD). In this study, the protein levels of copper-zinc SOD (CuZnSOD) in the cytosolic and nuclear fraction, manganese SOD (MnSOD) in the mitochondrial, and cytosolic fraction and cytochrome c (cyt c) in the liver of male rats exposed to 2 h of acute immobilization (IM) or Cold stress, 21 days chronic isolation or their combinations (chronic/acute stress) were examined. The serum corticosterone (CORT) level was measured, as an indicator of stress stimuli. Both acute stressors with elevated CORT levels caused a decrease of mitochondrial MnSOD, while acute IM resulted in redistribution of the CuZnSOD protein level between the cytosolic and nuclear fraction. Chronic isolation, during which the CORT level was close to control value, resulted in an increase of cytosolic CuZnSOD, whereas a decrease of MnSOD in mitochondrial and its corresponding increase in cytosol fraction was found. In both combined stress regimes, an increase of the CuZnSOD and MnSOD levels in the cytosolic fraction was recorded whereby increase of the CORT level was observed only in the chronic isolation followed by acute IM. The data indicate that acute and/or chronic stress models have different degrees of influence on serum CORT and SOD subcellular protein levels. Increased cytosolic CuZnSOD protein level under chronic isolation suggests that state of oxidative stress may also exist under CORT level similar to the basal value. The presence of MnSOD and cyt c in the cytosolic fraction could serve as useful parameters for mitochondrial dysfunction.

Immobilization stress elevates intron-containing transcripts for tyrosine hydroxylase in rat superior cervical ganglia indicating transcriptional activation

While both the adrenal medulla and sympathetic nervous system are important in mediating the catecholaminergic response to stress, there are crucial differences in the mechanism. Stress elevates tyrosine hydroxylase (TH) protein and mRNA levels in both the adrenal medulla and sympathetic ganglia. In the adrenal medulla, transcription of the TH gene is rapidly induced with immobilization (IMO) stress. Here, we examine whether IMO also increases TH transcription in the superior cervical ganglia (SCG). Quantitative real-time reverse transcription polymerase chain reaction was used to determine the changes in TH mRNA and in transcripts containing intron 2. As expected in the adrenal medulla following repeated IMO TH mRNA and intron containing transcripts were elevated about 5-fold. In the SCG, a significant increase in TH mRNA was observed following repeated 2 h IMO for 2 or 6 days, but not with single IMO. The intron 2 containing transcripts were elevated about 50% above controls with even single IMO, and were at similarly elevated levels after the 2nd or 6th repeated daily IMO. The results indicate, for the first time, that transcriptional mechanisms are involved in mediating the IMO stress triggered elevation in TH gene expression in the SCG.

Immobilization and stabilization of d-hydantoinase for production of N-carbamoyl-d-phenylglycine

Immobilization and stabilization of d-hydantoinase for production of N-carbamoyl-d-phenylglycine

G.P.2.04 Gene expression and muscle fiber function in a porcine AQM-ICU model

Skeletal muscle wasting and impaired muscle function in response to mechanical ventilation (MV), immobilization (IM) and various pharmacological treatments in intensive care unit (ICU) patients are clinically challenging partly due to the poorly understood intricate cellular and molecular networks and partly due to the unavailability of an animal model mimicking this condition in ICU patients. By employing a unique porcine model mimicking the conditions in the ICU with long-term mechanical ventilation and immobilization and exposed to various combinations of agents, i.e., pharmacological neuromuscular blockade (NMBA), corticosteroids (CS) and/or sepsis we have analyzed the expression profiles and physiological parameters in the muscle biopsies taken from the m.

Pituitary-Thyroid Function during Acute Immobilization Stress in Rats

In nine experiments a total of 394 male Wistar rats weighing about 300 g was used. In each experiment, one intact control group was used and several other groups were subjected to immobilization (IMO) for 2 to 240 min in a prone position by inserting their heads through steel wire loops fixed on a board and by fasting their limbs to four metal strips by adhesive tape. All animals were killed by decapitation and the levels of thyrotropin (TSH), thyroxine (T4), 3,5,3'-triiodothyronine (T3) and 3,3',5'-triiodothyronine (rT3) in plasma were estimated by radioimmunoassay. In one experiment, significant changes in the level of all hormones measured were found within 2 to 15 min of IMO, e.g. an increase of TSH, T4 and rT3 and a decrease of T3. Later (i.e. between 30 and 240 min) the level of TSH, T4 and T3 was repeatedly found to be significantly decreased almost in all experiments as compared to controls. In contrast, a significant increase of rT3 was found only in one out of 7 experiments, the values in other ones being unchanged. Corresponding changes of hormone levels were observed when IMO for 150 min was repeated daily for 7 or 40 days and the animals were sacrificed immediately after the last stress, while one day of rest after such procedures as well as after a single IMO for 150 min appeared to be sufficient for the levels to return to control values.

Effect of <i>Bupleurum falcatum</i> on the Stress-Induced Impairment of Spatial Working Memory in Rats

Many studies have shown that Bupleurum falcatum (BF), which is widely used in the treatment of various psychosomatic diseases in traditional Oriental medicine, is an effective therapeutic intervention for memory impairment. The purpose of this study was to examine the effect of BF on stress-induced alterations in learning and memory in rats using the Morris water maze (MWM) and elevated plus maze (EPM) behavioral tests. In addition, we examined the effects of BF treatment on the cholinergic system, as indicated by changes in neuronal choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) immunoreactivity in the hippocampus. BF (150, 300, or 600 mg/kg) was administered orally 30 min before exposure to repeated immobilization (IMO) stress (4 h/d for 14 d). The BF treatment produced a significant improvement in escape latency (time required to find the platform) in the MWM, and it also produced an anxiolytic-like effect in the EPM. Consistent with the behavioral data, BF treatment significantly attenuated the IMO stress-induced loss of cholinergic immunoreactivity in the hippocampus. These findings indicate that BF has a protective effect against repeated IMO stress-induced neuronal and cognitive impairments, and they suggest that BF may be useful in the treatment of stress-induced memory impairment.

Identifying the Stress Transcriptome in the Adrenal Medulla following Acute and Repeated Immobilization

Stress triggers changes in gene expression mediating important adaptive and maladaptive responses. The full repertoire of genes whose expression in the adrenal medulla is altered by stress has not been previously determined. In this study, gene profiling (RAE 230 2.0 Affymetrix) was applied to elucidate global changes in gene expression in adrenal medulla of rats exposed to 2-h immobilization (IMO) stress once or repeatedly for 6 consecutive days. The number of transcripts significantly (P < 0.01) altered with single IMO (651 up, 487 down) was more than with repeated IMO (370 up, 195 down). The annotated transcripts were further analyzed and categorized. The largest numbers of changes were in mRNA levels in the transcription factor and cell signaling categories. Robust changes were also observed in transcripts related to growth factors, apoptosis, neurosecretion/neuropeptides, heat shock proteins, structural proteins, chemokines, cytokines, metabolism/lipid-metabolism, and proteases. Many (>80%) were uniquely induced by single IMO. About half of transcripts changed by repeated IMO were also responsive to single IMO. Pathway analysis was applied to identify direct interactions and common targets among gene products altered by single and repeated IMO. In this paper, we briefly describe the most pronounced changes observed, with emphasis on those that may provide new insight into the common and distinct mechanisms whereby the adrenal medulla responses to a first encounter with stress compared to repeated exposure to the same stressor.

Adrenergic Responses to Stress

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.