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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 (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.

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.

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.

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.

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.