Peter Vargovic

Adipocytes as a new source of catecholamine production.

Catecholamines are an important regulator of lipolysis in adipose tissue. Here we show that rat adipocytes, isolated from mesenteric adipose tissue, express genes of catecholamine biosynthetic enzymes and produce catecholamines de novo. Administration of tyrosine hydroxylase inhibitor, alpha‐methyl‐p‐tyrosine, in vitro significantly reduced concentration of catecholamines in isolated adipocytes. We hypothesize that the sympathetic innervation of adipose tissues is not the only source of catecholamines, since adipocytes also have the capacity to produce both norepinephrine and epinephrine.

Catecholamine production is differently regulated in splenic T- and B-cells following stress exposure.

OBJECTIVES Stress is accompanied also by a rise in splenic catecholamines (CAs). However, indications about endogenous CA production in the spleen exist but there are no data about the cellular source of this production and possible modification by stress. Therefore, our aim was to investigate whether splenic T- and B-cells are one of main sources in the spleen expressing tyrosine hydroxylase (TH), enzyme crucial for CA biosynthesis, and phenylethanolamine N-methyltransferase (PNMT) which is necessary for epinephrine production. We also investigated whether stress is able to modify expression of both enzymes and CA levels within these cell fractions as well as tried to explain functional consequences of changes observed. RESULTS T-cells contain higher levels of TH mRNA than B-cells although protein levels appeared similar. On contrary, the PNMT mRNA and protein were higher in B-cells, which appeared to be the main source of PNMT in the spleen. T-cells increased TH and PNMT expression after acute stress while similar rise was observed in B-cells after repeated stress, most probably as a consequence of higher CA turnover in both cell populations. The rise in TH and PNMT was accompanied by an elevation of Bax/Bcl-2 mRNA ratio, number of apoptotic cells and also by a decline of IFN-γ mRNA in both cell types. Reduction of IL-2 and IL-4 mRNA was also observed in B-cells. CONCLUSION Stress-induced stimulation of endogenous CA biosynthesis in lymphocytes is dependent on the type of lymphocyte population and duration of stressor and leads to attenuated IFN-γ expression and induction of apoptosis. These changes might contribute to dysregulation of specific immune functions involving T- and B-cells and may decrease the ability to cope with intracellular agents following stress situations.

Repeated Stress Down-Regulates β2- and α2C-Adrenergic Receptors and Up-Regulates Gene Expression of IL-6 in the Rat Spleen

Catecholamines are among first compounds released during stress, and they regulate many functions of the organism, including immune system, via adrenergic receptors (ARs). Spleen, as an immune organ with high number of macrophages, possesses various ARs, from which β2-ARs are considered to be the most important for the modulation of immune functions. Nevertheless, little is known about the regulation and involvement of ARs in the splenic function by stress. Therefore, the aim of this work was to measure the gene expression of ARs and several cytokines in the spleen of rats exposed to a single and repeated (14×) immobilization stress (IMO). We have found a significant increase in β2-AR mRNA after a single IMO, but a significant decrease in β2-AR mRNA and protein level after repeated (14×) IMO. The most prominent decrease was detected in the gene expression of the α2A- and α2C-AR after repeated IMO. However, changes in mRNA were translated into protein levels only for the α2C-subtype. Other types of ARs remained unchanged during the stress situation. Since we proposed that these ARs might affect production of cytokines, we measured gene expression of pro-inflammatory (TNF-α, IL-1β, IL-6 and IL-18) and anti-inflammatory (IL-10 and TGF-β1) cytokines. We detected changes only in IL-6 and IL-10 mRNA levels. While IL-6 mRNA was increased, IL-10 mRNA dropped after repeated IMO. According to these results we suggest that changes of β2- and α2C-ARs participate in IL-6-mediated processes in the spleen, especially during chronic stress situations.

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.

Repeated immobilization stress induces catecholamine production in rat mesenteric adipocytes.

Catecholamines (CATs), the major regulator of lipolysis in adipose tissue, are produced mainly by the sympathoadrenal system. However, recent studies report endogenous CAT production in adipocytes themselves. This study investigated the effects of single and repeated (7–14 times) immobilization (IMO) stress on CAT production in various fat depots of the rat. Single IMO quickly induced a rise of norepinephrine (NE) and epinephrine (EPI) concentration in mesenteric and brown adipose depots. Adaptive response to repeated IMO included robust increases of NE and EPI levels in mesenteric and subcutaneous adipose tissue. These changes likely reflect the activation of sympathetic nervous system in fat depots by IMO. However, this process was also paralleled by an increase in tyrosine hydroxylase gene expression in mesenteric fat, suggesting regulation of endogenous CAT production in adipose tissue cells. Detailed time-course analysis (time course 10, 30, and 120 min) clearly showed that repeated stress led to increased CAT biosynthesis in isolated mesenteric adipocytes resulting in gradual accumulation of intracellular EPI during IMO exposure. Comparable changes were also found in stromal/vascular fractions, with more pronounced effects of single than repeated IMO. The potential physiological importance of these findings is accentuated by parallel increase in expression of vesicular monoamine transporter 1, indicating a need for CAT storage in adipocyte vesicles. Taken together, we show that CAT production occurs in adipose tissue and may be activated by stress directly in adipocytes.

Tauopathy in transgenic (SHR72) rats impairs function of central noradrenergic system and promotes neuroinflammation.

BackgroundBrain norepinephrine (NE) plays an important role in the modulation of stress response and neuroinflammation. Recent studies indicate that in Alzheimer’s disease (AD), the tau neuropathology begins in the locus coeruleus (LC) which is the main source of brain NE. Therefore, we investigated the changes in brain NE system and also the immune status under basal and stress conditions in transgenic rats over-expressing the human truncated tau protein.MethodsBrainstem catecholaminergic cell groups (LC, A1, and A2) and forebrain subcortical (nucleus basalis of Meynert), hippocampal (cornu ammonis, dentate gyrus), and neocortical areas (frontal and temporal association cortices) were analyzed for NE and interleukin 6 (IL-6) mRNA levels in unstressed rats and also in rats exposed to single or repeated immobilization. Moreover, gene expression of NE-biosynthetic enzyme, tyrosine hydroxylase (TH), and several pro- and anti-inflammatory mediators were determined in the LC.ResultsIt was found that tauopathy reduced basal NE levels in forebrain areas, while the gene expression of IL-6 was increased in all selected areas at the same time. The differences between wild-type and transgenic rats in brain NE and IL-6 mRNA levels were observed in stressed animals as well. Tauopathy increased also the gene expression of TH in the LC. In addition, the LC exhibited exaggerated expression of pro- and anti-inflammatory mediators (IL-6, TNFα, inducible nitric oxide synthases 2 (iNOS2), and interleukin 10 (IL-10)) in transgenic rats suggesting that tauopathy affects also the immune background in LC. Positive correlation between NE and IL-6 mRNA levels in cornu ammonis in stressed transgenic animals indicated the reduction of anti-inflammatory effect of NE.ConclusionsOur data thus showed that tauopathy alters the functions of LC further leading to the reduction of NE levels and exaggeration of neuroinflammation in forebrain. These findings support the assumption that tau-related dysfunction of LC activates the vicious circle perpetuating neurodegeneration leading to the development of AD.

Exaggerated phosphorylation of brain tau protein in CRH KO mice exposed to repeated immobilization stress.

Abstract Neuroendocrine and behavioral stress responses are orchestrated by corticotropin-releasing hormone (CRH) and norepinephrine (NE) synthesizing neurons. Recent findings indicate that stress may promote development of neurofibrillary pathology in Alzheimers disease. Therefore, we investigated relationships among stress, tau protein phosphorylation, and brain NE using wild-type (WT) and CRH-knockout (CRH KO) mice. We assessed expression of phosphorylated tau (p-tau) at the PHF-1 epitope and NE concentrations in the locus coeruleus (LC), A1/C1 and A2/C2 catecholaminergic cell groups, hippocampus, amygdala, nucleus basalis magnocellularis, and frontal cortex of unstressed, singly stressed or repeatedly stressed mice. Moreover, gene expression and protein levels of tyrosine hydroxylase (TH) and CRH receptor mRNA were determined in the LC. Plasma corticosterone levels were also measured. Exposure to a single stress increases tau phosphorylation throughout the brain in WT mice when compared to singly stressed CRH KO animals. In contrast, repeatedly stressed CRH KO mice showed exaggerated tau phosphorylation relative to WT controls. We also observed differences in extent of tau phosphorylation between investigated structures, e.g. the LC and hippocampus. Moreover, CRH deficiency leads to different responses to stress in gene expression of TH, NE concentrations, CRH receptor mRNA, and plasma corticosterone levels. Our data indicate that CRH effects on tau phosphorylation are dependent on whether stress is single or repeated, and differs between brain regions. Our findings indicate that CRH attenuates mechanisms responsible for development of stress-induced tau neuropathology, particularly in conditions of chronic stress. However, the involvement of central catecholaminergic neurons in these mechanisms remains unclear and is in need of further investigation.

Stress-Induced Activation of the Sympathoadrenal System is Determined by Genetic Background in Rat Models of Tauopathy

Stress may accelerate onset of neurodegenerative diseases in vulnerable subjects and, vice versa, neurodegeneration affects the responsiveness to stressors. We investigated the neuroendocrine response to immobilization stress in normotensive Wistar-Kyoto rats (WKY), spontaneously hypertensive rats (SHR), and transgenic rats of respective WKY and SHR strains overexpressing human truncated tau protein. Plasma levels of epinephrine, norepinephrine, and corticosterone were determined. An immobilization-induced elevation of epinephrine and norepinephrine was significantly reduced in WKY transgenic rats compared to WKY wild-type rats, while no differences were seen between SHR transgenic and SHR wild-type animals. Our data have shown that sympathoadrenal system response to stress strongly depends on both tau protein-induced neurodegeneration and genetic background of experimental animals.

Lipopolysaccharide induces catecholamine production in mesenteric adipose tissue of rats previously exposed to immobilization stress

Abstract Catecholamines (CAs) are mainly produced by sympathoadrenal system but their de novo production has been also observed in adipose tissue cells. The aim of this work was to investigate whether immune challenge induced by lipopolysaccharide (LPS) modulates biosynthesis of CAs in mesenteric adipose tissue (MWAT), as well as whether previous exposure to immobilization (IMO) stress could modulate this process. Sprague–Dawley rats were exposed to single (2 h) or repeated (2 h/7 days) IMO and afterwards injected with LPS (i.p., 100 μg/kg body weight) and sacrificed 3 h later. LPS did not alter CA biosynthesis in MWAT in control rats. Single and repeated IMO elevated CAs and expression of CA biosynthetic enzymes in MWAT, including adipocyte and stromal/vascular fractions (SVF). Repeated IMO followed by LPS treatment led to the up-regulation of CA-biosynthetic enzymes expression, elevation of CAs in SVF but depletion of norepinephrine and epinephrine in adipocyte fraction. Prior IMO caused a marked LPS-induced macrophage infiltration in MWAT as evaluated by F4/80 expression. A positive correlation between expression of tyrosine hydroxylase and F4/80 suggests macrophages as the main source of LPS-induced CA production in MWAT. Furthermore, prior exposure to the single or repeated IMO differently affected immune responses following LPS treatment by modulation of inflammatory cytokine expression. These data suggest that stress might be a significant modulator of immune response in MWAT via stimulation of the macrophage infiltration associated with cytokine response and de novo production of CAs.

Effect of a single and repeated stress exposure on gene expression of catecholamine biosynthetic enzymes in brainstem catecholaminergic cell groups in rats.

Brainstem catecholaminergic neurons significantly participate in the regulation of neuroendocrine system activity, particularly during stressful conditions. However, so far the precise quantitative characterisation of basal and stress‐induced changes in gene expression and protein levels of catecholaminergic biosynthetic enzymes in these neurons has been missing. Using a quantitative reverse transcription‐polymerase chain reaction method, we investigated gene expression of catecholamine biosynthetic enzymes in brainstem noradrenergic and adrenergic cell groups in rats under resting conditions as well as in acutely and repeatedly stressed animals. For the first time, we described quantitative differences in basal levels of catecholamine biosynthetic enzyme mRNA in brainstem catecholaminergic ascending and descending projecting cell groups. Moreover, we found and defined some differences among catecholaminergic cell groups in the time‐course of mRNA levels of catecholaminergic enzymes following a single and especially repeated immobilisation stress. The data obtained support the assumption that brainstem catecholaminergic cell groups represent a functionally differentiated system, which is highly (but specifically) activated in rats exposed to stress. Therefore, potential interventions for the treatment of stress‐related diseases need to affect the activity of brainstem catecholaminergic neurons not uniformly but with some degree of selectivity.