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Pheochromocytomas and paragangliomas (PPGLs) are rare tumors that secrete catecholamines and arise from the adrenal medulla or extra-adrenal sympathetic ganglia. These tumors secrete adrenaline and noradrenaline, but paragangliomas usually produce only noradrenaline because of the lack of phenylethanolamine N-methyltransferase (PNMT) expression. Composite paragangliomas, which are complex tumors consisting of multiple types of neuroblastic cells, are extremely rare. We present the case of a 46-year-old woman with an atypical catecholamine profile who was preoperatively diagnosed with pheochromocytoma. However, postoperative pathology revealed that the patient had an extra-adrenal paraganglioma accompanied by a ganglioneuroma, which led to the diagnosis of a composite tumor. Interestingly, PNMT is expressed in both paragangliomas and ganglioneuromas. In addition, we reviewed reported composite paragangliomas and compared their clinical features with those of composite pheochromocytomas. We also discuss various aspects of the etiology of composite paragangliomas and the mechanism by which PNMT is expressed in tumors.

Elucidating the function of specific cell types in a highly complex multicellular system such as the heart often requires detailed anatomic reconstruction. We recently described a distinctive class of phenylethanolamine n-methyltransferase (Pnmt+) cell-derived cardiomyocytes (PdCMs), a new cardiomyocyte population with a potential endocrine role. In this dataset, a 3D reconstruction was carried out to visualise the distribution of PdCMs throughout the murine heart. Rigid registration (stiff rotation and translation) was applied to properly align the fused heart slice images based on landmarks using TrakEM2, an open source plug-in in Fiji. The registered slices were then analysed and reconstructed using MATLAB (MATLAB®. Version 8.3.0.532). The final reconstructed 3D volume was 561×866×48 pixels (corresponding to spatial resolutions of 5.8, 8.9 and 2.5 mm in the x-, y- and z-direction respectively), and visualised in Paraview. The reconstruction allows for detailed analyses of morphology, projections and cellular features of different cell types, enabling further geometrical and topological analyses. Image data can be accessed and viewed through Figshare.

In the present study, channelrhodopsin 2 (ChR2) was specifically introduced into murine cells expressing the Phenylethanolamine n-methyltransferase (Pnmt) gene, which encodes for the enzyme responsible for conversion of noradrenaline to adrenaline. The new murine model enabled the identification of a distinctive class of Pnmt-expressing neuroendocrine cells and their descendants (i.e. Pnmt+ cell derived cells) within the heart. Here, we show that Pnmt+ cells predominantly localized to the left side of the adult heart. Remarkably, many of the Pnmt+ cells in the left atrium and ventricle appeared to be working cardiomyocytes based on their morphological appearance and functional properties. These Pnmt+ cell derived cardiomyocytes (PdCMs) are similar to conventional myocytes in morphological, electrical and contractile properties. By stimulating PdCMs selectively with blue light, we were able to control cardiac rhythm in the whole heart, isolated tissue preparations and single cardiomyocytes. Our new murine model effectively demonstrates functional dissection of cardiomyocyte subpopulations using optogenetics, and opens new frontiers of exploration into their physiological roles in normal heart function as well as their potential application for selective cardiac repair and regeneration strategies.

Pheochromocytomas and paragangliomas (PPGLs) are rare tumors that secrete catecholamines and arise from the adrenal medulla or extra-adrenal sympathetic ganglia. These tumors secrete adrenaline and noradrenaline, but paragangliomas usually produce only noradrenaline because of the lack of phenylethanolamine N-methyltransferase (PNMT) expression. Composite paragangliomas, which are complex tumors consisting of multiple types of neuroblastic cells, are extremely rare. We present the case of a 46-year-old woman with an atypical catecholamine profile who was preoperatively diagnosed with pheochromocytoma. However, postoperative pathology revealed that the patient had an extra-adrenal paraganglioma accompanied by a ganglioneuroma, which led to the diagnosis of a composite tumor. Interestingly, PNMT is expressed in both paragangliomas and ganglioneuromas. In addition, we reviewed reported composite paragangliomas and compared their clinical features with those of composite pheochromocytomas. We also discuss various aspects of the etiology of composite paragangliomas and the mechanism by which PNMT is expressed in tumors.

Phenylethanolamine N-methyltransferase (PNMT) is the terminal enzyme in the catecholamine biosynthetic pathway responsible for adrenaline biosynthesis. Adrenaline is involved in the sympathetic control of blood pressure; it augments cardiac function by increasing stroke volume and cardiac output. Genetic mapping studies have linked the PNMT gene to hypertension. This study examined the expression of cardiac PNMT and changes in its transcriptional regulators in the spontaneously hypertensive (SHR) and wild type Wistar-Kyoto (WKY) rats. SHR exhibit elevated levels of corticosterone, and lower levels of the cytokine IL-1β, revealing systemic differences between SHR and WKY. PNMT mRNA was significantly increased in all chambers of the heart in the SHR, with the greatest increase in the right atrium. Transcriptional regulators of the PNMT promoter show elevated expression of Egr-1, Sp1, AP-2, and GR mRNA in all chambers of the SHR heart, while protein levels of Sp1, Egr-1, and GR were elevated only in the right atrium. Interestingly, only AP-2 protein-DNA binding was increased, suggesting it may be a key regulator of cardiac PNMT in SHR. This study provides the first insights into the molecular mechanisms involved in the dysregulation of cardiac PNMT in a genetic model of hypertension. [ABSTRACT FROM AUTHOR]

Prenatal exposure to glucocorticoids (GCs) programs for hypertension later in life. The aim of the current study was to examine the impact of prenatal GC exposure on the postnatal regulation of the gene encoding for phenylethanolamine N-methyltransferase (PNMT), the enzyme involved in the biosynthesis of the catecholamine, epinephrine. PNMT has been linked to hypertension and is elevated in animal models of hypertension. Male offspring of Wistar-Kyoto dams treated with dexamethasone (DEX) developed elevated systolic, diastolic and mean arterial blood pressure compared to saline-treated controls. Plasma epinephrine levels were also elevated in adult rats exposed to DEX in utero. RT-PCR analysis revealed adrenal PNMT mRNA was higher in DEX exposed adult rats. This was associated with increased mRNA levels of transcriptional regulators of the PNMT gene: Egr-1, AP-2, and GR. Western blot analyses showed increased expression of PNMT protein, along with increased Egr-1 and GR in adult rats exposed to DEX in utero. Furthermore, gel mobility shift assays showed increased binding of Egr-1 and GR to DNA. These results suggest that increased PNMT gene expression via altered transcriptional activity is a possible mechanism by which prenatal exposure to elevated levels of GCs may program for hypertension later in life. [ABSTRACT FROM AUTHOR]

Pheochromocytomas and paragangliomas (PPGLs) are catecholamine-producing chromaffin cell tumors with diverse phenotypic features reflecting mutations in numerous genes, including MYC-associated factor X ( MAX). To explore whether phenotypic differences among PPGLs reflect a MAX-mediated mechanism and opposing influences of hypoxia-inducible factor (HIF)s HIF2α and HIF1α, we combined observational investigations in PPGLs and gene-manipulation studies in two pheochromocytoma cell lines. Among PPGLs from 140 patients, tumors due to MAX mutations were characterized by gene expression profiles and intermediate phenotypic features that distinguished these tumors from other PPGLs, all of which fell into two expression clusters: one cluster with low expression of HIF2α and mature phenotypic features and the other with high expression of HIF2α and immature phenotypic features due to mutations stabilizing HIFs. Max-mutated tumors distributed to a distinct subcluster of the former group. In cell lines lacking Max, re-expression of the gene resulted in maturation of phenotypic features and decreased cell cycle progression. In cell lines lacking Hif2α, overexpression of the gene led to immature phenotypic features, failure of dexamethasone to induce differentiation and increased proliferation. HIF1α had opposing actions to HIF2α in both cell lines, supporting evolving evidence of their differential actions on tumorigenic processes via a MYC/MAX-related pathway. Requirement of a fully functional MYC/MAX complex to facilitate differentiation explains the intermediate phenotypic features in tumors due to MAX mutations. Overexpression of HIF2α in chromaffin cell tumors due to mutations affecting HIF stabilization explains their proliferative features and why the tumors fail to differentiate even when exposed locally to adrenal steroids. [ABSTRACT FROM AUTHOR]

Sleep apnea syndrome (SAS), characterized by recurrent episodes of oxygen desaturation and reoxygenation (intermittent hypoxia (IH)), is a risk factor for hypertension and insulin resistance. We report a correlation between IH and insulin resistance/diabetes. However, the reason why hypertension is induced by IH is elusive. Here, we investigated the effect of IH on the expression of catecholamine-metabolizing enzymes using an in vitro IH system. Human and mouse neuroblastoma cells (NB-1 and Neuro-2a) were exposed to IH or normoxia for 24 h. Real-time RT-PCR revealed that IH significantly increased the mRNA levels of dopamine β-hydroxylase ( DBH ) and phenylethanolamine N-methyltransferase ( PNMT ) in both NB-1 and Neuro-2a. Western blot showed that the expression of DBH and PNMT in the NB-1 cells was significantly increased by IH. Reporter assays revealed that promoter activities of DBH and PNMT were not increased by IH. The miR-375 level of IH-treated cells was significantly decreased relative to that of normoxia-treated cells. The IH-induced up-regulation of DBH and PNMT was abolished by the introduction of the miR-375 mimic, but not by the control RNA. These results indicate that IH stress increases levels of DBH and PNMT via the inhibition of miR-375-mediated mRNA degradation, potentially playing a role in the emergence of hypertension in SAS patients.

C3 neurons constitute one of three known adrenergic nuclei in the rat central nervous system (CNS). While the adrenergic C1 cell group has been extensively characterized both physiologically and anatomically, the C3 nucleus has remained relatively obscure. This study employed a lentiviral tracing technique that expresses green fluorescent protein behind a promoter selective to noradrenergic and adrenergic neurons. Microinjection of this virus into the C3 nucleus enabled the selective tracing of C3 efferents throughout the rat CNS, thus revealing the anatomical framework of C3 projections. C3 terminal fields were observed in over 40 different CNS nuclei, spanning all levels of the spinal cord, as well as various medullary, mesencephalic, hypothalamic, thalamic, and telencephalic nuclei. The highest densities of C3 axon varicosities were observed in Lamina X and the intermediolateral cell column of the thoracic spinal cord, as well as the dorsomedial medulla (both commissural and medial nuclei of the solitary tract, area postrema, and the dorsal motor nucleus of the vagus), ventrolateral periaqueductal gray, dorsal parabrachial nucleus, periventricular and rhomboid nuclei of the thalamus, and paraventricular and periventricular nuclei of the hypothalamus. In addition, moderate and sparse projections were observed in many catecholaminergic and serotonergic nuclei, as well as the area anterior and ventral to the third ventricle, Lamina X of the cervical, lumbar, and sacral spinal cord, and various hypothalamic and telencephalic nuclei. The anatomical map of C3 projections detailed in this survey hopes to lay the first steps toward developing a functional framework for this nucleus. J. Comp. Neurol. 520:2352-2368, 2012. © 2012 Wiley Periodicals Inc. [ABSTRACT FROM AUTHOR]

Epinephrine (Epi), which initiates short-term responses to cope with stress, is, in part, stress-regulated via genetic control of its biosynthetic enzyme, phenylethanolamine N-methyltransferase (PNMT). In rats, immobilization (IMMO) stress activates the PNMT gene in the adrenal medulla via Egr-1 and Sp1 induction. Yet, elevated Epi induced by acute and chronic stress is associated with stress induced, chronic illnesses of cardiovascular, immune, cancerous, and behavioral etiologies. Major sources of Epi include the adrenal medulla and brainstem. Although catecholamines do not cross the blood-brain barrier, circulating Epi from the adrenal medulla may communicate with the central nervous system and stress circuitry by activating vagal nerve β-adrenergic receptors to release norepinephrine, which could then stimulate release of the same from the nucleus tractus solitarius and locus coeruleus. In turn, the basal lateral amygdala (BLA) may activate to stimulate afferents to the hypothalamus, neocortex, hippocampus, caudate nucleus, and other brain regions sequentially. Recently, we have shown that repeated IMMO or force swim stress may evoke stress resiliency, as suggested by changes in expression and extinction of fear memory in the fear-potentiated startle paradigm. However, concomitant adrenergic changes seem stressor dependent. Present studies aim to identify stressful conditions that elicit stress resiliency versus stress sensitivity, with the goal of developing a model to investigate the potential role of Epi in stress-associated illness. If chronic Epi over expression does elicit illness, possibilities for alternative therapeutics exist through regulating stress-induced Epi expression, adrenergic receptor function and/or corticosteroid effects on Epi, adrenergic receptors and the stress axis. [ABSTRACT FROM AUTHOR]

The aim of the present study was to reveal the effect of liver ischemia-reperfusion injury (LIRI) on the activity of selected neuronal phenotypes in rat brain by applying dual Fos-oxytocin (OXY), vasopressin (AVP), tyrosine hydroxylase (TH), phenylethanolamine N-methyltransferase (PNMT), corticoliberine (CRH), and neuropeptide Y (NPY) immunohistochemistry. Two liver ischemia-reperfusion models were investigated: (i) single ligation of the hepatic artery (LIRIa) for 30 min and (ii) combined ligation of the portal triad (the common hepatic artery, portal vein, and common bile duct) (LIRIb) for 15 min. The animals were killed 90 min, 5 h, and 24 h after reperfusion. Intact and sham operated rats served as controls. As indicated by semiquantitative estimation, increases in the number of Fos-positive cells mainly occurred 90 min after both liver reperfusion injuries, including activation of AVP and OXY perikarya in the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei, and TH, NPY, and PNMT perikarya in the catecholaminergic ventrolateral medullar A1/C1 area. Moreover, only PNMT perikarya located in the A1/C1 cell group exhibited increased Fos expression 5 h after LIRIb reperfusion. No or very low Fos expression was found 24 h after reperfusion in neuronal phenotypes studied. Our results show that both models of the LIRI activate, almost by the same effectiveness, a number of different neuronal phenotypes which stimulation may be associated with a complex of physiological responses induced by (1) surgery (NPY, TH, PNMT), (2) hemodynamic changes (AVP, OXY, TH, PNMT), (3) inflammation evoked by ischemia and subsequent reperfusion (TH), and (4) glucoprivation induced by fasting (NPY, PNMT, TH). All these events may contribute by different strength to the development of pathological alterations occurring during the liver ischemia-reperfusion injury. [ABSTRACT FROM AUTHOR]

Stress elicits adrenal epinephrine and cortisol release into the bloodstream to initiate physiological and behavioral responses to counter and overcome stress, the classic 'fight or flight' response (Cannon and De La Paz, Am J Physiol 28:64-70, ). Stress and the stress hormone epinephrine also contribute to the pathophysiology of illness, e.g., behavioral disorders, cardiovascular disease, and immune dysfunction. Epinephrine itself is regulated by stress through its biosynthesis by phenylethanolamine N-methyltransferase (PNMT, EC 2.1.1.28). Single and repeated immobilization (IMMO) stress in rats stimulates adrenal PNMT mRNA and protein expression via the transcription factors, Egr-1 and Sp1. Moderate hypoxic stress increases PNMT promoter-driven gene expression and endogenous PNMT mRNA and protein in PC12 cells. Induction is initiated through cAMP and PLC signaling, with PKA, PKC, PI3K, ERK1/2 MAPK, and p38 MAPK continuing downstream signal transduction, followed by activation of HIF1α, Egr-1, and Sp1. While functional Egr-1 and Sp1 binding sites exist within the proximal PNMT promoter, a putative hypoxia response element is a weak HIF binding site. Yet, HIF1α overexpression increases PNMT promoter-driven luciferase activity and endogenous PNMT. When the Egr-1 or Sp1 sites are mutated, HIF1α does not stimulate the PNMT promoter. siRNA knock down of Egr-1 or Sp1 prevents promoter activation while siRNA knock down of HIF1α inhibits Egr-1 and Sp1 induction. Findings suggest that hypoxia activates the PNMT gene indirectly via HIF1α stimulation of Egr-1 and Sp1. Thus, for stress-induced illnesses where adrenergic dysfunction is implicated, HIF1α may be an 'on-off' switch regulating adrenergic responses to stress and a potential target for therapeutic intervention. [ABSTRACT FROM AUTHOR]

Abstract: Hypoxia is shown to regulate the stress hormone epinephrine through its biosynthesis by phenylethanolamine N-methyltransferase (PNMT) via PNMT gene activation and transcription factors Egr-1 and Sp1 in adrenal medulla-derived PC12 cells. Moderate hypoxia (5% oxygen) markedly stimulates PNMT promoter-driven luciferase activity in the cells. Hypoxia increases Egr-1 and Sp1 mRNA and nuclear protein content and Egr-1 and Sp1 protein–DNA binding complex formation. Subsequent to transcription factor induction, endogenous PNMT mRNA and protein also increase. Egr-1 and Sp1 binding site inactivation or Egr-1 and Sp1 siRNA inhibit PNMT promoter stimulation by hypoxia. Hypoxia elevates protein kinase A (PKA), phospholipase C (PLC), phosphoinositide 3-kinase, protein kinase C, ERK1/2 mitogen-activated protein kinase and p38 mitogen-activated protein kinase expression while selective inhibitors of these signaling enzymes abrogate hypoxic induction of the PNMT promoter and the rise in Egr-1, Sp1 and PNMT mRNA and protein. PC12 cells lacking PKA or PLCγ-1 show significant reduction in PNMT promoter activation by hypoxia. Signaling inhibitors do not affect these responses or reduce hypoxic induction of the PNMT promoter to a lesser extent. Findings suggest that Egr-1 and Sp1 through synergistic interaction are critical transcriptional activators for hypoxic stress-regulated adrenergic function controlled via cAMP/PKA and PLC signaling. Identification of Sp1 as a mediator of hypoxia-induced transcriptional activation of PNMT has not been previously been shown. The effects of hypoxia on PNMT and thereby epinephrine may have important ramifications for the stress hormone epinephrine, its ability to regulate behavioral and physiological processes associated with stress and stress-elicited illness. [Copyright &y& Elsevier]

Abstract: Orexins are expressed in neurons of the dorsolateral hypothalamus and their axons widely distribute throughout the central nervous system. The noradrenergic cell groups of the lower brainstem belong to the targets of these orexin projections. Double immunostainings for orexin and phenylethanolamine N-methyltransferase (PNMT), as well as orexin and tyrosine hydroxylase (TH) were applied to demonstrate the orexinergic innervation of catecholamine cell groups in the lower brainstem of the mouse and the rat. In various densities, networks of orexin-positive fibers and terminals were present on neurons of each adrenaline (C1, C2, C3) and noradrenaline (locus coeruleus, A1, A2, A4, A5 and A7) cell groups. The most dense networks of orexin fibers and terminals were detected in the locus coeruleus, the subcoeruleus area, and in the nucleus of the solitary tract. By using confocal microscope to analyze triple immunostainings we could detect that about two-third of the orexin-PNMT or orexin-TH immunopositive close contacts contained synaptophysin (a presynapse-specific protein) in the C1, C2 and C3 adrenaline, or in the A1, A2 noradrenaline cell groups, respectively. Orexin–immunopositive axons in the C1, C2, as well as A1, A2 and A6 cell groups have been examined by an electron microscope. Relatively few asymmetrical (excitatory) synaptic contacts could be demonstrated between PNMT- or TH-positive dendrites and orexin terminals, although the vast majority of orexin-positive axons was located in juxtaposition to PNMT- or TH-positive neurons. [Copyright &y& Elsevier]

Background/Aims: The catecholaminergic pathway is important in the physical stress response; however, its role is not well understood in acute kidney injury (AKI). We studied single nucleotide polymorphisms (SNPs) of phenylethanolamine N-methyltransferase (PNMT), the terminal enzyme of the catecholaminergic pathway, and their association with adverse outcomes in AKI. Methods: We performed a case-control study of 961 Caucasian subjects (194 with AKI and 767 controls). The PNMT promoter G–161A (rs876493) and coding A+1543G (rs5638) SNPs were genotyped and haplotypes generated. The outcomes of interest were the development of AKI, in-hospital mortality, dialysis requirement, oliguria, and hemodynamic shock. Urine catecholamines were measured in cases to explore genotype-phenotype correlations. Results: The PNMT +1543 G allele was associated with AKI [odds ratio (OR) 2.19, 95% confidence interval (CI): 1.04–4.60]. For AKI cases, each PNMT –161 A allele was associated with lower mortality (OR 0.58, 95% CI: 0.35–0.99) and hemodynamic shock (OR 0.63, 95% CI: 0.40–1.00). The PNMT +1543 G allele was associated with oliguria (OR 3.35, 95% CI: 1.13–9.95). Urine adrenaline was associated with increased hemodynamic shock and mortality, but was lowest in PNMT –161 A/A carriers. Conclusion: In Caucasians, PNMT SNPs are associated with the development of AKI, disease severity, and in-hospital mortality. The adrenergic pathway provides another area of focus in the study of AKI. Copyright © 2010 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

To define the in vivo role of adrenergic catecholamines (CAs), we generated a mouse model whereby tyrosine hydroxylase (TH) was knocked out (KO) in phenylethanolamine N-methyltransferase–expressing cells. These adrenergic specific TH-KO mice were viable and grossly normal. Their resting heart rate and blood pressure, as monitored by telemetry, were unchanged. However, when challenged with treadmill exercise, their chronotropic responses were significantly reduced by 14% compared to wild-type mice. Thus, our data suggest that adrenergic CA is required for normal chronotropic responses to stress, but not required for prenatal and postnatal development or normal cardiovascular function at rest. [ABSTRACT FROM AUTHOR]

Abstract: The pithed rat model has been used extensively to study peripheral cardiovascular responses to electrical stimulation of the sympathetic nervous system, as pithing eliminates central and reflex effects. However, since the transgenic mouse has become a standard and economical model organism, an electrically stimulated pithed mouse would facilitate a variety of studies. We have developed surgical techniques, drug doses and stimulation parameters for an electrically stimulated pithed mouse to study peripheral sympathetic nerve effects on blood pressure. Similar to the pithed rat, the pithed mouse showed voltage and frequency-dependent blood pressure responses to a pulsed train of electrical stimuli. In addition, α-adrenergic stimulation with phenylephrine gave a marked systolic pressor response, while the β2 agonist salbutamol lowered diastolic blood pressure. Furthermore, pithed transgenic mice unable to synthesize catecholamines in adrenergic cells displayed smaller pressor responses than pithed control mice. In summary, the electrically stimulated pithed mouse can be used to study peripheral effects of the sympathetic system on cardiovascular dynamics unencumbered by central responses. [Copyright &y& Elsevier]

We report a case of an autopsy of unusual retroperitoneal sympathetic paraganglioma (SPG) that directly invaded the duodenum and showed expansive growth mimicking a submucosal tumor. The tumor was clinically suspected to be a gastrointestinal stromal tumor (GIST) of the duodenum because of its location and extension to the retroperitoneum without catecholamine-associated symptoms. However, a small biopsy specimen of the tumor showed diffuse proliferation of large basophilic cells that were negative for C-kit and CD34, ruling out GIST and indicating an epithelial malignancy. An autopsy revealed that the tumor was mainly in the retroperitoneum, measuring 7.5 × 9.5 cm, weighing 600 g and extending into the duodenum, adjacent to the pancreas but free of the adrenal glands. On cut section, the tumor involved the entire wall of the duodenum. There were no metastases in any organs. For differential diagnosis, endocrine tumors of the duodenum or pancreas and extra-adrenal SPG were considered. The tumor cells were immunohistochemically strongly positive for chromogranin A and were surrounded by cells positive for S100 protein. The Ki67-labeling index was under 1%. The four catecholamine-synthesizing enzymes were detected in the tumor cells. We report this case of SPG with emphasis on differential diagnosis and the significance of its local invasion. [ABSTRACT FROM AUTHOR]

Pheochromocytomas are catecholamine-producing tumors of the adult adrenal medulla. They are rare in humans and most other species but common in laboratory rats. However, the relevance of rat pheochromocytomas as a model for their human counterparts is uncertain. Previous studies of spontaneous and drug-induced rat pheochromocytomas and the PC12 pheochromocytoma cell line suggested a distinctive noradrenergic phenotype, possibly reflecting origin from a progenitor not present in the adult human adrenal. In this study, we studied 31 pheochromocytomas derived from test and control male and female rats in toxicologic studies for expression of the epinephrine-synthesizing enzyme phenylethanolamine- N-methyltransferase (PNMT) and the receptor tyrosine kinase Ret. PNMT, which defines adrenergic chromaffin cells, is frequently expressed in human pheochromocytomas, often in tumors that also overexpress RET. We also tested for the expression of the cell cycle checkpoint protein p27Kip1, which recently was reported absent in pheochromocytomas from a strain of rats with a hereditary mixed multiple endocrine neoplasia (MEN)-like syndrome. Using immunoblots, we demonstrated PNMT expression in almost 50% of the 31 tumors, although often at lower levels than in normal rat adrenal medulla. The majority of tumors overexpressed Ret. There was no apparent correlation between PNMT and Ret. However, in this study, PNMT expression was strongly associated with tumors arising in female rats, while overexpression of Ret did not show a sex predilection. Robust expression of p27Kip1 was seen in all tumors from the toxicologic studies and also in a small sample of pheochromocytomas from Long–Evans rats, which were reported to have a mixed MEN-like syndrome in the 1980s. The present results show that rat pheochromocytomas have greater phenotypic diversity than previously believed and greater similarity to their human counterparts with respect to these two important markers. Loss of p27Kip1 does not appear to account for the high frequency of pheochromocytomas in commonly utilized rat strains. [ABSTRACT FROM AUTHOR]

Abstract: A series of substituted 4,5,6,7-tetrahydrothieno[3,2-c]pyridines (THTPs) was synthesized and evaluated for their human phenylethanolamine N-methyltransferase (hPNMT) inhibitory potency and affinity for the α2-adrenoceptor. The THTP nucleus was suggested as an isosteric replacement for the 1,2,3,4-tetrahydroisoquinoline (THIQ) ring system on the basis that 3-thienylmethylamine (18) was more potent as an inhibitor of hPNMT and more selective toward the α2-adrenoceptor than benzylamine (15). Although the isosterism was confirmed, with similar influence of functional groups and chirality in both systems on hPNMT inhibitory potency and selectivity, the THTP compounds proved, in general, to be less potent as inhibitors of hPNMT than their THIQ counterparts, with the drop in potency being primarily attributed to the electronic properties of the thiophene ring. A hypothesis for the reduced hPNMT inhibitory potency of these compounds has been formed on the basis of molecular modeling and docking studies using the X-ray crystal structures of hPNMT co-crystallized with THIQ-type inhibitors and S-adenosyl-l-homocysteine as a template. [Copyright &y& Elsevier]

Abstract: The hypothalamic dorsomedial nucleus (DMN) represents an important coordinate center for regulation of autonomic and neuroendocrine systems, especially during stress response. The present study was focused on the gene expression of catecholamine-synthesizing enzymes and the protein levels of tyrosine hydroxylase in DMN, both in control and stressed rats. Moreover, pathways modulating the gene expression of tyrosine hydroxylase in DMN during immobilization (IMO) stress were also investigated. Gene expressions of all catecholamine-synthesizing enzymes were detected in DMN samples. While the levels of tyrosine hydroxylase and phenylethanolamine N-methyltransferase mRNA were increased in IMO rats, aromatic l-amino acid decarboxylase and dopamine-β-hydroxylase mRNA remained unchanged. Tyrosine hydroxylase protein levels were significantly elevated in the DMN only after repeated IMO stress. Postero-lateral deafferentations of the DMN, or transections of the ascending catecholaminergic pathways originating in the lower brainstem abolished the IMO-induced increase of tyrosine hydroxylase gene expression in the DMN. Nevertheless, postero-lateral deafferentations of the hypothalamic paraventricular nucleus (PVN), which separate the DMN from the PVN, had no effect on IMO-induced elevation of tyrosine hydroxylase mRNA in the DMN. The present data indicate that certain DMN neurons synthesize mRNA of catecholamine enzymes. The stress-induced increase of tyrosine hydroxylase and phenylethanolamine N-methyltransferase mRNA in DMN neurons indicates the involvement of these catecholaminergic neurons in stress response. The gene expression of tyrosine hydroxylase in DMN is modulated by lower brainstem and/or spinal cord, but not by PVN afferents. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

This review summarizes knowledge on the effects of stress on two catecholamine biosynthetic enzymes, tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT). Information is presented on differential responses of the enzymes to a variety of stressors as well as differential responses of the enzymes localized to the central nervous system vs. peripheral nervous system and tissues. Changes in mRNA and protein or activity are described, including species- and stressor-specific effects. While temporal changes in these parameters may differ for the particular stressor or enzyme, in general, maximal changes in mRNA and protein content occur at 6-8 and 24 h after stressor exposure, respectively. Elevation of TH and PNMT transcriptional activators prior to mRNA induction and nuclear run-on assays show that stress activates the genes encoding these enzymes. Yet, extents of induction of mRNA, protein and enzyme activity are often discordant depending on the stress, its duration and repetition of exposure. The extremes are concordant changes in mRNA and protein/activity vs. highly elevated mRNA with no change in protein/activity. Post-transcriptional and/or post-translational regulatory influences that may contribute to the complex effects of stress on TH, PNMT and the stress hormone epinephrine are explored. [ABSTRACT FROM AUTHOR]

Immobilization (IMMO) stress was used to examine how stress alters the stress hormone epinephrine (EPI) in the adrenal medulla in vivo. In rats subjected to IMMO for 30 or 120 min, adrenal corticosterone increased to the same extent. In contrast, EPI changed very little, suggesting that EPI synthesis replenishes adrenal pools and sustains circulating levels for the heightened alertness and physiological responses of the ‘flight or fight’ response. In part, stress activates EPI via the phenylethanolamine N-methyltransferase (PNMT) gene as single or repeated IMMO elevated PNMT mRNA. The rise in PNMT mRNA was preceded by induction of the PNMT gene activator, Egr-1, with increases in Egr-1 mRNA, protein, and protein–DNA binding complex apparent. IMMO also evoked changes in Sp1 mRNA, protein, and Sp1–DNA complex formation, although for chronic IMMO changes were not entirely coincident. In contrast, glucocorticoid receptor and AP-2 mRNA, protein, and protein–DNA complex were unaltered. Finally, IMMO stress elevated PNMT protein. However, with seven daily IMMOs for 120 min and delayed killing, protein stimulation did not attain the highly elevated levels expected based on mRNA changes. The latter may perhaps suggest initiation of adrenergic desensitization to prolonged and repeated IMMO stress and/or dissociation of transcriptional and post-transcriptional regulatory mechanisms. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: 1,2,3,4-Tetrahydrobenz[h]isoquinoline (THBQ, 11) is a potent, inhibitor of phenylethanolamine N-methyltransferase (PNMT). Docking studies indicated that the enhanced PNMT inhibitory potency of 11 (hPNMT K i =0.49μM) versus 1,2,3,4-tetrahydroisoquinoline (5, hPNMT K i =5.8μM) was likely due to hydrophobic interactions with Val53, Met258, Val272, and Val269 in the PNMT active site. These studies also suggested that the addition of substituents to the 7-position of 11 that are capable of forming hydrogen bonds to the enzyme could lead to compounds (14–18) having enhanced PNMT inhibitory potency. However, these compounds are in fact less potent at PNMT than 11. Furthermore, 7-bromo-THBQ (19, hPNMT K i =0.22mM), which has a lipophilic 7-substituent that cannot hydrogen bond to the enzyme, is twice as potent at PNMT than 11. This once again illustrates the limitations of docking studies for lead optimization. [Copyright &y& Elsevier]

Pheochromocytomas in multiple endocrine neoplasia type 2 (MEN-2) express phenylethanolamine N-methyltransferase (PNMT), the enzyme that catalyzes conversion of norepinephrine to epinephrine, whereas those in von Hippel–Lindau (VHL) syndrome do not. Consequently, pheochromocytomas in MEN-2 produce epinephrine, whereas those in VHL syndrome produce mainly norepinephrine. This study examined whether transcription factors known to regulate expression of PNMT explain the different tumor phenotypes in these syndromes. Quantitative polymerase chain reaction (PCR) and Western blotting were used to assess levels of mRNA and protein for the glucocorticoid receptor, early growth response 1 (Egr-1), the Sp1 transcription factor (Sp1), and MYC-associated zinc finger protein (MAZ) in 6 MEN-2 and 13 VHL tumors. Results were cross-checked with data obtained using microarray gene expression profiling in a further set of 10 MEN-2 and 12 VHL tumors. Pheochromocytomas in MEN-2 and VHL syndrome did not differ in expression of the glucocorticoid receptor, Egr-1, Sp1, or MAZ as assessed by quantitative PCR and Western blotting. Microarray data also indicated no relevant differences in expression of the glucocorticoid receptor, Egr-1, MAZ, and the AP2 transcription factor. Thus, our results do not support a role for the above transcription factors in determining differences in expression of PNMT in pheochromocytomas from patients with VHL syndrome and MEN-2. Microarray analysis, however, did indicate differences in expression of genes involved in neural crest cell lineage and chromaffin cell development, consistent with differential survival of PNMT-expressing cells in the two syndromes. [ABSTRACT FROM AUTHOR]

Sporadic and syndrome-associated human pheochromocytomas exhibit a spectrum of common and distinctive phenotypic markers. Animal models may contribute to understanding of common denominators leading to development and progression of pheochromocytoma, and to mechanisms that underlie distinctive phenotypes. Rat pheochromocytomas are common, in contrast to their human counterparts, and their frequency is increased by a variety of genotoxic or nongenotoxic agents. Toxicological studies of rats are therefore a potentially rich source of information on pheochromocytoma biology. To compare the molecular profiles of rat and human pheochromocytomas and to identify pathways potentially involved in pathogenesis of rat pheochromocytomas, we conducted a gene expression profiling study comparing 31 pheochromocytomas obtained from the National Toxicology Program to normal adult rat adrenal medulla. The microarray chips were generated from 31,769-oligomer set representing over 27,200 unique Mouse Ensembl genes. The analysis showed over 1,900 genes that were up- or downregulated in the tumors. More than half of the former are involved in protein synthesis and signal transduction, including oncogenes of the RAS family and several heat shock proteins and chaperones. Downregulated genes included receptors and tumor-suppressor genes, including NF2 and Dmbt1. Specific genes related to neuroendocrine function were either upregulated or downregulated in subsets of tumors. Cross-comparison with a human pheochromocytoma database showed greater than 60% correlation. Results of this study reveal both generic and specific parallels between rat and human pheochromocytomas. [ABSTRACT FROM AUTHOR]