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The sympathetic nervous system participates in the development and progression of several cancer types and this effect is mediated mainly via β-adrenergic signaling. However, the potential of β-adrenergic signaling blockade to prevent cancer development after exposure to carcinogens has not been investigated, yet. Therefore, in our study, we determined the effect of the β-blocker propranolol on the development and progression of mammary cancer induced in female rats by administration of the chemical carcinogen N-methyl-N-nitrosourea (MNU). The propranolol treatment (20 mg/kg body weight) started 12 days after MNU administration and lasted 10 weeks. We found that both saline and propranolol treatment significantly increased gene expression of the catecholamine-synthesizing enzyme tyrosine hydroxylase, indicating that repeated injection of saline or propranolol-induced stress in these two groups. However, compared to the vehicle-treated group, propranolol slightly delayed the development and moderately reduced the incidence of mammary carcinoma in animals. To evaluate the mechanisms mediating the effect of propranolol on the development of MNU-induced cancer, we investigated several parameters of the tumor microenvironment and found that propranolol increased gene expression of Casp3. Our data indicate that propranolol treatment that starts after exposure to carcinogens might represent a new, useful approach for preventing the development of cancer, especially in stressed individuals. However, the potential efficiency of propranolol treatment for preventing cancer development and progression in individuals exposed to carcinogens needs further investigation.

The corticotropin-releasing hormone family of peptides is involved in regulating the neuroendocrine stress response. Also, the vagus nerve plays an important role in the transmission of immune system-related signals to brain structures, thereby orchestrating the neuroendocrine stress response. Therefore, we investigated gene expression of urocortin 2 (Ucn2) and c-fos, a markers of neuronal activity, within the hypothalamic paraventricular nucleus (PVN), a brain structure involved in neuroendocrine and neuroimmune responses, as well as in the adrenal medulla and spleen in vagotomized rats exposed to immune challenge. In addition, markers of neuroendocrine stress response activity were investigated in the adrenal medulla, spleen, and plasma. Intraperitoneal administration of lipopolysaccharide (LPS) induced a significant increase of c-fos and Ucn2 gene expression in the PVN, and adrenal medulla as well as increases of plasma corticosterone levels. In addition, LPS administration induced a significant increase in the gene expression of tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) in the adrenal medulla. In the spleen, LPS administration increased gene expression of c-fos, while gene expression of TH and PNMT was significantly reduced, and gene expression of Ucn2 was not affected. Subdiaphragmatic vagotomy significantly attenuated the LPS-induced increases of gene expression of c-fos and Ucn2 in the PVN and Ucn2 in the adrenal medulla. Our data has shown that Ucn2 may be involved in regulation of the HPA axis in response to immune challenge. In addition, our findings indicate that the effect of immune challenge on gene expression of Ucn2 is mediated by vagal pathways.

Aberrant neurogenesis in the subventricular zone (SVZ) and hippocampus (HIP) contributes to schizophrenia pathogenesis. Haloperidol (HAL) and olanzapine (OLA), commonly prescribed antipsychotics for schizophrenia treatment, affect neurogenesis too. The effect of HAL and OLA on an mHippoE-2 cell line was studied in vitro where we measured the cell number and projection length. In vivo, we studied the gene expression of DCX, Sox2, BDNF, and NeuN in the SVZ and HIP in an MK-801-induced animal schizophrenia model. Cells were incubated with HAL, OLA, and MK-801 for 24, 48, and 72 h. Animals were injected for 6 days with saline or MK801 (0.5 mg/kg), and from the 7th day with either vehicle HAL (1 mg/kg) or OLA (2 mg/kg), for the next 7 days. In vitro, HAL and OLA dose/time-dependently suppressed cells’ proliferation and shortened their projection length. HAL/OLA co-treatment with MK-801 for 24 h reversed HAL’s/OLA’s inhibitory effect. In vivo, HAL and OLA suppressed DCX and NeuN genes’ expression in the HIP and SVZ. MK-801 decreased DCX and NeuN genes’ expression in the HIP and OLA prevented this effect. The data suggest that subchronic HAL/OLA treatment can inhibit DCX and NeuN expression. In an MK-801 schizophrenia model, OLA reversed the MK-801 inhibitory effect on DCX and NeuN and HAL reversed the effect on DCX expression; however, only in the HIP.

The mechanisms responsible for the anti-inflammatory effects of antidepressants are only partially understood. Published data indicate that the vagal anti-inflammatory pathway could be involved in mediating this effect. Therefore, we investigated the influence of subdiaphragmatic vagotomy on the anti-inflammatory effect of fluoxetine in rats injected with lipopolysaccharide (LPS) to induce an inflammatory response. The extent of this response was determined by measurement of TNF-α, IL-1β, and IL-6 plasma levels, along with gene expression of TNF-α, IL-1β, and IL-6 in the spleen and selected structures of the brain. To evaluate possible central mechanisms, c-fos mRNA levels were determined in the nucleus of the solitary tract, dorsal motor nucleus of the vagus, paraventricular hypothalamic nucleus, basolateral amygdala, central nucleus of the amygdala, hippocampus, and frontal cortex. We found that pretreatment with fluoxetine substantially prevented LPS-induced increases of pro-inflammatory cytokines in plasma and gene expression in the spleen and brain in animals with an intact vagus nerve. However, in vagotomized animals, fluoxetine pretreatment only partially attenuated the LPS-induced increase in these markers of peripheral inflammation. Our data has shown that fluoxetine exerts potent anti-inflammatory effects in both the periphery and brain. Moreover, we found that the peripheral anti-inflammatory action of fluoxetine is mediated, at least partially, by activation of a vagal anti-inflammatory pathway. The role of the vagus nerve in mediating the anti-inflammatory effects of antidepressants has been marginally explored and our findings highlight its potential contribution to this mechanism of action of antidepressants.

The corticotropin-releasing hormone family of peptides is involved in regulating the neuroendocrine stress response. Also, the vagus nerve plays an important role in the transmission of immune system-related signals to brain structures, thereby orchestrating the neuroendocrine stress response. Therefore, we investigated gene expression of urocortin 2 (Ucn2) and c-fos, a markers of neuronal activity, within the hypothalamic paraventricular nucleus (PVN), a brain structure involved in neuroendocrine and neuroimmune responses, as well as in the adrenal medulla and spleen in vagotomized rats exposed to immune challenge. In addition, markers of neuroendocrine stress response activity were investigated in the adrenal medulla, spleen, and plasma. Intraperitoneal administration of lipopolysaccharide (LPS) induced a significant increase of c-fos and Ucn2 gene expression in the PVN, and adrenal medulla as well as increases of plasma corticosterone levels. In addition, LPS administration induced a significant increase in the gene expression of tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) in the adrenal medulla. In the spleen, LPS administration increased gene expression of c-fos, while gene expression of TH and PNMT was significantly reduced, and gene expression of Ucn2 was not affected. Subdiaphragmatic vagotomy significantly attenuated the LPS-induced increases of gene expression of c-fos and Ucn2 in the PVN and Ucn2 in the adrenal medulla. Our data has shown that Ucn2 may be involved in regulation of the HPA axis in response to immune challenge. In addition, our findings indicate that the effect of immune challenge on gene expression of Ucn2 is mediated by vagal pathways.

CRH system integrates responses to stress challenges, whereas antipsychotics may impinge on this process. Effect of haloperidol (HAL) and aripiprazole (ARI) on chronic mild stress (CMS) induced neurobehavioral and CRH/CRHR1 system changes was studied in functionally interconnected rat brain areas including prefrontal cortex (PFC), bed nucleus of the stria terminalis (BNST), hypothalamic paraventricular nucleus (PVN), hippocampus (HIP), and amygdala (AMY). Animals were exposed to CMS for 3-weeks and since the 7th day of CMS injected with vehicle (VEH), HAL (1 mg/kg) or ARI (10 mg/kg) for 4-weeks. Expression levels of CRH, CRHR1, and c-fos genes and anxiety-like and anhedonia behavioural patterns were evaluated. CMS in VEH animals suppressed CRH gene expression in the PFC and BNST, c-fos expression in all areas, except HIP, and increased CRHR1 gene expression in the HIP. Antipsychotics decreased CRH gene expression in all areas, except HIP and by CMS elevated CRHR1 expression in the HIP (ARI also in AMY). CMS and antipsychotics decreased the sucrose preference. Aripiprazole prevented CRH expression decrease in the BNST and sucrose preference induced by CMS. Haloperidol increased time spent in the EPM open arms. These data indicate that HAL and ARI selectively influenced behavioural parameters and CRH/CRHR1 gene expression levels in CMS animals.

The brain exerts complex effects on the initiation and progression of cancer in the body. However, the influence of cancer localized in peripheral tissues on the brain has been only partially described. Therefore, we investigated gene expression in brain structures that participate in transmitting viscerosensory signals, regulating autonomic functions and food intake, as well as cognition in C57Bl/6J mice with B16-F10 melanoma. In addition, we investigated the relationship between peripheral inflammation and neuroinflammation. We found increased neuronal activity in the nucleus of the solitary tract of tumor-bearing mice, whereas neuronal activity in the A1/C1 catecholaminergic cell group, parabrachial nucleus, lateral hypothalamic area, ventromedial nucleus of the hypothalamus, paraventricular nucleus of the hypothalamus, and hippocampus was decreased. In the majority of investigated brain structures, we found increased gene expression of IL-1β, whereas gene expression of IL-6 and NF-κB was reduced or unchanged compared with controls. Melanoma-bearing mice also showed increased gene expression of tyrosine hydroxylase in the A1/C1 catecholaminergic cell group, nucleus of the solitary tract, and locus coeruleus, as well as reduced mRNA levels of hypocretin neuropeptide precursor protein in the lateral hypothalamic area, and proopiomelanocortin in the arcuate nucleus. In addition, we found reduced mRNA levels of Bcl-2, brain-derived neurotrophic factor, and doublecortin in the hippocampus. Our data indicate that skin melanoma induces complex changes in the brain, and these changes are most probably caused by cancer-related signals mediated by pro-inflammatory cytokines.

Objectives. Previously we have shown that 20 days after the tumor cells injection smaller melanomas have been developed in chemically sympathectomized mice in comparison with animals having intact sympathetic nervous system. However, it is known that chemical sympathectomy reduces the sympathetic neurotransmission only temporarily. In the present study, we monitored the survival of the sympathectomized mice with melanoma with an attempt to find out how long the suppressing effect of sympathectomy on the melanoma growth may endure. Methods. The chemical sympathectomy was performed by intraperitoneal injection of neurotoxin 6-hydroxydopamine in male C57BL/6J mice. Seven days later, the animals were injected subcutaneously with B16-F10 melanoma cells. Then, melanoma development, survival of the tumor-bearing mice and weight of the developed tumor mass were analyzed. Results. Sympathectomy delayed the development of the palpable tumors (18th day vs.14th day) and significantly prolonged the survival of the tumor-bearing mice (median 34 days vs. 29 days). However, the weight of the developed melanoma was significantly increased in the sympathectomized mice in comparison with the animals having intact sympathetic nervous system. Conclusions. The data of the present study showed that effect of the chemical sympathectomy, performed before the tumor growth induction, persisted even at the time when sympathetic nerves started to regenerate that resulted in a prolonged survival of the mice with melanoma. However, comparing to our previous study, in which we have shown a reduced tumor mass in earlier stages of the tumor growth, specifically 20 days after melanoma cells injection, now we indicate that in later stages of the melanoma progression, the tumor mass was significantly increased in sympathectomized animals. These contra-intuitive findings may indicate that interventions affecting the sympathetic nervous system may exert complex effect on the tumor progression. Based on these data we may suggest that the potential therapeutic interventions affecting the sympathetic signaling in the tumor tissue and its microenvironment should attenuate the sympathetic neurotransmission not only temporarily but till the complete regression of the tumor tissue.

Neuroendocrine stress response is regulated by several feedback loops. Since it has been suggested that afferent vagal pathways contribute to these feedback loops, we examined the effect of surgical subdiaphragmatic vagotomy on both baseline and stress-induced increases in plasma epinephrine, norepinephrine, and corticosterone levels in vagotomized and sham-operated Sprague Dawley rats. On either the 3rd or 14th day following vagotomy, the animals were exposed to acute immobilization stress and blood from the jugular vein was collected both before and during stress exposure. We found that vagotomy significantly enhanced immobilization-induced increases of plasma epinephrine, norepinephrine, and corticosterone levels on the 3rd day following surgery. However, on the 14th day following surgery, vagotomy enhanced only increase of plasma epinephrine levels in stressed rats. Our data indicate that afferent pathways of the vagus nerve are involved in negative feedback regulation of epinephrine secretion from the adrenal medulla during stressful conditions. We hypothesize that this feedback mechanism might be mediated by the binding of circulating epinephrine on β2-adrenergic receptors localized on sensory endings of the vagus nerve.

While the parasympathetic nervous system appears to be involved in the regulation of tumor progression, its exact role is still unclear. Therefore, using a rat BP6-TU2 fibrosarcoma tumor model, we investigated the effect of (1) reduction of vagal activity produced by subdiaphragmatic vagotomy; and (2) enhancement of vagal activity produced by continuous delivery of electric impulses to the cervical part of the vagus nerve on tumor development and survival of tumor-bearing rats. We also evaluated the expression of cholinergic receptors within in vitro cultivated BP6-TU2 cells. Interestingly, we found that both, vagal stimulation and subdiaphragmatic vagotomy slightly reduced tumor incidence. However, survival of tumor-bearing rats was not affected by any of the experimental approaches. Additionally, we detected mRNA expression of the α1, α2, α5, α7, and α10 subunits of nicotinic receptors and the M1, M3, M4, and M5 subtypes of muscarinic receptors within in vitro cultivated BP6-TU2 cells. Our data indicate that the role of the vagus nerve in modulation of fibrosarcoma development is ambiguous and uncertain and requires further investigation.

Recent data indicate that peripheral, as well as hypothalamic pro-inflammatory cytokines play an important role in the development of cancer cachexia. However, there are only a few studies simultaneously investigating the expression of inflammatory molecules in both the periphery and hypothalamic structures in animal models of cancer cachexia. Therefore, using the Yoshida ascites hepatoma rat's model of cancer cachexia we investigated the gene expression of inflammatory markers in the spleen along with the paraventricular and arcuate nuclei, two hypothalamic structures that are involved in regulating energy balance. In addition, we investigated the effect of intracerebroventricular administration of PS-1145 dihydrochloride (an Ikβ inhibitor) on the expression of selected inflammatory molecules in these hypothalamic nuclei and spleen. We observed significantly reduced food intake in tumor-bearing rats. Moreover, we found significantly decreased expression of IL-6 in the spleen as well as decreased NF-κB in the paraventricular nucleus of rats with Yoshida ascites hepatoma. Similarly, expression of TNF-α, IL-1β, NF-κB, and COX-2 in the arcuate nucleus was significantly reduced in tumor-bearing rats. Administration of PS-1145 dihydrochloride reduced only the gene expression of COX-2 in the hypothalamus. Based on our findings, we suggest that the growing Yoshida ascites hepatoma decreased food intake by mechanical compression of the gut and therefore this model is not suitable for investigation of the inflammation-related mechanisms of cancer cachexia development.

The mechanisms underlying stress-related modulation of immune function via urocortin 1 and urocortin 2 have been only vaguely described. Therefore, we investigated the effect of LPS injection or immobilization stress on gene expression of urocortin 1 and urocortin 2 in the rat spleen, along with the potential involvement of glucocorticoids. Our data showed: a) different regulation of urocortin 1 and urocortin 2 gene expression in the rat spleen under different stressful conditions (LPS vs. immobilization stress) and b) diverse effects of stress-induced adrenal glucocorticoids on this process. Our findings indicate a specific, rather than general regulation of splenic immune function by urocortins during stressful conditions.

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.

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.

Exposure to severe stress leads to development of neuropsychiatric disorders, including depression and Post-Traumatic Stress Disorder (PTSD) in at-risk individuals. Neuropeptide Y (NPY) is associated with resilience or improved recovery. Therefore exogenous administration to the brain has therapeutic potential although peripheral administration can trigger undesirable side effects. Here, we established conditions with intranasal (IN) NPY infusion to rats to obtain CSF concentrations in the proposed anxiolytic range without significant change in plasma NPY. Rats were pretreated with IN NPY or vehicle before exposure to single prolonged stress (SPS) animal model of PTSD and compared to untreated controls. The IN NPY appeared to lessen the perceived severity of stress, as these animals displayed less time immobile in forced swim part of the SPS. Thirty minutes after SPS the elevation of plasma adrenocorticotropic hormone (ACTH) and corticosterone was not as pronounced in NPY-infused rats and the induction of tyrosine hydroxylase (TH) in locus coeruleus (LC) was attenuated. Seven days after SPS, they displayed lower depressive-like behavior on Forced Swim Test and reduced anxiety-like behavior on Elevated Plus Maze. The prolonged effect of SPS on Acoustic Startle Response was also lower in NPY-infused rats. Plasma ACTH, corticosterone, and hippocampal glucocorticoid receptor levels were significantly above controls only in the vehicle – but not IN NPY-treated group 1 week after SPS. Baseline TH mRNA levels in LC did not differ among groups, but increased with forced swim in the vehicle – but not NPY-pretreated animals. Administration of IN NPY after exposure to SPS led to similar, but not identical, reduction in development of anxiety, depressive-like behavior and hyperarousal. The results show that single IN NPY can alter stress-triggered dysregulation of the hypothalamic–pituitary–adrenal (HPA) axis and activation of central noradrenergic activity. These findings provide proof of concept for potential of IN NPY for non-invasive prophylactic treatment or early intervention in response to traumatic stress.

The corticotropin-releasing hormone (CRH) family regulates the endocrine stress response. Here, we examined the effect of immobilization stress (IMO) on gene expression of adrenomedullary CRH family members. Urocortin 2 (Ucn2) has the highest basal gene expression and is increased by > 30-fold in response to single IMO and about 10-fold after six daily repeated IMO. IMO also induced a smaller rise in CRH (six-fold) and CRH receptor type 1 (CRHR1; two-fold with single IMO). The influence of glucocorticoids was examined. Dexamethasone (DEX) or corticosterone greatly increased Ucn2 mRNA levels in PC12 cells in a dose-dependent manner. The DEX elicited rise in Ucn2 was abolished by actinomycin D pre-treatment, indicating a transcriptionally mediated response. DEX also triggered a rise in CRHR1 and lowered CRH mRNA levels. In CRH-knockout mice, where the IMO-induced rise in corticosterone was attenuated, the response of IMO on Ucn2, as well as CRHR2 mRNAs was absent. Overall, the results suggest that the stress-triggered rise in glucocorticoids is involved in the large induction of Ucn2 mRNA levels by IMO, which may allow Ucn2 to act in an autocrine/paracrine fashion to modulate adrenomedullary function, or act as an endocrine hormone.

Accumulated evidence indicates that sympathetic nerves may potentiate tumor growth, including melanoma. To elucidate possible mechanisms for this effect, we performed chemical sympathectomy by intraperitoneal (i.p.) injection of the neurotoxin 6-hydroxydopamine hydrobromide (100 mg/kg of body weight); in nine adult male C57BL/6J mice; nine control mice received i.p. vehicle (VEH). Seven days later, all mice were injected subcutaneously with 3 × 10(3) B16-F10 melanoma cells. Mice were euthanized 20 d after injection of melanoma cells, for measurement of tumor weight and expression of genes related to sympathetic signaling, apoptosis, hypoxia and angiogenesis in tumor tissue. To assess potential involvement of the hypothalamo-pituitary-adrenocortical axis in the effect of sympathectomy on melanoma growth, concentrations of plasma corticosterone and level of glucocorticoid receptor mRNA in tumor tissue were determined. We found that sympathectomy significantly attenuated melanoma growth (tumor weight 0.29 ± 0.16 g versus 1.02 ± 0.30 g in controls; p 0.05). In tumor tissue from sympathectomized mice, we found significantly increased gene expression (measured by real-time PCR), relative to VEH-injected controls, of tyrosine hydroxylase, neuropeptide Y and glucocorticoid receptor (all p 0.05), and alpha1, beta1 and beta3 adrenergic receptors (all p 0.025), and factors related to apoptosis (Bcl-2 and caspase-3; p 0.05) and hypoxia (hypoxia inducible factor 1 alpha) (p = 0.005). Plasma corticosterone concentrations were significantly elevated (p 0.05) in these mice. Our findings indicate that sympathectomy induces complex changes in the tumor microenvironment reducing melanoma growth. Such complex changes should be considered in the prediction of responses of cancer patients to interventions affecting sympathetic signaling in tumor tissue and its environment.

Decapitation induces excessive increases in the plasma catecholamine levels in the blood obtained from the body of sacrificed animals. However, the mechanisms responsible for this phenomenon remain unknown. We investigated the neuronal pathways responsible for such increases in the plasma epinephrine and norepinephrine levels. To investigate this, we completely or partially transected the spinal cord at the upper thoracic level in Sprague–Dawley rats. Functionally, the successfulness of interruption of interconnections between brain and spinal preganglionic neurons was confirmed by almost complete blockade of immobilization-induced increases in the catecholamine plasma levels. After complete transection of the spinal cord and subsequent decapitation of rats, we found significant reduction of decapitation-induced effects on the catecholamine release from the adrenal medulla and sympathetic nerve endings. Our data indicate that the activity of the sympathoadrenal system is under significant inhibitory influence from the brain structures regulating the activity of sympathetic preganglionic neurons.

Hypertension is a serious risk factor for cardiovascular disease, and the angiotensinogen (AGT) gene locus is associated with human essential hypertension. The human AGT (hAGT) gene has an A/G polymorphism at -6, and the -6A allele is associated with increased blood pressure. However, transgenic mice containing 1.2 kb of the promoter with -6A of the hAGT gene show neither increased plasma AGT level nor increased blood pressure compared with -6G. We have found that the hAGT gene has three additional SNPs (A/G at -1670, C/G at -1562, and T/G at -1561). Variants -1670A, -1562C, and -1561T almost always occur with -6A, and variants -1670G, -1562G, and -1561G almost always occur with -6G. Therefore, the hAGT gene may be subdivided into either -6A or -6G haplotypes. We show that these polymorphisms affect the binding of HNF-1α and glucocorticoid receptor to the promoter, and a reporter construct containing a 1.8-kb hAGT gene promoter with -6A haplotype has 4-fold increased glucocorticoid-induced promoter activity as compared with -6G haplotype. In order to understand the physiological significance of these haplotypes in an in vivo situation, we have generated double transgenic mice containing either the -6A or -6G haplotype of the hAGT gene and the human renin gene. Our ChIP assay shows that HNF-1α and glucocorticoid receptor have stronger affinity for the chromatin obtained from the liver of transgenic mice containing -6A haplotype. Our studies also show that transgenic mice containing -6A haplotype have increased plasma AGT level and increased blood pressure as compared with -6G haplotype. Our studies explain the molecular mechanism involved in association of the -6A allele of the hAGT gene with hypertension.

Vesicular monoamine transporters (VMATs) mediate transmitter uptake into neurosecretory vesicles. There are two VMAT isoforms, VMAT1 and VMAT2, encoded by separate genes and displaying different cellular distributions and pharmacological properties. We examined the effect of immobilization stress (IMO) on expression of VMATs in the rat adrenal medulla. Under basal conditions, VMAT1 is widely expressed in all adrenal chromaffin cells, while VMAT2 is co-localized with tyrosine hydroxylase (TH) but not phenylethanolamine N-methyltransferase (PNMT), indicating its expression in norepinephrine (NE)-, but not epinephrine (Epi)-synthesizing chromaffin cells. After exposure to IMO, there was no change in levels of VMAT1 mRNA. However, VMAT2 mRNA was elevated after exposure of rats to 2 h IMO once (1× IMO) or daily for 6 days (6× IMO). The changes in VMAT2 mRNA were reflected by increased VMAT2 protein after the repeated IMO. Immunofluorescence revealed an increased number of cells expressing VMAT2 following repeated IMO and its colocalization with PNMT in many chromaffin cells. The findings suggest an adaptive mechanism in chromaffin cells whereby enhanced catecholamine storage capacity facilitates more efficient utilization of the well-characterized heightened catecholamine biosynthesis with repeated IMO stress.

Corticotropin-releasing factor is well known activator of the hypothalamic-pituitary-adrenocortical axis, that represents crucial system participating on stress response of the organism. Urocortins are members of the corticotropin-releasing factor family of peptides with proposed effects on neuroendocrine and behavioral stress response mechanisms. Urocortin 2, one of three known urocortins, is present in central and peripheral stress response system and its expression can be augmented by glucocorticoids. In the present study we have examined how glucocorticoid withdrawal affects urocortin 2 gene expression after acute immobilization in the adrenal medulla and selected brain areas in rats. We used pharmacological adrenalectomy to block synthesis of corticosterone. Our results show that the immobilization-induced rise in urocortin 2 mRNA levels in rat adrenal medulla was not inhibited by glucocorticoid withdrawal. On the other hand, observed changes in the brain indicate that the effect of stress and pharmacological adrenalectomy on urocortin 2 gene expression is site-specific. While in the paraventricular nucleus and locus coeruleus the immobilization induced rise of urocortin 2 was not inhibited by pharmacological adrenalectomy in the arcuate nucleus and central amygdala it was. Moreover, we have seen a significant depletion of urocortin 2 plasma levels after immobilization. The immobilization induced rise of urocortin 2 gene expression in rat adrenal medulla and brain areas regulating stress response pathways and preservation of its induction after adrenalectomy suggests a role of urocortin 2 in the neuroendocrine stress response of an organism. This article is protected by copyright. All rights reserved.

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.

Although stress is tightly connected with elevated levels of catecholamines, stress effects on target structures of catecholamine action--adrenoceptors (ARs)--has not been deeply studied yet. Similarly, very little is known about changes of muscarinic receptors (MRs) during stress. We determined changes in these receptors in the individual parts of the heart (right atria and ventricles) of animals (rats and mice) exposed to a single and repeated immobilization stress. Changes of tissue catecholamines, beta(2)-AR gene expression, protein levels, and binding sites were determined in rat right ventricles, and changes in beta(1)-, beta(2)-, and beta(3)-AR gene expression were followed in murine right atria. Tissue catecholamines were elevated, while beta(2)-AR mRNA levels and beta(2)-AR proteins and binding were decreased, in rat right ventricles. In murine right atria, beta(1)- and beta(2)-AR gene expression was elevated, while beta(3)-AR mRNA levels and M(2)-MR were reduced. Taken together, our data show that interaction of AR and MR is important for the organism coping with stress and that different heart regions reveal distinct reactions to stress.

This study aimed to contribute to the knowledge of cardiovascular regulation associated with repeated exercise by evaluating untraditional parameters in the model of voluntary wheel running. Possible changes in cardiac phenylethanolamine N-methyltransferase (PNMT) gene expression were evaluated using running for 3 weeks in four rat strains, and the hypothesis that voluntary wheel running modifies mean arterial pressure (MAP) responses to oxytocin administration was verified. Running activity increased gradually and was high in spontaneously hypertensive rats (SHR) and Sprague-Dawley rats, while low in Wistar rats. The levels of PNMT mRNA in the left but not right atrium increased significantly in rat strains exhibiting high physical activity. Concentrations of PNMT mRNA were significantly higher in SHR and Sprague-Dawley compared to those in Wistar rats, which ran much shorter distances. MAP was found to be higher in rats exposed to voluntary running, which might be the result of the cessation of the exercise 24 h before the measurements. Oxytocin treatment (5 microg/kg and 30 microg/kg i.v.) resulted in significant increase in MAP in both control and running animals in a dose-dependent manner. In conclusion, voluntary wheel running failed to modify sensitivity to cardiovascular action of oxytocin but resulted in increased gene expression of PNMT in left, but not right, heart atrium in a running activity-dependent manner.

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.

Subcutaneous injection of diluted formalin (0.2 ml of 4% solution/100 g BW) can influence the increase of plasma epinephrine levels in rats exposed to exteroceptive (handling, immobilization), as well as to interoceptive stressors (insulin-induced hypoglycemia), without having any effect on norepinephrine release. In the present studies, the effect of the above-mentioned stressors has been investigated on formalin-induced prolactin (PRL) and corticosterone secretion. Administrations of formalin via chronically implanted subcutaneous cannula into the hind limb without handling induce an immediate increase in both plasma PRL and corticosterone levels. While PRL concentration reaches its peak value within 5 min then returns to the basal level by the end of the 30th min, corticosterone level also starts to rise immediately after formalin administration reaching its highest concentration within 15–30 min, but it remains at this high level during the next 60 min, then it declines and returns to the pre-injection level. Application of formalin to animals exposed to different heterotypic stressors (like handling or insulin-induced hypoglycemia) produces an attenuated PRL response, while plasma corticosterone levels induced by the same nociceptive component remained unchanged. Combinations of formalin injection with immobilization also show an attenuated PRL response. The present data indicate that plasma PRL response to formalin is related to its acute nociceptive phase, and application of different stressors prior to formalin injection significantly attenuate plasma PRL levels, while it does not influence corticosterone responses.

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.

We have previously described the development of substantial, but reversible obesity in Wistar rats fed with palatable liquid nutrition (Fresubin). In this study, we investigated changes in serum hormone levels, glycemia, fat mass, adipocyte size, and gene expression of adipokines and inflammatory markers in adipose tissue of Wistar rats fed by Fresubin (i) for 5 months, (ii) up to 90 days of age, or (iii) after 90 days of age to characterize metabolic alterations and their reversibility in rats fed with Fresubin. An intra-peritoneal glucose tolerance test was also performed to determine levels of serum leptin, adiponectin, insulin, and C-peptide in 2- and 4-month-old animals. In addition, mesenteric and epididymal adipose tissue weight, adipocyte diameter, and gene expression of pro- and anti-inflammatory adipokines and other markers were determined at the end of the study. Chronic Fresubin intake significantly increased adipocyte diameter, reduced glucose tolerance, and increased serum leptin, adiponectin, insulin, and C-peptide levels. Moreover, gene expression of leptin, adiponectin, CD68, and nuclear factor kappa B was significantly increased in mesenteric adipose tissue of Fresubin fed rats. Monocyte chemotactic protein 1 messenger RNA (mRNA) levels increased in mesenteric adipose tissue only in the group fed Fresubin during the entire experiment. In epididymal adipose tissue, fatty acid binding protein 4 mRNA levels were significantly increased in rats fed by Fresubin during adulthood. In conclusion, chronic Fresubin intake induced complex metabolic alterations in Wistar rats characteristic of metabolic syndrome. However, transition of rats from Fresubin to standard diet reversed these alterations.

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.

The involvement of the nonclassical renin-angiotensin system (RAS) in the adrenomedullary response to stress is unclear. Therefore, we examined basal and immobilization stress (IMO)-triggered changes in gene expression of the classical and nonclassical RAS receptors in the rat adrenal medulla, specifically the angiotensin II type 2 (AT2) and type 4 (AT4) receptors, (pro)renin receptor [(P)RR], and Mas receptor (MasR). All RAS receptors were identified, with AT2receptor mRNA levels being the most abundant, followed by the (P)RR, AT1Areceptor, AT4receptor, and MasR. Following a single IMO, AT2and AT4receptor mRNA levels decreased by 90 and 50%, respectively. Their mRNA levels were also transiently decreased by repeated IMO. MasR mRNA levels displayed a 75% transient decrease as well. Conversely, (P)RR mRNA levels were increased by 50% following single or repeated IMO. Because of its abundance, the function of the (P)RR was explored in PC-12 cells. Prorenin activation of the (P)RR increased phosphorylation of extracellular signal-regulated kinase 1/2 and tyrosine hydroxylase at Ser31, likely increasing its enzymatic activity and catecholamine biosynthesis. Together, the broad and dynamic changes in gene expression of the nonclassical RAS receptors implicate their role in the intricate response of the adrenomedullary catecholaminergic system to stress.

The sympathetic nervous system regulates many immune functions and modulates the anti-tumor immune defense response, too. Therefore, we studied the effect of 6-hydroxydopamine induced sympathectomy on selected hematological parameters and inflammatory markers in rats with Yoshida AH130 ascites hepatoma. We found that chemically sympathectomized tumor-bearing rats had significantly increased neutrophil-to-lymphocyte ratio, leukocyte-to-lymphocyte ratio, and plasma levels of tumor necrosis factor alpha. Although our findings showed that sympathetic denervation in tumor-bearing rats led to increased neutrophil-to-lymphocyte ratio, that is an indicator of the disease progression, we found no significant changes in tumor growth and survival of sympathectomized tumor-bearing rats.

Bradykinin, acting via the bradykinin B2 receptor (B2R), is a potent stimulator of adrenomedullary catecholamine biosynthesis and release and likely plays an important role in the adrenomedullary stress response. However, the effects of stress on the expression of this receptor in the adrenal medulla are currently unclear. Here, we examined the changes in adrenomedullary B2R gene expression in male rats in response to single (1 time) and repeated (6 times) exposure to 2 hours immobilization stress (IMO). Immediately after 1 or 6 times IMO, B2R mRNA levels were increased by 9-fold and 7-fold, respectively, and returned to unstressed control levels 3 hours later. This large, but transient, increase in mRNA elicited a doubling of protein levels 3 hours after the stress exposure. Next, the role of the hypothalamic-pituitary-adrenocortical axis in the stress-induced upregulation of B2R gene expression was examined. Treatment with endogenous (corticosterone) and synthetic (dexamethasone) glucocorticoids dose-dependently increased B2R mRNA levels in adrenomedullary-derived PC12 cells. Furthermore, cortisol supplementation at levels mimicking stress exposure elevated B2R mRNA levels in the adrenal medulla of hypophysectomized rats. In response to 1 exposure to IMO, the stress-triggered rise in plasma corticosterone and adrenomedullary B2R mRNA levels was attenuated in CRH-knockout mice and absent in pharmacologically adrenalectomized rats, indicating a requirement for glucocorticoids in the upregulation of B2R gene expression with stress. Overall, the increase in B2R gene expression in response to the stress-triggered rise in glucocorticoids likely enhances catecholamine biosynthesis and release and may serve as an adaptive response of the adrenomedullary catecholaminergic system to stress.

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.

While the renin–angiotensin system is important for adrenomedullary responses to stress, the involvement of specific angiotensin II (Ang II) receptor subtypes is unclear. We examined gene expression changes of angiotensin II type 1A (AT1A) and type 2 (AT2) receptors in rat adrenal medulla in response to immobilization stress (IMO). AT2receptor mRNA levels decreased immediately after a single 2-h IMO. Repeated IMO also decreased AT2receptor mRNA levels, but the decline was more transient. AT1Areceptor mRNA levels were unaltered with either single or repeated IMO, although binding was increased following repeated IMO. These effects of stress on Ang II receptor expression may alter catecholamine biosynthesis, as tyrosine hydroxylase and dopamine β-hydroxylase mRNA levels in PC12 cells are decreased with Ang II treatment in the presence of ZD7155 (AT1receptor antagonist) or with CGP42112 (AT2receptor agonist) treatment. Involvement of stress-triggered activation of the hypothalamic–pituitary–adrenocortical or sympathoadrenal axis in AT2receptor downregulation was examined. Cultured cells treated with the synthetic glucocorticoid dexamethasone displayed a transcriptionally mediated decrease in AT2receptor mRNA levels. However, glucocorticoids are not required for the immediate stress-triggered decrease in AT2receptor gene expression, as demonstrated in corticotropin-releasing hormone knockout (CrhKO) mice and hypophysectomized rats, although they can regulate basal gene expression. cAMP and pituitary adenylate cyclase-activating polypeptide also reduced AT2receptor gene expression and may mediate this response. Overall, the effects of stress on adrenomedullary AT1Aand AT2receptor expression may contribute to allostatic changes, such as regulation of catecholamine biosynthesis.

With acute stress, the release of adrenomedullary catecholamines is important for handling the emergency situation. However, when chronic or repeated, stress alters the allostatic load and leads to a hyperadrenergic state, resulting in the development or worsening of a wide range of diseases. To help elucidate the mechanism, we examined the effects of single and repeated immobilization stress on gene expression of components of neurosecretory vesicles in the adrenal medulla. Male Sprague–Dawley rats were exposed to immobilization stress once for 2 h (1× IMO) or daily for six consecutive days (6× IMO). Compared to unstressed controls, 1× IMO elevated gene expression of vesicular monoamine transporter 2 (VMAT2). In response to 6× IMO, not only was VMAT2 mRNA still elevated, but chromogranin A (CgA) and chromogranin B (CgB) mRNAs were also increased two to three-fold above basal levels. To investigate the possible role of the hypothalamic–pituitary–adrenal axis in the induction of VMAT2, PC12 cells were treated with the synthetic glucocorticoid dexamethasone, which was found to elevate VMAT2 mRNA expression. The findings suggest that following repeated stress, elevations of various components of neurosecretory vesicles in the adrenal can facilitate more efficient utilization of the well-characterized heightened catecholamine levels.

Rats subjected to various accelerations (+G) exhibited increased levels of plasma epinephrine (EPI), norepinephrine (NE), and corticosterone. However, the collection of blood was performed after a centrifugation finished, and therefore the levels could be affected by the process of deceleration. The aim of this study was to evaluate plasma EPI, NE, and corticosterone levels in blood collected directly during centrifugation after reaching different G (2-6), using newly developed remote-controlled equipment. Animals placed into the centrifuge cabins had inserted polyethylene tubing in the tail artery, which was connected with a preprogrammed device for blood withdrawals. Plasma EPI, NE, and corticosterone levels were measured at different time intervals of hypergravity of 2-6G. Plasma EPI levels showed a huge, hypergravity-level-dependent increase. After the last blood collection was completed during hypergravity, the centrifuge was turned off and another blood sampling was performed immediately after the centrifuge stopped (10 min). In these samples, plasma EPI showed significantly lower levels compared to centrifugation intervals. Plasma NE levels were significantly increased after 6G only. The increase in plasma corticosterone was dependent on level of G, however after the centrifuge stopped, corticosterone levels remained elevated. Thus, our data show that hypergravity highly activates the adrenomedullary and hypothalamo-pituitary-adrenocortical systems, whereas the sympathoneural system is activated only at high hypergravity. Immediately after centrifugation is over, EPI levels quickly return to control values. Our technique of blood collection during centrifugation allows assessment of the real hormonal levels at the particular hypergravity value.

Immobilization represents a strong stressor inducing a profound increase in plasma epinephrine and norepinephrine levels. We have previously demonstrated that a subcutaneous injection of formalin (0.2 mL of 4% solution/100 g bw) attenuated the immobilization-induced elevation of plasma epinephrine levels in rats. In the present study, we investigated whether other painful and stressful stimuli, such as capsaicin, hydrochloric acid, mechanical pressure, heat, and cold, might also attenuate the increase of plasma epinephrine in rats exposed to acute immobilization stress. With the exception of formalin, all of the painful stimuli applied failed to attenuate the increase of plasma epinephrine levels in immobilized animals. Our data suggest that the attenuation of an immobilization-induced increase in plasma epinephrine levels is specific for subcutaneous formalin administration.

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.