Phenylethanolamine N-methyltransferase mRNA Levels Research Articles

Overexpression of miR-375 and L-type Amino Acid Transporter 1 in Pheochromocytoma and Their Molecular and Functional Implications.

Pheochromocytoma (Pheo) is a tumor derived from chromaffin cells. It can be studied using 18F-dihydroxyphenylalanine (DOPA)—positron emission tomography (PET) due to its overexpression of L-type amino acid transporters (LAT1 and LAT2). The oncogenic pathways involved are still poorly understood. This study examined the relationship between 18F-DOPA-PET uptake and LAT1 expression, and we explored the role of miR-375 and putative target genes. A consecutive series of 58 Pheo patients were retrospectively analyzed, performing 18F-DOPA-PET in 32/58 patients. Real-time quantitative PCR was used to assess the expression of LAT1, LAT2, phenylethanolamine N-methyltransferase (PNMT), miR-375, and the major components of the Hippo and Wingless/Integrated pathways. Principal germline mutations associated with hereditary Pheo were also studied. Pheo tissues had significantly higher LAT1, LAT2, and PNMT mRNA levels than normal adrenal tissues. MiR-375 was strongly overexpressed. Yes-associated protein 1 and tankyrase 1 were upregulated, while beta-catenin, axin2, monocarboxylate transporter 8, and Frizzled 8 were downregulated. A positive relationship was found between 18F-DOPA-PET SUV mean and LAT1 gene expression and for 24 h-urinary norepinephrine and LAT1. This is the first experimental evidence of 18F-DOPA uptake correlating with LAT1 overexpression. We also demonstrated miR-375 overexpression and downregulated (Wnt) signaling and identified the Hippo pathway as a new potentially oncogenic feature of Pheo.

Phenylethanolamine N-methyltransferase downregulation is associated with malignant pheochromocytoma/paraganglioma.

Malignant pheochromocytoma/paraganglioma (PCC/PGL) is defined by the presence of metastases at non-chromaffin sites, which makes it difficult to prospectively diagnose malignancy. Here, we performed array CGH (aCGH) and paired gene expression profiling of fresh, frozen PCC/PGL samples (n = 12), including three malignant tumors, to identify genes that distinguish benign from malignant tumors. Most PCC/PGL cases showed few copy number aberrations, regardless of malignancy status, but mRNA analysis revealed that 390 genes were differentially expressed in benign and malignant tumors. Expression of the enzyme, phenylethanolamine N-methyltransferase (PNMT), which catalyzes the methylation of norepinephrine to epinephrine, was significantly lower in malignant PCC/PGL as compared to benign samples. In 62 additional samples, we confirmed that PNMT mRNA and protein levels were decreased in malignant PCC/PGL using quantitative real-time polymerase chain reaction and immunohistochemistry. The present study demonstrates that PNMT downregulation correlates with malignancy in PCC/PGL and identifies PNMT as one of the most differentially expressed genes between malignant and benign tumors.

Gene Regulation of Catecholamine Biosynthetic Enzymes by Nitric Oxide in PC12 Cells

Nitric oxide (NO) regulates a wide range of physiological processes. Recent studies show that NO can regulate the release of catecholamines (CA) from the adrenal medulla. In the current study, the PC12 cell line was used to examine the effect of NO on the regulation of the CA biosynthetic enzymes: tyrosine hydroxylase (TH), dopamine-β-hydroxylase (DBH) and phenylethanolamine Nmethyltransferase (PNMT). Treatment of PC12 cells with the NO donor, sodium nitroprusside (SNP) for 6 hours significantly increased TH, DBH and PNMT mRNA levels. In addition, NO potentiates the regulation of gene expression of all three CA biosynthetic enzymes by glucocorticoids and cholinergic agonists. The signaling pathways involved in NO regulation of CA biosynthetic enzymes were investigated with the use of specific kinase activators and inhibitors, with results supporting a contributing role of PKA, PKC and PKG in SNP-mediated induction for all three CA genes (p

Regulation of Catecholamine-Synthesising Enzymes and ß-Adrenoceptors Gene Expression in Ventricles of Stressed Rats

Stress exposure activates the sympathoneural system, resulting in catecholamine release. Chronic stress is associated with development of numerous disorders, including cardiovascular diseases. Here we investigated the expression of mRNAs for catecholamine biosynthetic enzymes tyrosine-hydroxylase, dopamine-beta-hydroxylase and phenylethanolamine N-methyl-transferase, and for beta(1)- and beta(2)-adrenoceptors in the right and left ventricles of rats exposed to chronic unpredictable mild stress. The tyrosine-hydroxylase and dopamine-beta-hydroxylase mRNA levels were not affected by stress, whereas the phenylethanolamine N-methyltransferase mRNA levels significantly increased in both right and left ventricles. No changes in beta(1)-adrenoceptor mRNA levels in either right or left ventricles were observed. At the same time, stress produced a significant increase of beta(2)-adrenoceptor mRNA levels in left ventricles. These results suggest that elevated expression of phenylethanolamine N-methyltransferase in both ventricules and beta(2)-adrenoceptor genes in left ventricles could provide a molecular mechanism that leads to altered physiological response, which is important for the organism coping with stress.

Effect of immobilization stress on gene expression of catecholamine biosynthetic enzymes in heart auricles of socially isolated rats

Chronic stress is associated with the development of cardiovascular diseases. The sympathoneural system plays an important role in the regulation of cardiac function both in health and disease. In the present study, the changes in gene expression of the catecholamine biosynthetic enzymes tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT) and protein levels in the right and left heart auricles of naive control and long-term (12 weeks) socially isolated rats were investigated by Taqman RT-PCR and Western blot analysis. The response of these animals to additional immobilization stress (2 h) was also examined. Long-term social isolation produced a decrease in TH mRNA level in left auricles (about 70%) compared to the corresponding control. Expression of the DBH gene was markedly decreased both in the right (about 62%) and left (about 81%) auricles compared to the corresponding control, group-maintained rats, whereas PNMT mRNA levels remained unchanged. Exposure of group-housed rats to acute immobilization for 2 h led to a significant increase of mRNA levels of TH (about 267%), DBH (about 37%) and PNMT (about 60%) only in the right auricles. Additional 2-h immobilization of individually housed rats did not affect gene expression of these enzymes in either the right or left auricle. Protein levels of TH, DBH and PNMT in left and right heart auricles were unchanged either in both individually housed and immobilized rats. The unchanged mRNA levels of the enzymes examined after short-term immobilization suggest that the catecholaminergic system of the heart auricles of animals previously exposed to chronic psychosocial stress was adapted to maintain appropriate cardiovascular homeostasis.

Regulation of catecholamine biosynthetic enzymes by nitric oxide

The neurotransmitters/neurohormones catecholamines (CA) are involved in the sympathetic control of arterial blood pressure and cardiac function. Recent studies show that nitric oxide (NO) can regulate the release of CA from the adrenal medulla. The in vitro PC12 cell line was used to examine the effect of NO on the regulation of the CA biosynthetic enzymes: tyrosine hydroxylase (TH), dopamine‐β‐hydroxylase (DBH) and phenylethanolamine N‐methyltransferase (PNMT). Results obtained from drug treatments with the NO donor, sodium nitroprusside (SNP) constitute an assessment of its effects in PC12 cells. Treatment of cells with SNP for 6 hours significantly increased TH and PNMT mRNA levels. Combination drug studies with SNP and intracellular kinase activators (forskolin, PMA, 8‐Br‐cGMP) and inhibitors for PKA (H‐89), cGMP (6‐anilinoquinone) PKG (DT‐2) and PKC (GF109203X) were also conducted. Increases in transcript levels for TH and PNMT were obtained under combination drug treatments of SNP and activators of PKA and PKG (p<0.05). mRNA transcript levels of TH, DBH and PNMT showed significant decreases when cells were pre‐treated with PKA, PKC and PKG inhibitors. Furthermore, preliminary transfections showed activation of the PNMT promoter by SNP treatment. Results from this study suggest that NO is capable of regulating CA biosynthetic enzymes, TH and PNMT via activation of the PKA and PKG pathways.

Phenylethanolamine N‐Methyltransferase Gene Expression in the Heart and Blood Pressure Response to Oxytocin Treatment in Rats Exposed to Voluntary Wheel Running

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.

Regulation of Gene Expression of Catecholamine Biosynthetic Enzymes in Dopamine‐β‐Hydroxylase‐ and CRH‐Knockout Mice Exposed to Stress

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.

The effects of oxidative stress on phenylethanolamine N‐methyltransferase gene regulation

Reactive oxygen species (ROS) are an unavoidable by-product of cellular respiration; however cells are able to respond to high levels through regulation of a number of genes. ROS are known to regulate catecholamine gene expression, however, currently little is known regarding the role of ROS in the regulation of the phenylethanolamine N-methyltransferase (PNMT) gene. PNMT is the terminal enzyme in the catecholamine biosynthetic pathway involved in the synthesis of the neurotransmitter, epinephrine. To investigate the effects of ROS, an in vitro cell culture model employing the oxygen-sensitive rat pheochromocytoma (PC12) cell line is treated with hypoxic agents cobalt chloride (CoCl2), iron chelator deferoxamine mesylate (DM) and proteasome inhibitor (MG132). CoCl2 treatment increased expression of PNMT mRNA levels at 6 and 24 h, whereas DM and MG132 treatments increased expression at 24 h. Western blot analysis showed an increase in nuclear protein levels of both the immediate early gene transcription factor (Egr1), and the hypoxia inducible factor 1α (HIF1α) following 3 h CoCl2 treatment. In contrast, only increased levels of HIF1α were observed following 3 h treatments with DM or MG132. Analysis of the wild-type PNMT promoter-driven luciferase activity revealed maximum activity at 24 h CoCl2, DM, and MG132 treatments. These findings provide evidence that oxidative stress can regulate PNMT gene expression.

Transcriptional silencing of glucocorticoid-inducible phenylethanolamine N-methyltransferase expression by sequential signaling events

Specific arrays and timing of environmental cues including glucocorticoids, neurotrophic factors and intracellular messengers influence phenotype expression in developing chromaffin cells or sympathetic neurons. Although the two lineages are closely related, only adrenergic chromaffin cells express phenylethanolamine N-methyltransferase (PNMT), the enzyme that synthesizes epinephrine, while neurons and noradrenergic chromaffin cells are PNMT-negative. It remains unclear to what extent the ability to express PNMT is determined by environmental cues versus intrinsic heterogeneity already present in ganglionic and adrenal precursors. Mouse pheochromocytoma (MPC) cell lines are a model for studying adrenergic differentiation. In two MPC lines that exhibit up to 1000-fold induction of PNMT mRNA by dexamethasone, pretreatment with glial cell line-derived neurotrophic factor (GDNF) and/or the cyclic AMP analog cpt-cAMP markedly blunts or abrogates PNMT inducibility. PNMT suppression occurs without apparent neuronal differentiation in one of the MPC lines and in normal adult mouse chromaffin cell cultures. Our results establish transcriptional suppression by cAMP as a mechanism for regulating PNMT expression in both normal and neoplastic mouse chromaffin cells. However, contrast between large increases in PNMT mRNA levels and low stimulation of promoter activity suggests that modulation of mRNA degradation also plays an important role. Clarification of mechanisms that regulate these two processes in MPC cells may provide insight into developmental mechanisms governing expression and maintenance of the adrenergic phenotype.

Modulation by 6-hydroxydopamine of expression of the phenylethanolamine N-methyltransferase (PNMT) gene in the rat heart during immobilization stress

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.

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.

COMPARATIVE STUDIES OF PC12 AND MOUSE PHEOCHROMOCYTOMA–DERIVED RODENT CELL LINES AS MODELS FOR THE STUDY OF NEUROENDOCRINE SYSTEMS

We have compared PC12 cell lines derived from different laboratories and the newly developed mouse pheochromocytoma (MPC) cell line. Morphologically, there were distinct differences in size, shape, adherence, and clumping behavior, which varied in response to different culture media, growth substrates, and nerve growth factor. Quantitative messenger ribonucleic acid (mRNA) analysis showed significant variability in the expression of the catecholaminergic biosynthetic enzymes tyrosine hydroxylase (TH), phenylethanolamine N-methyltransferase (PNMT), the noradrenaline transporter (NAT), and neuron-specific enolase (NSE) between all lines examined. Of most significance were the increased levels of PNMT mRNA in the MPC cells, which were to 15-fold greater than in the PC12 cell lines grown under the same conditions in Dulbecco modified Eagle medium (P < or = 0.05). Growth of MPC cells in Roswell Park Memorial Institute media induced a further significant increase in PNMT gene expression (P < or = 0.05). Immunohistochemistry for TH, PNMT, and NAT was generally consistent with mRNA analysis, with the MPC cells demonstrating strong immunoreactivity for PNMT. The MPC cells showed the highest levels of desipramine-sensitive [(3)H] noradrenaline uptake activity (threefold > than PC12 American Type Culture Center line, P < or = 0.05), despite relatively low levels of NAT mRNA. These results indicate that PC12 cell lines should be carefully chosen for optimal utility in the study of chromaffin cell or sympathetic neuron biology and that cell features will be influenced by type of media and substrate chosen. Furthermore, they confirm that the new MPC cell line is likely a useful model for the study of adrenergic mechanisms or studies involving NAT.

Modulation of hypoglycemia‐induced increases in plasma epinephrine by estrogen in the female rat

Clinical studies have demonstrated that estrogen replacement therapy suppresses stress-induced increases in plasma catecholamines. The present study determined whether normal circulating levels of estrogen can modulate hypoglycemia-induced increases in plasma epinephrine (EPI). In anesthetized female rats, insulin-induced hypoglycemia (0.25 U/kg) increased plasma EPI concentration to a significantly greater extent in 14-day ovariectomized (OVEX) rats compared to that in sham-operated controls. In 17beta-estradiol (E2)-replaced OVEX rats, the hypoglycemia-induced rise in plasma EPI was reduced significantly when compared to that in vehicle-replaced OVEX rats. OVEX and E2 replacement had no effect on tyrosine hydroxylase or phenylethanolamine N-methyltransferase mRNA levels in the adrenal medulla. In isolated adrenal medullary chromaffin cells, agonist-induced increases in intracellular Ca2+ were unaffected by 48-hr exposure to 10 nM E2. In contrast, acute (3-min) exposure to micromolar concentrations of E2 dose-dependently and reversibly inhibited agonist-induced Ca2+ transients. In addition, in OVEX rats, a constant infusion of E2 significantly reduced the insulin-induced increase in plasma EPI concentration compared to that in vehicle-infused controls. These data demonstrate that physiologic levels of circulating E2 can modulate hypoglycemia-induced increases in plasma EPI. This effect seems independent of steroid influence on adrenal medullary secretion or biosynthesis. In contrast, acute exposure to high levels of E2 can also suppress hypoglycemia-induced increases in plasma epinephrine, due at least in part to inhibition of stimulus-secretion coupling.

Stressor Specificity and Effect of Prior Experience on Catecholamine Biosynthetic Enzyme Phenylethanolamine N‐Methyltransferase

The specific activation of two components of the sympathoadrenal system (adrenomedullary and sympathoneural) by various stressors was recently described. The aim of this work was to investigate changes in catecholamine (CA) biosynthetic enzyme phenylethanolamine N-methyltransferase (PNMT) gene expression, protein level, and activity in the adrenal medulla of rats after a single or repeated exposure to various homotypic or novel heterotypic stressors. Immobilization for 2 h (IMO), cold 4 degrees C (COLD), administration of insulin 5I U (INS), or 2-deoxyglucose 500 mg/kg (2DG) were used as stressors. Plasma epinephrine (EPI) and norepinephrine (NE) levels clearly showed that these stressors specifically activate the aforementioned systems. A single exposure to IMO, COLD, INS, or 2DG induced increases in PNMT mRNA levels in the adrenal medulla. Besides PNMT mRNA, repeated exposure to IMO also elevated activity and protein levels of the enzyme; however, chronic cold exposure did not show PNMT changes compared to control animals at room temperature. PNMT gene expression was also investigated in rats adapted to repeated immobilization stress or to chronic cold exposure after a single exposure to various heterotypic novel stressors. Cold-adapted rats responded to heterotypic novel stressors (IMO, INS) by exaggerated responses of PNMT mRNA levels compared to responses in naive rats exposed to the same stressors at room temperature. Immobilization-adapted rats did not show exaggerated responses of PNMT mRNA after exposure to novel stressors. Therefore, observed differences in plasma CA and adrenomedullary mRNA levels suggest a specific regulation of CA release, synthesis, and gene expression of CA biosynthetic enzymes, which depends on the quality of the stressor. Exposure of adapted rats to novel stressors induces exaggerated responses, but this process also depends on the specificity of the stressor used. Different stressors regulate PNMT gene expression by specific mechanisms especially in chronically stressed rats. These mechanisms remain to be elucidated. It is the ability of the long-term stressed organism to respond differently to novel heterotypic stressors that we consider an important adaptive phenomenon of catecholaminergic systems in rats.

PHENYLETHANOLAMINE N-METHYLTRANSFERASE GENE EXPRESSION IN TRANSPLANTED HUMAN HEART

P643 Background: Phenylethanolamine N-methyltransferase (PNMT) is an enzyme involved in the epinephrine synthesis, and its gene expression is a sign of local epinephrine production Aim: This study was focused to i) detect the possible PNMT gene expression in transplanted human heart and ii) investigate changes in gene expression of the PNMT depending on time after heart transplantation (HTx) and iii) evaluate relations of the PNMT expression with some clinical and physiological variables. Patients and methods: The mRNA levels of PNMT in the myocardial tissue were determined in 22 (21 males) patients, 0 – 12 years after HTx. Mean age of patients was 45.7 ± 13.2 years. Myocardial tissue was obtained at regular endomyocardial biopsy (EMB) performed routinely for diagnosis of the graft rejection. No histological sings of cellular rejection were observed in these patients. For PNMT mRNA estimation one half of the biopsy punch was used, weighting about 1-3 mg. Relative quantification of mRNA levels were performed using reverse transcription and polymerase chain reaction (RT-PCR). As a housekeeper, beta-actin was used. Heart rate (HR), blood pressure (BP), parameters of heart rate variability, graft systolic and diastolic functions were recorded during 48 hours after EMB. Results: During the first three years after HTx, PNMT mRNA levels were higher (mean +/−S.E.M) (0.75 ± 0.06, n=12), compared to further years (0.33 ± 0.062, n=10) (p<0,01). Heart rate variability in low frequency band of power spectrum (LF) was lower in patients during the first three years after HTx (2.73 ± 0.31 vs. 5.67 ± 0.69; p<0.01; Significant negative linear correlation was found between PNMT mRNA and LF (p=0,026, r = -0,54). No association was observed between heart rate, blood pressure, left ventricular ejection fraction, peak early mitral valve flow velocity (E) to the peak velocity after left atrial systole (A) ratio (E/A),deceleration time (DT), 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 on the presence of local PNMT transcription in human heart after HTx. Appearance of PNMT transcription in myocardium after HTx could reflect autonomous “sympathicotrophy”. Decrease in PNMT expression with time following HTx suggests graft reinervation.

Localization and regulation of phenylethanolamine N-methyltransferase gene expression in the heart of rats and mice during stress.

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.

Catecholamine synthesizing enzymes and their modulation by immobilization stress in knockout mice.

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.

Modulation of catecholamine-synthesizing enzymes in the rat heart by repeated immobilization stress.

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.

Changes in gene expression of phenylethanolamine N-methyltransferase in the transplanted human 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.