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Haloperidol is an antipsychotic agent that primarily acts as an antagonist of D2 dopamine receptors. Besides other receptor systems, it targets sigma 1 receptors (σ1Rs) and inositol 1,4,5-trisphosphate receptors (IP3Rs). Aim of this work was to investigate possible changes in IP3Rs and σ1Rs resulting from haloperidol treatment and to propose physiological consequences in differentiated NG-108 cells, i.e., effect on cellular plasticity. Haloperidol treatment resulted in up-regulation of both type 1 IP3Rs (IP3R1s) and σ1Rs at mRNA and protein levels. Haloperidol treatment did not alter expression of other types of IP3Rs. Calcium release from endoplasmic reticulum (ER) mediated by increased amount of IP3R1s elevated cytosolic calcium and generated ER stress. IP3R1s were bound to σ1Rs, and translocation of this complex from ER to nucleus occurred in the group of cells treated with haloperidol, which was followed by increased nuclear calcium levels. Haloperidol-induced changes in cytosolic, reticular, and nuclear calcium levels were similar when specific σ1 blocker -BD 1047- was used. Changes in calcium levels in nucleus, ER, and cytoplasm might be responsible for alterations in cellular plasticity, because length of neurites increased and number of neurites decreased in haloperidol-treated differentiated NG-108 cells.

Haloperidol is an antipsychotic agent and acts as dopamine D2 receptor (D2R) antagonist, as a prototypical ligand of sigma1 receptors (Sig1R) and it increases expression of type 1 IP3 receptors (IP3R1). However, precise mechanism of haloperidol action on cardiomyocytes through dopaminergic signaling was not described yet. This study investigated a role of dopamine receptors in haloperidol-induced increase in IP3R1 and Sig1R, and compared physiological effect of melperone and haloperidol on basic heart parameters in rats.We used differentiated NG-108 cells and H9c2 cells. Gene expression, Western blot and immunofluorescence were used to evaluate haloperidol-induced differences; proximity ligation assay (PLA) and immunoprecipitation to determine interactions of D1/D2 receptors. To evaluate cardiac parameters, Wistar albino male rats were used.We have shown that antagonism of D2R with either haloperidol or melperone results in upregulation of both, IP3R1 and Sig1R, which is associated with increased D2R, but reduced D1R expression. Immunofluorescence, immunoprecipitation and PLA support formation of heteromeric D1/D2 complexes in H9c2 cells. Treatment with haloperidol (but not melperone) caused decrease in systolic and diastolic blood pressure and significant increase in heart rate.Because D1R/D2R complexes can engage Gq-like signaling in other experimental systems, these results are consistent with the possibility that disruption of D1R/D2R complex in H9c2 cells might cause a decrease in IP3R1 activity, which in turn may account for the increase expression of IP3R and Sig1R. D2R is probably not responsible for changes in cardiac parameters, since melperone did not have any effect.

The left and right ventricles fulfill different role in heart function. Here we compare cham - ber specific changes in local catecholamine concentrations; gene expression and the receptor protein amount of all three β-adrenoceptors (β-AR) in rat right heart ventricles exposed to acute (1 session) and repeated (7 sessions) immobilization stress (IMMO) vs. previously observed changes in left ventricles. Density of muscarinic receptors as main cardio-inhibitive receptors was also measured. In the right ventricles, noradrenaline and adrenaline were increased. No β1-AR changes were observed, in spite of the increased sympathetic activity. On the other hand, we have found a decrease of β2- AR gene expression (reduction to 30%) after 7 IMMO and protein (to 59%) after 1 IMMO . β3-AR gene expression was increased after 7 IMMO. Muscarinic receptor density was not changed. When comparing correlation in left and right ventricles, there was strong correlation between adrenaline and β2-AR gene expression, protein and β3-AR gene expression in the left ventricles while only cor - relation between adrenaline and β2-AR mRNA and protein in the right ventricles was found. Our results show that maintenance of cardiac homeostasis under stress conditions are to a great extent achieved by a balance between different receptors and also by a balanced receptor changes in left vs. right ventricles. Taken together, decrease of cardio-stimulating β2-AR represents a new important mechanism by which β2-AR contributes to the heart physiology.

Hydrogen sulfide (H2S) as a novel gasotransmitter regulates variety of processes, including calcium transport systems. Sodium calcium exchanger (NCX) is one of the key players in a regulation calcium homeostasis. Thus, the aims of our work were to determine effect of sulfide signaling on the NCX type 1 (NCX1) expression and function in HeLa cells, to investigate the relationship of β-adrenergic receptors with the NCX1 in the presence and/or absence of H2S, and to determine physiological importance of this potential commu- nication. As a H2S donor, we used morpholin-4-ium-4- methoxyphenyl(morpholino) phosphinodithioate—GYY4137. We observed increased levels of the NCX1 mRNA, protein, and activity after 24 h of GYY4137 treatment. This increase was accompanied by elevated cAMP due to the GYY4137 treatment, which was completely abolished, when NCX1 was silenced. Increased cAMP levelswould point to upregulation of β-adrenergic receptors. Indeed, GYY4137 increased expres- sion of β 1a ndβ 3( but notβ2) adrenergic receptors. These receptors co-precipitated, co-localized with the NCX1, and induced apoptosis in the presence of H2S. Our results suggest that sulfide signaling plays a role in regulation of the NCX1, β1 and β3 adrenergic receptors, their co-localization, and stimula- tion of apoptosis, which might be of a potential importance in cancer treatment.

Stress is a contributor of many cardiovascular diseases. Positive inotropic and chronotropic effects of catecholamines are regulated via β-adrenergic receptors (ARs). Many reports exist concerning changes of cardiac β1- and β2-ARs in stress, but only a few deal with modulation of cardiac β3-AR. Our aim was to analyze the expression and binding sites of β1-, β2- and β3-ARs and adenylyl cyclase activity in the left ventricle, and β3-AR expression and binding in the left atrium of rats exposed to acute and chronic immobilization stress (IMO). The concentration of noradrenaline in the ventricle decreased, while adrenaline increased, especially after repeated IMO. The mRNA and protein levels, and binding sites of β3-subtype significantly rose following chronic IMO, while all parameters for β2-AR dropped after single and repeated exposure. Similarly, the mRNA levels and binding sites for β3-subtype increased in the left atrium as a consequence of chronic IMO. The rise in β3-subtypes and a drop in β2-subtypes resulted in inhibition of adenylyl cyclase activity within the left ventricle. Taken together, among other factors, up-regulation of β3-AR could represent an adaptation mechanism, which might be related to altered physiological function of the left ventricle and atrium during prolonged emotional stress and might serve cardioprotective function during catecholamine overload. Copyright © 2013 John Wiley & Sons, Ltd.

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.

Pheochromocytomas (PHEOs) and paragangliomas (PGLs) are specific types of neuroendocrine tumors that originate in the adrenal medulla or sympathetic/parasympathetic paraganglia, respectively. Although these tumors are intensively studied, a very effective treatment for metastatic PHEO or PGL has not yet been established. Preclinical evaluations of novel therapies for these tumors are very much required. Therefore, in this study we tested the effect of triptolide (TTL), a potent nuclear factor-kappaB (NF-κB) inhibitor, on the cell membrane norepinephrine transporter (NET) system, considered to be the gatekeeper for the radiotherapeutic agent 131I-metaiodobenzylguanidine (131I-MIBG). We measured changes in the mRNA and protein levels of NET and correlated them with proapoptotic factors and metastasis inhibition. The study was performed on three different stable PHEO cell lines. We found that blocking NF-κB with TTL or capsaicin increased both NET mRNA and protein levels. Involvement of NF-κB in the upregulation of NET was verified by mRNA silencing of this site and also by using NF-κB antipeptide. Moreover, in vivo treatment with TTL significantly reduced metastatic burden in an animal model of metastatic PHEO. The present study for the first time shows how NF-κB inhibitors could be successfully used in the treatment of metastatic PHEO/PGL by a significant upregulation of NET to increase the efficacy of 131I-MIBG and by the induction of apoptosis.

Although c-Fos protein is one of the principal molecules in intracellular signaling, c-fos gene disruption is associated with alterations in neuronal functions that do not correspond to its importance in function. The aim of the study was to evaluate the changes of dopaminergic system together with acetylcholinesterase (AChE) in c-fos disruption (KO). KO male mice showed an increase in D₁-like receptor (279% of WT) and D₂-like receptor (345% of WT) binding sites in the cortex. On the gene expression level (assessed by real-time PCR), lower quantities of D₁R-mRNA (0.64) and D₅R-mRNA (0.6) were found in females when compared to males in the frontal cortex, higher D₂R-mRNA in the parietal (1.43) and temporal (2.64) cortex and lower AChE-mRNA (0.67). On the contrary, female striatum contained higher level of D₂R-mRNA (1.62) and AChE-mRNA (1.57) but lower level of D₃R-mRNA (0.73). Hypothalamic D₁R-mRNA, D₂R-mRNA and D₄R-mRNA were higher in females (1.38, 1.63, and 1.68, respectively). Disruption of c-fos increased selectively D₅R-mRNA (1.31) in male parietal cortex, D₂R-mRNA (1.72) in male temporal cortex, and cerebellar D₂R-mRNA in both males (1.43) and females (1.42), respectively. In females, we found rather decrease in DR-mRNA. Multiple correlations in mRNA quantities (in WT mice) were found, which changed considerably upon c-fos KO. Main interactions in WT were inter-regional, CNS of KO underwent an extensive restructuring comprising intraregional interactions in the frontal cortex, hypothalamus, and cerebellum. These changes in DR (between others) could be considered as one of the adaptive mechanisms in c-fos KO mice.

The present study addresses the hypothesis that adrenergic regulation modulates the effect of apoptosis. Therefore we studied, whether α1-adrenergic receptor's agonist phenylephrine (PE) can affect or induce apoptosis in rat pheochromocytoma (PC12) cells. We have shown that PE treatment did not increase level of the apoptosis, or level of the caspase 3 mRNA. When apoptosis was induced in the presence of PE, caspase 3 mRNA was significantly increased, while the percentage of apoptotic cells remained unchanged compared to apoptotic group without PE. During this process, α1D-, β2- and β3-adrenergic receptors (ARs) were upregulated. Since all these three types of ARs are differently localized in the cell, we assume that mutual communication of all three ARs is crucial to participate in this signaling and during development of apoptosis, some of these systems might translocate. Another important system in handling noradrenaline during apoptosis might be noradrenaline transporter (NET), since it was downregulated in apoptotic cells treated with PE, compared to untreated apoptotic cells. However, precise mechanism of mutual communication among all these systems remains to be elucidated.

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 β(2)-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 β(2)-AR mRNA after a single IMO, but a significant decrease in β(2)-AR mRNA and protein level after repeated (14×) IMO. The most prominent decrease was detected in the gene expression of the α(2A)- and α(2C)-AR after repeated IMO. However, changes in mRNA were translated into protein levels only for the α(2C)-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 β(2)- and α(2C)-ARs participate in IL-6-mediated processes in the spleen, especially during chronic stress situations.

Stress, if exaggerated, modulates a variety of metabolic pathways and results in development of serious health consequences. The cell membrane sodium-calcium exchanger (NCX) is a major calcium extrusion system and is also modulated by stress. It has been shown previously that mRNA, protein levels and activity of the type 1 NCX (NCX1) in the left ventricle of the rat heart are increased by stressors, such as immobilization or hypoxia. In this study we investigated whether exposure to a subsequent different stressor can affect gene expression, protein level and activity of the NCX1 in rat kidney compared to exposure to only one type of stressor. In these experiments, we used immobilization and cold as the model stressors.We found that cold exposure at 4 degrees C for 24 h, when applied after immobilization repeated seven times, completely abolished the immobilization-induced increase in NCX mRNA level and after 7 days cold exposure the increases in NCX1 protein and activity in rat kidney were also abolished. Permanently increased NCX1 expression can result in imbalance of cellular calcium homeostasis and thus contribute to kidney dysfunction. Based on our results, we conclude that exposure to a cold stressor can have a protective effect on the kidney in rats exposed previously to repeated immobilization stress. This might be explained by differential stimulation of sympathetic neural and adrenal medullary responses by these different stressors.

The supposed role of cardiac sigma receptors is fine tuning of contractility. Sigma receptors affect several ionic channels and hence their signaling is reflected by the electrophysiological properties of the heart. Numerous ligands of sigma receptors are known to prolong the QT interval and therefore cause a variety of arrhythmias, including severe ones. The effects of the prototypical sigma ligand haloperidol have been studied extensively in humans as well as in various animal models, primarily after acute administration. We examined the incidence of arrhythmias, changes in heart rate, and prolongation of QT interval in isolated Langendorff-perfused rat and guinea pig hearts after they were exposed to nanomolar concentrations of haloperidol. Hearts from both untreated (acute) and pretreated (chronic) animals were investigated. While QT prolongation and arrhythmias due to haloperidol administration were observed in untreated rat and guinea pig hearts, arrhythmias were completely prevented in both species of chronically treated animals. In treated guinea pigs, the results were generally less convincing. Since the hearts were exposed to nanomolar concentration of haloperidol, we conclude that our data may be explained by desensitization of sigma receptors.

Adrenergic regulation of the heart function is well documented by many studies. Catecholamines act through alpha(1)-, beta(1)-, beta(2)-, and beta(3)-adrenoceptors (ARs) in the heart. There are many findings about the changes of beta(1)- and beta(2)-AR in heart failure (HF). On the other hand, the role of other AR subtypes is not clear yet. We focused on determining how HF could affect gene expression and specific ligand binding to alpha(1A)-, alpha(1B)-, alpha(1D)-, beta(1)-, beta(2)-, and beta(3)-AR. Hearts from 11 patients with HF subjected to transplantation were investigated. As a control, corresponding parts from hearts not suitable for transplantation were used. We have found significantly higher mRNA levels of alpha(1A)-, alpha(1B)-,beta(1)-, and beta(2)-AR in the left ventricle of failing hearts compared to the levels in controls. beta(3)-AR mRNA levels in the left ventricle of failing hearts were not changed. No changes in mRNA levels of all receptors studied in other cardiac areas were found. On the other hand, binding studies showed a substantial decrease in left ventricles of failing hearts in all alpha(1)-AR subtypes and in beta(1)- and beta(2)-AR. However, the binding to beta(3)-AR was not changed. Our results suggest that alpha(1)-AR changes might be part of a compensatory mechanism, by which the heart suffering from the HF tries to secure its function, and it could be hypothesized that ineffective beta(3)-AR regulation might be involved in development of HF. According to our knowledge, this is the first report about the beta(3)-AR binding in HF.

Norepinephrine-deficient mice harbor a disruption of the gene for dopamine-beta-hydroxylase (DBH-KO). Corticotropin-releasing hormone knockout mice (CRH-KO) have markedly reduced HPA activity. The aim of the present work was to study how deficiency of DBH and CRH would affect tyrosine hydroxylase (TH), DBH, and phenylethanolamine N-methyltransferase (PNMT) gene expression and protein levels in the adrenal medulla (AM) and stellate ganglia (SG) of control and stressed mice. Both in AM and SG, single immobilization significantly increased TH and DBH mRNA and protein levels both in wild-type (WT) and CRH-KO mice. On the other hand, the stress-triggered increase in PNMT mRNA and protein levels seen in WT mice was absent in CRH-KO mice. DBH-KO mice are more sensitive to stress but survive a single 2 h restraint stress in a tube. The increase in TH mRNA levels induced by restraint stress in WT was not observed in DBH-KO mice. PNMT mRNA and especially PNMT protein levels were significantly elevated in AM of DBH-KO mice. In SG of DBH-KO mice, TH mRNA levels were not affected; however, PNMT gene expression was highly elevated. Thus, disruption of the DBH gene surprisingly blocks the stress-induced elevation of TH mRNA levels in AM but increases PNMT gene expression in both AM and SG. Our data indicate that adrenergic signaling is required for stress-induced increase in TH mRNA and that this signaling restrains stress-induced increase in PNMT mRNA. They also confirm that the HPA system plays a crucial role in the stress-induced regulation of PNMT gene expression.

Changes in the heart rate and force of contraction are regulated by catecholamines via adrenoceptors (AR). In this work, we measured gene expression of AR in left heart atria and ventricles in rats exposed to cold stress and in cold-acclimated rats exposed to a novel stressor (immobilization). We found a significant increase in beta(3)-AR in left ventricle of rats exposed to acute (1 day) and long-term (28 days) cold, but no changes in beta(1)- and beta(2)-AR mRNA levels. However, single immobilization significantly decreased beta(2)-AR mRNA levels both in left atria and ventricles compared to acute cold stress. Application of a novel stressor (immobilization) to previously cold-acclimated animals did not show decrease of beta(2)-AR mRNA levels as seen in intact animals. Moreover, beta(1)- and beta(2)-AR did not show any significant changes. Surprisingly, the most prominent changes in the heart were detected for alpha(1B)-AR gene expression. We found decreased levels of alpha(1B)-AR mRNA in the heart of rats exposed to cold and immobilization. We also found that exposure of cold-acclimated rats to immobilization is responsible for additional decrease of alpha(1B)-AR mRNA levels in heart. It seems that while beta-AR undergoes adaptation, alpha(1B)-AR is probably prepared to modulate heart functions. Proposed mechanism of beta-AR adaptation needs to be elucidated. Thus, we have shown that gene expression of different AR subtypes in the heart is regulated differently by various stressors. A protective role of beta(2)-, beta(3)-AR, and alpha(1B)-AR in the process of heart adaptation to chronic stress exposure is proposed.

Hypertensive TGR(mRen2)27 (TGR) rats represent a strain with genetically upregulated renin-angiotensin-aldosterone system. Simultaneously with development of hypertension, a daily profile in blood pressure (BP) inverts and in mature TGR rats BP is higher during the lighttime (L) than the darktime (D). Physiological mechanisms of inverted BP rhythm generation are not understood. In our study we determined circadian profiles of plasma hormones related to BP control (aldosterone, corticosterone, melatonin, prolactin) in TGR and control Sprague-Dawley (SD) rats over 24 h and expression of genes encoding catecholamine synthesizing enzymes, tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH), and phenylethanolamine-N-methyltransferase (PNMT) in adrenals and stellate ganglia. Plasma levels of corticosterone and aldosterone were higher in TGR than SD rats but acrophases of their rhythms were not changed. Darktime peak of prolactin in TGR rats was decreased in comparison with SD animals and pineal melatonin levels started to rise earlier in TGR than in SD rats. In adrenals we found upregulated expression of TH, DBH, and PNMT mRNA at the beginning of the lighttime in TGR compared to SD rats. Expression of TH and DBH in stellate ganglia was not different in TGR rats in comparison with SD, but PNMT expression was higher during L compared to D in TGR rats. We hypothesize that upregulated adrenal medulla functioning in the morning and disturbed communication between circadian oscillators and mechanisms involved in BP control can explain the reversed BP profile in TGR rats.

Aging process is accompanied by various biological dysfunctions including altered calcium homeostasis. Modified calcium handling might be responsible for changed cardiac function and potential development of the pathological state. In the present study we compared the mRNA and protein levels of the intracellular Ca(2+)-handling proteins--inositol 1,4,5-trisphosphate receptor (IP(3)R), ryanodine receptor (RyR), sarcoplasmic reticulum Ca(2+) pump (SERCA2), and also transient receptor potential C (TRPC) channels in cardiac tissues of 5-, 15-, and 26-month-old rats. Aging was accompanied by significant increase in the mRNA levels of IP(3)R and TRPC channels in both ventricles and atria, but mRNA level of the type 2 RyR was unchanged. Protein content of the IP(3)R1 correlated with mRNA levels, in the left ventricle of 15- and 26-month-old rats the value was approximately 1.8 and 2.8-times higher compared to 5-month-old rats. No significant differences were observed in mRNA and protein levels of the SERCA2 among 5-month-old and aged rats. However, Ca(2+)-ATPase activity significantly decreased with age, activities in 5-, 15-, and 26-month-old rats were 421.2 +/- 13.7, 335.5 +/- 18.1 and 304.6 +/- 14.8 nmol P(i) min(-1) mg(-1). These results suggest that altered transporting activity and/or gene expression of Ca(2+)-handling proteins of intracellular Ca(2+) stores might affect cardiac function during aging.

There is strong evidence about the importance of catecholamines and calcium signaling in heart function. Also, interaction of these two systems is well documented. Catecholamines signal through adrenergic receptors, and further activate calcium transport either from the extracellular space, or from the intracellular calcium stores. This review summarizes current knowledge on catecholamine production in the heart, with special focus on the final enzyme in the catecholamine synthesizing pathway, phenylethanolamine N-methyltransferase (PNMT), in different cell types in the heart. Further, signaling through different types of adrenergic receptors in physiological conditions and after exposure to different stressors is discussed. Also, part of this review considers activation of an intracellular calcium transport system via inositol 1,4,5-trisphosphate receptor and to possible functional consequences in control and stress conditions.

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.

1. 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. 2. 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. 3. 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. 4. 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.

Previously we have shown that inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) are abundantly expressed in the atria of rat hearts. Since arrangement of atria is very heterogeneous, in this work we focused on the precise localization of IP3 receptors in the left atrium, where the gene expression of the type 1 IP3R was the highest. The mRNA levels of the IP3 type 1 receptors in the left atrium, left ventricle and myocytes were determined using real-time polymerase chain reaction and Taqman probe. For precise localization, immunohistochemistry with the antibody against type 1 IP3Rs was performed. The mRNA of type 1 IP3 receptor was more than three times higher in the left atrium than in the left ventricle, as determined by real-time PCR. Expression of the type 1 IP3 receptor mRNA was higher in the atria, especially in parts containing cardiac ganglion cells. The atrial auricles, which are particularly free of ganglion cells, and the ventricles (wall of the right and left ventricle and ventricular septum) contained four to five times less IP3 receptors than atrial samples with ganglia. IP3R type 1 immunoreactivity detected by a confocal microscope attributed the most condensed signal on ganglionic cells, although light immunoreactivity was also seen in cardiomyocytes. These results show that type 1IP3 receptors predominate in intrinsic neuronal ganglia of cardiac atria.

Inositol 1,4,5-trisphosphate (IP3) receptors are calcium-releasing channels localized on the sarcoplasmic reticulum. IP3 receptors mediate the calcium mobilizing effect of a wide range of hormones, cytokines, and neurotransmitters and play an important role in variety of cell functions. The aim of this work was to study, how partial depletion of catecholamines affects the gene expression and protein levels of the type 1 IP3 receptors in rat heart. The type 1 IP3 receptor mRNA levels were studied in the left cardiac atrium and ventricle of rats treated with 6-hydroxydopamine (6-OHDA) in control and stressed conditions. The 6-OHDA produces anatomical and functional denervation resulting in decreased levels of noradrenaline and adrenaline. We also used corticoliberin (CRH) knockout mice, where secretion of adrenaline is significantly suppressed. Administration of 6-OHDA significantly decreases mRNA levels of the type 1 IP3 receptor in both, the left atrium and the left ventricle, while the gene expression of the sarcoplasmic reticular Ca2+-ATPase (SERCA 2) was unaffected. CRH knockout mice possess markedly lower levels of the type 1 IP3 receptor mRNA compared to wild-type mice in both, control and stressed conditions. These data point to the adrenergic modulation of the type 1 IP3 receptors in the rat hearts.

Inositol 1,4,5-trisphosphate (IP(3)) receptors belong to the intracellular calcium channels that release calcium from the intracellular stores after binding IP(3). Three types of IP(3) receptors occurred in a tissue specific manner and different promoters direct their gene expression. Thus, understanding of the transcriptional regulation is the first step towards comprehension of the function of these receptors. Since the retinoic acid activates RARE and AP2 transcription factors, the present study focuses on determination of whether or not expression of type 1 and 2 IP(3) receptors is modulated by retinoic acid in selected brain areas. We have found that mRNA levels of the type 1 IP(3) receptors were decreased significantly in cerebellum and hypothalamus, but not in the brain stem of rats treated with retinoic acid, compared to untreated littermates. The mRNA levels of the type 2 IP(3) receptor were significantly decreased in all tested tissues, cerebellum, hypothalamus, and also in brain stem after the treatment with retinoic acid. These results show that gene expression of both type 1 and 2 IP(3) receptors is regulated by retinoic acid, although the effect of retinoic acid on mRNA levels of the type 1 IP(3) receptors is dependent on brain area.

It has been previously shown that modification of thyroid hormone levels have a profound impact on cardiac function, predominantly through a direct regulation of the sarcoplasmic reticulum protein levels. Nevertheless, little is known about the regulation of calcium transport systems in skeletal muscle due to the altered concentration of thyroid hormones. Thus, the goal of our study was to find out whether altered thyroid status could change the gene expression of the Na(+)/Ca(2+) exchanger (NCX), the inositol 1,4,5-trisphosphate (IP(3)) receptors and ryanodine receptors (RyRs) in slow and fast skeletal muscles of rats. A hyperthyroid state was maintained in rats by triiodothyronine (T(3)) administration, while methimazole was employed for inducing hypothyroidism. After a period of 2-10 months of T(3) treatment we observed a significant increase in mRNA levels of the NCX, RyRs and IP(3) receptors. This increase was more pronounced in the slow soleus than in the fast extensor digitorum longus (EDL) muscle. It is tempting to speculate that thyroid hormones also alter calcium concentration and thus influence the process of excitation-contraction coupling in the skeletal muscle.

IP(3) receptors are intracellular calcium channels, releasing calcium from the sarcoplasmic reticulum. In the heart, IP(3) receptors of type 1 and 2 were found. These receptors predominate in atria, although they occur also in ventricles, as determined by real-time PCR and Western blot analysis. Single-immobilization stress was found to increase mRNA and/or protein levels of types 1 and 2 IP(3) receptors in cardiac atria. However, in stellate ganglia, which innervate the heart, no changes in the mRNA of the type 1 IP(3) receptors were observed after single-immobilization stress. In adrenal medulla, a moderate decrease in both mRNA and protein levels of IP(3) receptors was observed after single-immobilization exposure. After repeated immobilization, mRNA and protein levels of types 1 and 2 IP(3) receptors decreased significantly in all tested tissues. Our results point to different processing of the single stress in different tissues, while repeated stress results in rapid and significant decrease of the IP(3) receptors.

Stress is one of the major risk factors responsible for the increased incidence of a number of common life-threatening disorders, predominantly of cardiovascular origin. The aim of the present study was to establish the effect of repeated immobilization stress on gene expression and protein levels of aromatic L-amino acid decarboxylase (AADC) and phenylethanolamine N-methyltransferase (PNMT) in cardiac left and right atria. In the process of repeated immobilization, rats were immobilized 2 h daily for 7 days and killed 3 h after the last immobilization. Gene expression was determined by regular and real-time reverse transcription with subsequent polymerase chain reaction, and protein levels were determined by Western blot analysis. In cardiac atria, we identified mRNA for AADC and PNMT. Repeated immobilization stress did not affect AADC mRNA levels. However, repeated immobilization significantly increased PNMT mRNA levels compared with unstressed control animals. No further increase was observed compared with adapted control rats (rats immobilized six times for 2 h daily and decapitated 24 h after the sixth immobilization). AADC protein levels corresponded with mRNA levels of this enzyme. However, we were not able to detect PNMT immunoreactive protein. The observed elevation in the gene expression of PNMT mRNA levels in the heart may be involved in the increased risk of cardiovascular diseases with stress.

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.

Social stressors, like other stressors, are powerful activators of the sympathoadrenomedullary system. Differential housing (single vs. group) and social defeat of rats is known to alter the activity of catecholamine-synthesizing enzymes in the medulla. The present studies examined the effect of 70 days of triad (3 rats per large cage) and individual housing of male rats on adrenal mRNA levels of tyrosine hydroxylase (TH), dopamine-β-hydroxylase (DBH) and phenylethanolamine-N-methyltransferase (PNMT) and on TH protein levels. Behavioral ratings carried out at the triad formation indicated that dominant rats exhibited mostly offensive aggressive behaviors. By contrast, subordinate rats expressed primarily defensive behaviors, while the subdominant rats displayed intermediate levels of these behaviors. Overall, compared with single housing, triad housing resulted in lower gene expression for TH, DBH and PNMT and lower TH protein in the adrenals. Within triads, gene expression for these enzymes and TH protein concentration were higher in subordinate compared with dominant and subdominant rats. The dominant rats tended to have the lowest gene expression of these enzymes. These data indicate that in rodents, individual housing and a subject’s social rank have a differential impact on the regulation of catecholamine biosynthesis already during the process of gene expression of catecholamine biosynthetic enzymes in the adrenals.

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-s-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 2 h, 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/μg 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/μg 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 meausured 22 h after the termination of the stress stimulus. Repeated immobilization (7 days, 2 h 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, 2 h 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.

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, 5IU), 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.

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.

Calcium homeostasis is crucial for the proper function of cardiac cells. Since the Na(+)/Ca(2+) exchanger is an important modulator of calcium homeostasis especially in the heart, the objective of this study was to investigate the effect of immobilization stress on the high capacity Na(+)/Ca(2+) exchanger in rat heart ventricles and atria. Repeated immobilization stress increased both the mRNA and the protein level and the activity of the Na(+)/Ca(2+) exchanger in the left, but not the right ventricle of rat heart. Since corticosterone is rapidly increased during the stress stimulus, it might be assumed that mRNA of the Na(+)/Ca(2+) exchanger is increased through a glucocorticoid responsive element. However, we have found that cortisol did not change the Na(+)/Ca(2+) exchanger at the mRNA or protein levels. These results clearly show that this effect of stress is not mediated via cortisol.

Capsaicin, the pungent agent in chili peppers, has been shown to act as a tumor-suppressor in cancer. In our previous study, capsaicin was shown to induce apoptosis in the rat pheochromocytoma cell line (PC12 cells). Thus, the aim of the present study was to determine the potential mechanism by which capsaicin induces apoptosis. We treated PC12 cells with 50, 100 and 500 µM capsaicin and measured the reticular calcium content and expression of the reticular calcium trans- port systems. These results were correlated with endoplasmic reticulum (ER) stress markers CHOP, ATF4 and X-box binding protein 1 (XBP1), as well as with apoptosis induction. We observed that capsaicin decreased reticular calcium in a concentration-dependent manner. Simultaneously, expression levels of the sarco/endoplasmic reticulum pump and ryanodin receptor of type 2 were modified. These changes were accom - panied by increased ER stress, as documented by increased stress markers. Thus, from these results we propose that in PC12 cells capsaicin induces apoptosis through increased ER stress.

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.

Numerous ligands of sigma receptors are known to prolong the QT interval and therefore cause a variety of arrhythmias. High affinity binding sites for the prototypical sigma ligand haloperidol were found in membranes of cardiac myocytes from adult rats. Activation of sigma 1 receptor leads to a release of calcium from the endoplasmic reticulum that follows increased synthesis of inositol 1,4,5-trisphosphate (IP3). We studied the effect of long-term haloperidol treatment on the expression of sigma 1 receptors, IP3 receptors of type 1 and 2 in the individual parts of the rat heart, in isolated rat cardiomyocytes and in PC12 cells. We have found that prolonged treatment with haloperidol significantly increased mRNA levels of sigma 1 receptors in both atria and ventricles. Sigma 1 receptor's mRNA was increased also in isolated cardiomyocytes. Haloperidol treatment affects the expression of IP3 receptors of type 1 and 2 in cardiac atria, but not in cardiac ventricles. We observed increase in IP3 receptors in differentiated PC12 cells, but not in isolated cardiomyocytes. We propose that this increase might participate in triggering cardiac arrhythmias during haloperidol treatment, which has to be further verified.

The spontaneously hypertensive rats (SHR) were fed with nitric oxide synthase (NOS) blocker 7-nitroindazole (7-NI, 10 mg/kg/day) for 6 weeks and an expression of intracellular calcium channels, SERCA and proapoptotic agents was evaluated in kidney. Treatment of rats with 7-NI resulted in a significant increase in mRNA and protein levels of the IP3 receptors type 1 and type 2, while mRNA levels of the IP3 receptor type 3 remained unchanged. The mRNA of other intracellular calcium channels, ryanodine receptors type 1 and type 2 was also upregulated by 7-NI treatment. Gene expression of the SERCA2a, calcium pump responsible for loading intracellular stores with calcium, revealed increased gene expression due to 7-NI as well. Interestingly, proapoptotic agents caspase 3 and Bax were also upregulated by the 7-NI treatment. These results may indicate that nNOS blocker 7-NI modifies intracellular calcium transport system, which may have impact on altered calcium handling and regulation of various metabolic pathways.

Type 1 and 2 inositol 1,4,5-trisphosphate (IP3) receptors have been found in cardiac tissue, although they are localized in different types of cells. While the type 1 predominates in neuronal cells and cardiac ganglia, type 2 IP3 receptor is localized mainly in cardiomyocytes. In the heart, gene expression of the type 1 IP3 receptor is modulated by catecholamines, while type 2 is not affected. In this study we compared the effects of two stressors--cold exposure and hypoxia--on gene expression and protein levels of type 1 and 2 IP3 receptors. We found that both types of IP3 receptors were increased by these stressors, although the response of the type 2 IP3 receptor was delayed. We propose that regulation of these types of IP3 receptors in the heart might differ in control conditions and during stress.

Retinoic acids (RAs) and also their analogs (synthetic retinoids and rexinoids) have been regarded as major therapeutic and/or chemopreventive agents and can regulate a number of diverse processes-such as immune system, hormonal systems. In this work we describe different effects of short-term treatment of Wistar male rats with 13-cis retinoic acid on the regulation of retinoic acid receptors (RARs), retinoid-X receptors (RXRs), thyroid hormone receptors (TRs), ERs, 5'-DI, EGFR and erb-B2/neu genes in liver and/or spleen. Using RT-PCR analysis we have found that administration of 13-cis retinoic acid enhanced expression of RARbeta and PPARgamma mRNA, and decreased expression of RARalpha, RARgamma, RXRbeta and TRbeta mRNA in liver. On the other hand, in spleen this treatment resulted in decreased expression of RARalpha, RARbeta, RARgamma, TRalpha and ERbeta mRNA. Our findings indicate distinct modulation of various signal pathways by short-term administration of 13cRA, which also differ in spleen when compared to liver. We suggest that even a short-term treatment of rats with 13cRA may affect a reasonable number of steps in retinoid signaling pathways, a number of which might be very likely extended by long-term treatment of mammals by 13-cis retinoic acid.

Ca 2+ -dependent and Ca 2+ -independent nitric oxide synthase (NOS) activity, and neuronal and inducible NOS immunoreactivity (nNOS-IR and iNOS-IR), were investigated in the rabbit lower lumbar spinal cord after i) sciatic nerve transection and survival of experimental animals for 2 weeks, ii) treatment of animals with N-nitro-L-arginine (NNLA), an inhibitor of nNOS dosed at 20 mg/b.w. for 12 days, and iii) after treatment of animals with the inducible NOS (iNOS) inhibitor, aminoguanidine, dosed at 100 mg/b.w. for 4 and 12 days. Our attention was focused on the dorsal part of L4-L6 segments receiving sensory inputs from the sciatic nerve, and on the ventral part consisting of sciatic nerve motor neurons. Sciatic nerve transection increased Ca 2+ -dependent NOS activity and the density of nNOS in the dorsal part of the spinal cord on the ipsilateral side. NNLA treatment effectively reduced nNOS-IR in both the dorsal horn and the dorsal column, and decreased Ca 2+ -dependent NOS activity in the lower lumbar segments. Immunocytochemical analysis disclosed the up-regulation of iNOS immunoreactive staining after peripheral axotomy in α-motoneurons. The changes in iNOS expression and Ca 2+ -independent NOS activity were not significantly corrected by aminoguanidine treatment for 4 days. Long-lasting iNOS inhibition decreased Ca 2+ -independent NOS activity, but caused motor neuron degeneration and mediated small necrotic foci in the ventrolateral portion of the ventral horn. The results of the present study provide evidence that constitutive NOS inhibition by NNLA is more effective than specific long-lasting inhibition of iNOS by aminoguanidine treatment.

Aim: The Na+/Ca2+ exchanger (NCX) is a major Ca2+ extrusion system in the plasma membrane of cardiomyocytes and an important component participating on the excitation-contraction coupling process in muscle cells. NCX1 isoform is the most abundant in the heart and is known to be changed after development of ischaemia or myocardial infarction. Objective of this study was to investigate the effect of stress factors (immobilization, cold and short-term hypoxia) on the expression of NCX1, in vivo, in the heart of rat and mouse. Methods: We compared gene expression and protein levels of control and stressed animals. The activity of NCX was measured by the whole cell configuration using the patch clamp. We also measured physiological parameters of the heart in physiological conditions and under ischaemiareperfusion to compare response of control and stressed hearts. Results: We have found that only strong stress stimulus (hypoxia, immobilization) applied repeatedly for several days elevated the NCX1 mRNA level. Cold, which is a weaker stressor that activates mainly sympathoneural, and only marginally adrenomedullary system did not affect the gene expression of NCX1. Thus, from these results it appears that hormones produced by the adrenal medulla (mainly adrenaline) might be involved in this process. To study possible mechanism of the NCX1 regulation by stress, we focused on the possible role of the hypothalamo-pituitary-adrenocortical pathway in the activation of catecholamine synthesis in the adrenal medulla. We have already published that cortisol affects activity, but not the gene expression of NCX1. In this work, we used corticotropin-releasing hormone (CRH) knockout mice, where secretion of corticosterone and subsequently adrenaline is significantly suppressed. As no increase in NCX1 mRNA was observed in CRH knockout mice due to immobilization stress, we proposed that adrenaline (probably regulated via corticosterone) is involved in the regulation of NCX1 gene expression during stress. Conclusions: The gene expression and protein levels of the NCX1 are increased by the strong stress stimuli, e.g. hypoxia, or immobilization stress. The activity of NCX1 is decreased. Based on these results, we assume that the gene expression of NCX is increased as a consequence of suppressed activity of this transport system.

International audience; The regulation of cell growth and differentiation and also expression of a number of genes by retinoids are mediated by nuclear retinoid receptors (RARs and/or RXRs). In this study we investigated age-related alteration in both RAR and RXR receptor subtypes gene expression and tissue transglutaminase (tTG) activity before and after supplementation with 13-cis retinoic acid (13cRA) in human peripheral blood mononuclear cells (PBMCs). Healthy men (40) were divided in two groups according to their age (young group: 26.1 +/- 4.1 years and old group: 65.4 +/- 3.8 years). Each volunteer received 13cRA (Curacne (R), 0.5 mg/(kg day)) during a period of 4 weeks. We have shown that RXR beta expression was decreased significantly (p = 0.0108) in PBMCs of elderly men when compared to that of young volunteers. Distribution of retinoic acid receptor subtype expression in PBMCs was found in the order: RXRP > RAR gamma > RXR alpha > RAR alpha. The tTG activity in PBMCs reflected a trend to be enhanced after 13-cis retinoic acid supplementation. In conclusion, we demonstrate a significant decrease in the expression of RXRP subtype of rexinoid receptors in PBMCs of healthy elderly men. Our data suggest that in healthy elderly men reduction of RXRP expression in PBMCs might be a common feature of physiological senescence.

Phenylethanolamine N-methyltransferase (PNMT) is the final enzyme in the catecholamine synthesizing cascade that converts noradrenaline (NA) to adrenaline (Adr). Both of these catecholamines are physiologically important hormones and neurotransmitters in mammals with profound influence on the activity of the cardiovascular system. Although PNMT activity and gene expression have been reported in the neonatal and also adult rat heart, little is known about the identity of the cells expressing PNMT mRNA. In this study, we have shown that besides PNMT in neuronal and intrinsic cardiac cells, this enzyme is expressed also in rat cardiomyocytes, as shown by immunofluorescence in isolated cardiomyocytes. To determine which cells in the heart more sensitively show stress-induced changes in PNMT mRNA expression, we performed chemical sympathectomy by administration of 6-hydroxydopamine (6-OHDA), which destroys catecholaminergic terminals. We determined PNMT mRNA levels in the left atria and ventricles of control and stressed rats. In the rats treated with 6-OHDA, PNMT mRNA levels were not changed under normal, physiological conditions compared to vehicle treated rats. Similar results were observed on isolated cardiomyocytes from control and 6-OHDA treated rats. However, 6-OHDA treatment prevented immobilization-induced increase in PNMT mRNA expression. The results allow us to propose that in the heart, the immobilization-induced increase in PNMT gene expression is probably not in cardiomyocytes, but in neuronal cells.

Aim: This study was performed to examine thyrotropin-releasing hormone (TRH) secretion and regulation in rat heart. Methods: Expression of prepro-TRH gene in left atrium and left ventricle was studied by RT-PCR. TRH secretion from slices of left auricle and left ventricle in response to cell swelling (induced by hypotonic medium or ethanol in isosmotic medium), angiotensin II and losartan and their combinations was studied. Results: RT-PCR revealed two times higher prepro-TRH expression in left auricle than left ventricle. In transgenic rats with extra copy of mouse renin gene a marked increase of prepro-TRH expression in the heart was noted but the relative difference between left atrium and left ventricle persisted. The swelling stimulated TRH release from both left auricle and left ventricle and this stimulation could not be inhibited by bumetanide. Angiotensin II (10 nmol L -1 ) added into medium significantly decreased basal secretion of TRH. The inhibiting effect of Angiotensin II was prevented by 1 μmol L -1 losartan, an angiotensin II AT1 receptor blocker. When angiotensin II and hypotonicity were applied simultaneously, swelling-induced secretion persisted. Conclusion: TRH secretion from heart slices has attributes of regulated secretion - depending on the stimulus it could be either stimulated or inhibited. Renin positively affects prepro-TRH expression in the heart. Angiotensin II inhibits TRH secretion from heart tissue by a mechanism involving AT1 receptors. Swelling-induced TRH secretion overrides inhibitory effect of angiotensin II. Swelling could be a useful tool when natural or pharmacological secretagogue is unknown. Peptides and proteins released by swelling could be mediators of local and remote ischaemic preconditioning protecting from subsequent ischaemia.

Both A5 and A7 brainstem noradrenergic cell groups innervate dorsal horns of the spinal cord. Moreover, A5 cell group directly innervates sympathetic preganglionic neurons. Thus, A5 and A7 noradrenergic neurons could modulate the sympathoadrenal system (SAS) activity. We investigated the role of A5 and A7 noradrenergic cell groups in regulation of the SAS activity under control and stressful conditions. We evaluated the effect of electrolytical lesions of A5 or A7 cell groups and also the effect of bilateral brainstem cuts interrupting brainstem pathways on tyrosine hydroxylase gene expression in A5 and A7 areas and on the SAS activity measured by plasma epinephrine and norepinephrine levels. We have found that immobilization stress increases activity of the A5 and A7 brainstem areas and also levels of the gene expression of tyrosine hydroxylase, the rate-limiting catecholamine biosynthetic enzyme. Immobilization of sham-operated and brainstem pathways transected or A5 or A7 lesioned animals induced a similar, highly significant increase in plasma epinephrine and norepinephrine levels in both sham-operated and A5 or A7 destroyed or transected groups. Our data suggest that both A5 and A7 noradrenergic cell groups are activated during immobilization stress. However, transection of brainstem pathways innervating A5 and A7 neurons or lesion of A5 or A7 cell groups is not sufficient enough for changes in immobilization stress-induced activation of the SAS. We suggest that neither A5 and A7 noradrenergic neurons nor the transected brainstem pathways represent structures crucial for an activation of the SAS during immobilization stress. We hypothesize that during regulation of the stress response, various areas and pathways are involved and the elimination just one of them might be compensated by the remained intact areas and pathways.

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

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.

Most alcohol researchers do not address the effects of intoxication on the sympatho-adrenomedullary system response to stressful situations. We previously determined that rats consuming nearly 9 g ethanol (EtOH) per kg body weight per day in liquid diet form for 1 week increased adrenal gene expression of enzymes for catecholamine synthesis that was further elevated by acute IMMO. We hypothesized that the response to chronic mild stressors would also be altered after consumption of lower concentrations of EtOH in drinking water. Two experiments were conducted: 10% w/v for 4 weeks or 6% w/v for 7 weeks +/- wire mesh restraint (WMR). These were compared with ad libitum (adlib) and pair-fed control rats. Adrenal gene expression of catecholamine synthesizing enzymes was assayed. Tyrosine hydroxylase gene expression was elevated 80% to 90% by alcohol consumption in both experiments (P0.001) compared with adlib control rats. Dopamine betab-hydroxylase and phenylethanolamine-N-methyl transferase gene expressions were unaffected by 10% alcohol (P0.05) but were increased by 6% alcohol (P0.01). WMR decreased already elevated gene expression of all three enzymes. Pair feeding to 6% EtOH drinkers also increased gene expression for the three enzymes but was decreased by WMR, although not to levels of adlib rats. Increased gene expression for adrenal synthesis of catecholamines in response to repeated alcohol consumption increases the likelihood that the subject can respond physiologically to acute or chronic stress. This may have life-saving consequences in humans and in animals known to consume fermented materials and may contribute to increased aggressive behavior.

Stress is one of the major contributors to the development of cardiovascular disorders and psychiatric illnesses. Immobilization stress belongs to severe stressors and is known to activate several calcium transport systems. The aim of this work was to determine whether repeated immobilization stress changes mRNA and protein levels of the type 1 and 2 inositol-1,4,5-trisphosphate (IP 3 ) receptors in cardiac tissue. Rats were immobilized for 7 days, 2 h daily. After repeated immobilization, increased numbers of collagen fibers were accumulated in the heart atria compared to hearts of the control group of rats. Gene expression was determined after reverse transcription and subsequent real-time polymerase chain reaction, using SYBR Green fluorescent dye. Protein levels were determined by Western blot and hybridization with the primary antibody against IP 3 receptors. Contrary to single immobilization, repeated immobilization decreased a gene expression of the type 1 and 2 IP 3 receptors, and also protein levels of the IP 3 receptors. Although the physiologic relevance of our observations remains to be elucidated, we propose that the decrease in IP 3 receptors may have an impact on the development of the pathophysiologic changes in the heart.

Heart transplantation (HTx) is an accepted treatment for precisely defined patients with chronic congestive heart failure; however, as a result of the procedure, the graft is completely denervated. Our study focused on the catecholamine biosynthetic pathway, that is, the production of epinephrine, which is known to have positive chronotropic and inotropic effects on the heart. mRNA levels of the phenylethanolamine N-methyltransferase (PNMT), the enzyme catalyzing epinephrine synthesis in myocardial tissue, were determined in 18 patients (0 to 10 yr after HTx). Samples of myocardium were obtained from the right ventricle at the time of a routine endomyocardial biopsy performed for the diagnosis of graft rejection. Results were correlated with the following clinical parameters: heart rate, heart rate variability, blood pressure, graft systolic function, and the presence of the rejection. We observed that heart PNMT mRNA levels were significantly higher during the first 3 yr as compared to longer periods after HTx. Also, a decrease in the average heart rate and an increase in the heart rate variability were documented. Levels of the PNMT mRNA do not correlate with blood pressure, left ventricular systolic function at rest, and rejection. Thus, a gradual decrease of the heart rate and an increase in the heart rate variability after HTx is considered to be a sign of cardiac graft reinervation. We speculate that the increased PNMT transcription in human myocardium in early intervals after HTx reflects "autonomous sympathicotrophy." A decrease in the PNMT gene expression with the number of years after HTx could be a consequence of the reinnervation process.

Recently we have described the existence of phenylethanolamine N-methyltransferase (PNMT) mRNA in the heart of adult rats. In this study, we report the first data on distribution of the PNMT protein in rat hearts, which follows the distribution of PNMT mRNA (high levels in the atria and low levels in ventricles). The main aim of this study was to determine the localization of the PNMT mRNA in the heart and to examine whether gene expression of this enzyme is affected by immobilization (IMO) stress in a time-dependent manner. PNMT mRNA levels were detected in all seven studied parts of the heart (atria without and with intramural ganglion cells, ventricles, and septum), with the highest levels in the left atrium and its ganglionic part. Both Southern blot and sequencing verified the specificity of PNMT detected by RT-PCR. Single IMO for 2-h increased gene expression of PNMT, as determined by both RT-PCR and Real-Time PCR in the right and left atria. Surprisingly, the ganglionic parts of the atria did not respond to stress stimulation. Peak levels of PNMT mRNA were found in the 3-h interval after the IMO terminated, and also 24 h after the first or sixth IMO. Expression of aromatic L-amino acids decarboxylase and dopamine-beta-hydroxylase has also been detected in the heart of control and stressed rats. In the atria, the effect of stress is clearly modulated by glucocorticoids, since in mice with corticotrophin-releasing hormone knocked out gene the immobilization-induced increase in the PNMT mRNA levels seen in wild-type animals was abolished. Thus, our data have shown that gene expression of the PNMT is localized, not predominantly in cardiac ganglion cells, but in a wide range in atrial cardiomyocytes. Mechanism responsible for the regulation of stress-induced increase of PNMT gene expression in cardiac atria is clearly dependent on the presence of glucocorticoids.