Identification of phenylethanolamine N-methyltransferase gene expression in stellate ganglia and its modulation by stress.

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.

Gene expression of phenylethanolamine N-methyltransferase in corticotropin-releasing hormone knockout mice during stress exposure.

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. 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. 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. 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.

Phenylethanolamine N-methyltransferase gene expression in transplanted human heart.

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 < .01). HR variability in the low frequency band of the power spectrum (LF) was lower among patients during the first 3 years after HTx (2.73 +/- 0.31 vs 5.67 +/- 0.69 ms; P < .01). A significant negative linear correlation was observed between PNMT mRNA and LF (P = .05; r = -0.47). No association was noted between HR, BP, parameters of systolic or diastolic function, standard deviation of all RR intervals, or high frequency (0.15-0.40 Hz) bands of the 24-hour RR interval power spectrum. CONCLUSION: This work provides the first evidence of the presence of local PNMT transcription in human heart after HTx. Appearance of PNMT transcription in the myocardium after HTx may reflect autonomous "sympathicotrophy." Decreased PNMT expression with time elapsed after HTx suggests graft reinervation.

Quantitative evaluation of catecholamine enzymes gene expression in adrenal medulla and sympathetic Ganglia of stressed rats.

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.

Repeated immobilization stress reduces the gene expression of the type 1 and 2 IP3 receptors in stellate ganglia.

Inositol 1,4,5-trisphosphate (IP(3)) is one of the second messengers produced by phosphoinositid hydrolysis and triggers IP(3) receptor (IP(3)R) mediated calcium release from intracellular pools. To determine whether immobilization stress affects the gene expression and protein level of IP(3)R in stellate ganglia, animals were immobilized once for 2h and/or for 7 days, 2h daily. After decapitation, stellate ganglia were extirpated and the gene expression of IP(3) receptors was evaluated. Protein levels of IP(3) receptor were measured by Western blot analysis using the antibody against IP(3) receptor. In the present work, we clearly show that type 1 and 2 IP(3) receptors, but not the type 3 IP(3) receptor, are expressed in stellate ganglia. Both types, type 1 and 2 IP(3) receptors, are not significantly affected by single 2h immobilization stress on mRNA and protein level. However, gene expression of both these types is significantly reduced by repeated immobilization stress for 7 days, 2h daily. The IP(3) receptor protein is reduced as well. Physiological relevance of our observations remains to be elucidated.

Quantitation of changes in gene expression of norepinephrine biosynthetic enzymes in rat stellate ganglia induced by stress.

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-ss-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 2h, 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/microg 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/microg 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 measured 22 h after the termination of the stress stimulus. Repeated immobilization (7 days, 2h 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, 2h 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.

[Catecholamine synthesis and expression of enzymes of the catecholamine pathway in the rat heart at rest and during stress]. / Syntéza katecholamínov a expresia enzýmov katecholaminergnej dráhy v srdci potkana v kl'ude a za stresu.

Catecholamines participate in a variety of cell regulations and physiological mechanisms, but also in the development of neurological, psychiatrical, endocrine and cardiovascular diseases. Diseases of the cardiovascular system, such as hypertension, myocardial infarction, ischaemia, etc. represent a serious medical problem and a frequent cause of the human death. It is alarming that development of these diseases depends preferentially on the wrong life-style and affect lower age groups. One of the factors that participate on unwilling increase of the incidence of cardiovascular diseases is the stress. Under the stress, organism mobilizes its energetical sources, increases cardiac performance and activates other processes, which enable to handle this load. Catecholamines belong to the important mediators of the stress response. On the one side they are required to buffer the stress situation, but the pathological changes in the regulation of their synthesis, secretion and action significantly participate on the development of several diseases (e.g. also of the cardiovascular origin). The aim of this review is to show the role of catecholamines in the heart not only under the physiological, but also in pathophysiological conditions. Stress as a modern civilization factor participates on the development of several diseases. Understanding of the mechanism of development of these diseases is the first step to the development of an appropriate therapy. Changes in the catecholamine levels during stress, as well as enzymes, which participate on the synthesis of catecholamines, are undoubtedly the important part of this mechanism.

Identification of tyrosine hydroxylase gene expression in rat spleen.

This study was aimed to identify tyrosine hydroxylase (TH) gene expression in the rat spleen under basal and stress conditions. Using the reverse transcription polymerase chain reaction we did not detect TH mRNA in rat spleen either in control, or immobilized animals. Semi-nested PCR revealed a clear signal, demonstrating that TH mRNA is formed in the spleen, although in low abundance. We also detected both, TH immunoreactive protein and TH activity in the rat spleen that were in higher abundance than expected from the mRNA levels. This study identifies, for the first time, TH gene expression in rat spleen. Since TH protein and activity are present in the spleen in much higher abundance compared to corresponding mRNA, the majority of TH protein is most probably supplied by the sympathetic innervation of spleen.

Existence of cardiac PNMT mRNA in adult rats: elevation by stress in a glucocorticoid-dependent manner.

Phenylethanolamine N-methyltransferase (PNMT) is the enzyme that synthesizes epinephrine from norepinephrine. The aim of this study was to determine potential PNMT gene expression in the cardiac atria and ventricles of adult rats and to examine whether the gene expression of this enzyme is affected by immobilization stress. PNMT mRNA levels were detected in all four parts of the heart, with the highest level in the left atrium. Both Southern blot and sequencing verified the specificity of PNMT detected by RT-PCR. Single immobilization for 2 h increased gene expression of PNMT in both atria and ventricles. In atria, this effect was clearly modulated by glucocorticoids, because either adrenalectomy or hypophysectomy prevented the increase in PNMT mRNA levels in response to immobilization stimulus. This study establishes, for the first time, that PNMT gene expression occurs in cardiac atria and also, to a small extent, in ventricles of adult rats. Immobilization stress increases gene expression in atria and ventricles. This increase requires an intact hypothalamus-pituitary-adrenocortical axis, indicating the involvement of glucocorticoids.

Gene expression of catecholamine synthesizing enzymes in A5 cell group and modulation of tyrosine hydroxylase mRNA by immobilization stress.

OBJECTIVE: The A5 group of noradrenergic neurons plays a key role in autonomic mechanisms like cardiovascular regulation, nociception and respiration. The aim of this work was to detect the gene expression of catecholamine synthesizing enzymes in A5 brain nuclei. METHODS: The gene expression of. tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH) and phenylethanolamine N-methyl-transferase (PNMT) in A5 brain nuclei was estimated. We also investigated various time intervals after the end of the single two-hour immobilization, as well as the effect of short-term repeated immobilization (120 min daily for 7 days) on tyrosine hydroxylase gene expression, the rate-limiting enzymes in catecholamines biosynthesis, in the A5 cell group. For all experiments, reverse transcription with subsequent polymerase chain reaction (RT-PCR) was used. RESULTS: As expected, we detected a clear signal for TH and DBH mRNA but no signal for PNMT mRNA. Both, single and repeated immobilization stress exposure increased significantly the gene expression of TH in A5 area. Maximal elevation in TH mRNA levels occurred after single immobilization for two hours and subsequent decapitation 24 hours later. CONCLUSIONS: In this study we detected for the first time the presence of DBH mRNA in micro dissected A5 cell group. We also showed how the gene expression of tyrosine hydroxylase changed with the function of time after the single immobilization exposure. Thus, TH mRNA in A5 cell group is modulated by immobilization stress in a time-dependent manner.