Role of Phenylethanolamine-N-methyltransferase on Nicotine-Induced Vasodilation in Rat Cerebral Arteries.

OBJECTIVE: The sympathetic-parasympathetic (or axo-axonal) interaction mechanism mediated that neurogenic relaxation, which was dependent on norepinephrine (NE) releases from sympathetic nerve terminal and acts on ß2-adrenoceptor of parasympathetic nerve terminal, has been reported. As NE is a weak ß2-adrenoceptor agonist, there is a possibility that synaptic NE is converted to epinephrine by phenylethanolamine-N-methyltransferase (PNMT) and then acts on the ß2-adrenoceptors to induce neurogenic vasodilation. METHODS: Blood vessel myography technique was used to measure relaxation and contraction responses of isolated basilar arterial rings of rats. RESULTS: Nicotine-induced relaxation was sensitive to propranolol, guanethidine (an adrenergic neuronal blocker), and Nω-nitro-l-arginine. Nicotine- and exogenous NE-induced vasorelaxation was partially inhibited by LY-78335 (a PNMT inhibitor), and transmural nerve stimulation depolarized the nitrergic nerve terminal directly and was not inhibited by LY-78335; it then induced the release of nitric oxide (NO). Epinephrine-induced vasorelaxation was not affected by LY-78335. However, these vasorelaxations were completely inhibited by atenolol (a ß1-adrenoceptor antagonist) combined with ICI-118,551 (a ß2-adrenoceptor antagonist). CONCLUSIONS: These results suggest that NE may be methylated by PNMT to form epinephrine and cause the release of NO and vasodilation. These results provide further evidence supporting the physiological significance of the axo-axonal interaction mechanism in regulating brainstem vascular tone.

Composite paraganglioma-ganglioneuroma with atypical catecholamine profile and phenylethanolamine N-methyltransferase expression: a case report and literature review.

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.

Intermittent Hypoxia Increased the Expression of DBH and PNMT in Neuroblastoma Cells via MicroRNA-375-Mediated Mechanism.

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 ß-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.

The noradrenergic profile of plasma metanephrine in neuroblastoma patients is reproduced in xenograft mice models and arise from PNMT downregulation.

Metanephrines (MNs; normetanephrine (NMN), metanephrine (MN) and methoxytyramine (MT)) detected in urine or plasma represent the best biomarker for neuroblastoma (NB) diagnosis, however the metabolism of both catecholamine (CAT) and MNs remains enigmatic in NB. Using patient-derived xenograft (PDX) models derived from primary NB cells, we observed that the plasma levels of MNs in NB-PDX-bearing mice were comparable as in patients. Interestingly, murine plasma displayed an elevated fraction of glucuronidated forms of MNs relative to human plasma where sulfonated forms prevail. In tumors, the concentration ranges of MNs and CAT and the expression levels of the main genes involved in catecholamine metabolism were similar between NB-PDX and human NB tissues. Likewise, plasma and intratumoral profiles of individual MNs, with increased levels of MT and NMN relative to MN, were also conserved in mouse models as in patients. We further demonstrated the downregulation of the Phenylethanolamine N-Methyltransferase gene in NB biopsies and in NB-PDX explaining this biochemical phenotype, and giving a rational to the low levels of epinephrine and MN measured in NB affected patients. Thus, our subcutaneous murine NB-PDX models not only reproduce the phenotype of primary NB tumors, but also the metabolism of catecholamine as observed in patients. This may potentially open new avenues in preclinical studies for the follow up of novel therapeutic options for NB through the quantification of plasma MNs.

Epinephrine May Contribute to the Persistence of Traumatic Memories in a Post-traumatic Stress Disorder Animal Model.

The importance of catecholamines in post-traumatic stress disorder (PTSD) still needs to be explored. We aimed to evaluate epinephrine's (EPI) causal role and molecular mechanism for the persistence of PTSD traumatic memories. Wild-type (WT) and EPI-deficient mice (phenylethanolamine-N-methyltransferase-knockout mice, Pnmt-KO) were induced with PTSD and behavioral tests were performed. Some Pnmt-KO mice were administered with EPI or vehicle. Catecholamines were quantified by HPLC-ED. Nr4a1, Nr4a2, and Nr4a3 mRNA expression were evaluated by real-time PCR in hippocampus samples. It was observed an increase in EPI and freezing behavior, and a decrease in open arm entries in the elevated plus-maze test and time spent in the light in the light-dark test in WT mice in the PTSD-induction group compared to control. After induction of PTSD, Pnmt-KO mice showed a decrease in freezing, as well as an increase in open arm entries and transitions between compartments compared to WT. After PTSD induction, Pnmt-KO mice administered with EPI showed an increase in freezing compared with the vehicle. On day 0 of PTSD induction, it was observed an increase in mRNA expression of Nr4a2 and Nr4a3 genes in the hippocampus of WT mice compared to control, contrary to Pnmt-KO mice. In conclusion, our data suggest that EPI may be involved in the persistence of traumatic memories in PTSD, possibly through enhancement of the expression of Nr4a2 and Nr4a3 genes in the hippocampus. Peripheral administration of EPI restored contextual traumatic memories in Pnmt-KO mice, which suggests a causal role for EPI. The persistence of contextual traumatic memories may contribute to anxiety-like behavior and resistance of traumatic memory extinction in this PTSD mice model.

Epinephrine Released During Traumatic Events May Strengthen Contextual Fear Memory Through Increased Hippocampus mRNA Expression of Nr4a Transcription Factors.

Epinephrine (EPI) strengthens contextual fear memories by acting on peripheral ß2-adrenoceptors. Phenylethanolamine-N-methyltransferase-knockout (Pnmt-KO) mice are EPI-deficient mice and have reduced contextual fear learning. Our aim was to evaluate the molecular mechanisms by which peripheral EPI strengthens contextual fear memory and if a ß2-adrenoceptor antagonist can erase contextual fear memories. Pnmt-KO and wild-type (WT) mice were submitted to fear conditioning (FC) procedure after treatment with EPI, norepinephrine (NE), EPI plus ICI 118,551 (selective ß2-adrenoceptor antagonist), ICI 118,551 or vehicle (NaCl 0.9%). Catecholamines were separated and quantified by high performance liquid chromatography-electrochemical detection (HPLC-ED). Blood glucose was measured by coulometry. Real-time polymerase chain reaction (qPCR) was used to evaluate mRNA expression of nuclear receptor 4a1 (Nr4a1), Nr4a2 and Nr4a3 in hippocampus samples. In WT mice, plasma EPI concentration was significantly higher after fear acquisition (FA) compared with mice without the test. NE did not increase in plasma after FA and did not strengthen contextual fear memory, contrary to EPI. Freezing induced by EPI was blocked by ICI 118,551 in Pnmt-KO mice. In WT mice, ICI 118,551 blocked blood glucose release into the bloodstream after FA and decreased contextual fear memory. Nr4a1, Nr4a2 and Nr4a3 mRNA expression decreased in Pnmt-KO mice compared with WT mice after FC procedure. In Pnmt-KO mice, EPI induced an increase in mRNA expression of Nr4a2 compared to vehicle. In conclusion, EPI increases in plasma after an aversive experience, possibly improving long-term and old memories, by acting on peripheral ß2-adrenoceptors. Glucose could be the mediator of peripheral EPI in the central nervous system, inducing the expression of Nr4a transcription factor genes involved in consolidation of contextual fear memories.

Chronic exercise induces pathological left ventricular hypertrophy in adrenaline-deficient mice.

Adrenaline-deficient phenylethanolamine-N-methyltransferase-knockout mice (Pnmt-KO) have concentric heart remodeling and though their resting blood pressure is normal, it becomes higher during acute exercise. The aim of this study was to evaluate cardiac morphological, functional and molecular alterations after chronic exercise in adrenaline-deficient mice. Genotypes at the Pnmt locus were verified by polymerase chain reaction (PCR) of ear samples of Pnmt-KO and wild-type (WT) mice. These mice were submitted to chronic exercise training during 6weeks. Blood pressure was determined by a photoelectric pulse detector. Mice were anesthetized and cardiac morphology and function were evaluated by echocardiography and hemodynamics. IGF-1, IGF-1R, ANP and BNP mRNA were quantified by real-time PCR in left ventricle (LV) samples. Pnmt-KO mice showed increased systolic blood pressure compared with WT mice. A significant increase was found in LV mass, and LV posterior wall thickness in trained Pnmt-KO compared to trained WT mice, without significant differences in LV volumes. Acute ß1-adrenergic stimulation with dobutamine increased systolic function indexes in WT mice, but not in Pnmt-KO mice. LV expression of IGF-1 and ANP was increased in trained Pnmt-KO mice when compared to trained WT mice. In conclusion, in response to chronic exercise adrenaline-deficient Pnmt-KO mice show concentric LV hypertrophy and impaired response to dobutamine, suggesting an initial stage of pathological cardiac hypertrophic remodeling. These results support the need for an efficient partial conversion of noradrenaline into adrenaline for prevention of blood pressure overshoot and thus pathological cardiac hypertrophic remodeling in chronic exercise.

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.

Epinephrine increases contextual learning through activation of peripheral ß2-adrenoceptors.

RATIONALE: Phenylethanolamine-N-methyltransferase knockout (Pnmt-KO) mice are unable to synthesize epinephrine and display reduced contextual fear. However, the precise mechanism responsible for impaired contextual fear learning in these mice is unknown. OBJECTIVES: Our aim was to study the mechanism of epinephrine-dependent contextual learning. METHODS: Wild-type (WT) or Pnmt-KO (129x1/SvJ) mice were submitted to a fear conditioning test either in the absence or in the presence of epinephrine, isoprenaline (non-selective ß-adrenoceptor agonist), fenoterol (selective ß2-adrenoceptor agonist), epinephrine plus sotalol (non-selective ß-adrenoceptor antagonist), and dobutamine (selective ß1-adrenoceptor agonist). Catecholamines were separated by reverse-phase HPLC and quantified by electrochemical detection. Blood glucose was measured by coulometry. RESULTS: Re-exposure to shock context induced higher freezing in WT and Pnmt-KO mice treated with epinephrine and fenoterol than in mice treated with vehicle. In addition, freezing response in Pnmt-KO mice was much lower than in WT mice. Freezing induced by epinephrine was blocked by sotalol in Pnmt-KO mice. Epinephrine and fenoterol treatment restored glycemic response in Pnmt-KO mice. Re-exposure to shock context did not induce a significant difference in freezing in Pnmt-KO mice treated with dobutamine and vehicle. CONCLUSIONS: Aversive memories are best retained if moderately high plasma epinephrine concentrations occur at the same moment as the aversive stimulus. In addition, epinephrine increases context fear learning by acting on peripheral ß2-adrenoceptors, which may induce high levels of blood glucose. Since glucose crosses the blood-brain barrier, it may enhance hippocampal-dependent contextual learning.

Cardiac phenylethanolamine N-methyltransferase: localization and regulation of gene expression in the spontaneously hypertensive rat.

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-1ß, 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.

Prenatal glucocorticoid exposure programs adrenal PNMT expression and adult hypertension.

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.

Effect of a single and repeated stress exposure on gene expression of catecholamine biosynthetic enzymes in brainstem catecholaminergic cell groups in rats.

Brainstem catecholaminergic neurons significantly participate in the regulation of neuroendocrine system activity, particularly during stressful conditions. However, so far the precise quantitative characterisation of basal and stress-induced changes in gene expression and protein levels of catecholaminergic biosynthetic enzymes in these neurons has been missing. Using a quantitative reverse transcription-polymerase chain reaction method, we investigated gene expression of catecholamine biosynthetic enzymes in brainstem noradrenergic and adrenergic cell groups in rats under resting conditions as well as in acutely and repeatedly stressed animals. For the first time, we described quantitative differences in basal levels of catecholamine biosynthetic enzyme mRNA in brainstem catecholaminergic ascending and descending projecting cell groups. Moreover, we found and defined some differences among catecholaminergic cell groups in the time-course of mRNA levels of catecholaminergic enzymes following a single and especially repeated immobilisation stress. The data obtained support the assumption that brainstem catecholaminergic cell groups represent a functionally differentiated system, which is highly (but specifically) activated in rats exposed to stress. Therefore, potential interventions for the treatment of stress-related diseases need to affect the activity of brainstem catecholaminergic neurons not uniformly but with some degree of selectivity.

Dietary unsaturated fatty acids differently affect catecholamine handling by adrenal chromaffin cells.

Catecholamines (CA) play an important role in cardiovascular (CDV) disease risk. Namely, noradrenaline (NA) levels positively correlate whereas adrenaline (AD) levels negatively correlate with obesity and/or CDV disease. Western diets, which are tipically rich in Ω-6 fatty acids (FAs) and deficient in Ω-3 FAs, may contribute to the development of obesity, type 2 diabetes and/or coronary artery disease. Taking this into consideration and the fact that our group has already described that saturated FAs affect catecholamine handling by adrenal chromaffin cells, this work aimed to investigate the effect of unsaturated FAs upon catecholamine handling in the same model. Our results showed that chronic exposure to unsaturated FAs differently modulated CA cellular content and release, regardless of both FA series and number of carbon atoms. Namely, the Ω-6 arachidonic and linoleic acids, based on their effect on CA release and cellular content, seemed to impair NA and AD vesicular transport, whereas γ-linolenic acid selectively impaired AD synthesis and release. Within the Ω-9 FAs, oleic acid was devoid of effect, and elaidic acid behaved similarly to γ-linolenic acid. Eicosapentaenoic and docosahexaenoic acids (Ω-3 series) impaired the synthesis and release of both NA and AD. These results deserve attention and future development, namely, in what concerns the mechanisms involved and correlative effects in vivo.

Opposing effects of HIF1α and HIF2α on chromaffin cell phenotypic features and tumor cell proliferation: Insights from MYC-associated factor X.

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.

Association analysis of ERBB2 amplicon genetic polymorphisms and STARD3 expression with risk of gastric cancer in the Chinese population.

The purpose of this study was to investigate whether risk of gastric cancer (GC) was associated with single nucleotide polymorphisms (SNPs) in a gene cluster on the chromosome 17q12-q21 (ERBB2 amplicon) in the Chinese Han population. We detected twenty-six SNPs in this gene cluster containing steroidogenic acute regulatory-related lipid transfer domain containing 3 (STARD3), protein phosphatase 1 regulatory subunit 1B (PPP1R1B/DARPP32), titin-cap (TCAP), per1-like domain containing 1(PERLD1/CAB2), human epidermal growth factor receptor-2 (ERBB2/HER2), zinc-finger protein subfamily 1A 3 (ZNFN1A3/IKZF3) and DNA topoisomerase 2-alpha (TOP2A) genes in 311 patients with GC and in 425 controls by Sequenom. We found no associations between genetic variations and GC risk. However, haplotype analysis implied that the haplotype CCCT of STARD3 (rs9972882, rs881844, rs11869286 and rs1877031) conferred a protective effect on the susceptibility to GC (P=0.043, odds ratio [OR]=0.805, 95% confidence intervals [95% CI]=0.643-0.992). The STARD3 rs1877031 TC genotype endued histogenesis of gastric mucinous adenocarcinoma and signet-ring cell carcinoma (P=0.021, OR=2.882, 95% CI=1.173-7.084). We examined the expression of STARD3 in 243 tumor tissues out of the 311 GC patients and 20 adjacent normal gastric tissues using immumohistochemical (IHC) analysis and tissue microarrays (TMA). The expression of STARD3 was observed in the gastric parietal cells and in gastric tumor tissues and significantly correlated with gender (P=0.004), alcohol drinking (P<0.001), tumor location (P=0.007), histological type (P=0.005) and differentiation (P=0.023) in GC. We concluded that the combined effect of haplotype CCCT of STARD3 might affect GC susceptibility. STARD3 expression might be related to the tumorigenesis of GC in the Chinese population.

Therapeutic potential of Pnmt+ primer cells for neuro/myocardial regeneration.

Phenylethanolamine n-methyltransferase (Pnmt) catalyzes the conversion of norepinephrine into epinephrine, and thus serves as a marker of adrenergic cells. In adults, adrenergic cells are present in the adrenal medullae and the central and peripheral (sympathetic) nervous systems where they play key roles in stress responses and a variety of other functions. During early embryonic development, however, Pnmt first appears in the heart where it is associated with specialized myocytes in the pacemaking and conduction system. There is a transient surge in cardiac Pnmt expression beginning when the first myocardial contractions occur, before any nerve-like or neural crest cells appear in the heart. This early expression of Pnmt denotes a mesodermal origin of these "Instrinsic Cardiac Adrenergic" (ICA) cells. Interestingly, Pnmt+ cells are found in all four chambers of the developing heart, but by adult stages, are found primarily concentrated on the left side of the heart. This regionalized expression occurs in the left atrium and in specific regions of the left ventricle roughly corresponding to basal, mid, and apical sections. A second distinct population of Pnmt-expressing (Pnmt+) cells enters the embryonic heart from invading neural crest, and these "Neural Crest-Derived" (NCD) Pnmt+ cells appear to give rise to a subpopulation(s) of cardiac neurons. Pnmt expression thus serves as a marker not only for adrenergic cells, but also for precursor or "primer" cells destined to become specialized myocytes and neurons in the heart. This review discusses the distribution of Pnmt in the heart during development, including the types of cells where it is expressed, and their potential use for regenerative medicine therapies for cardiovascular disease.

Missing fragments: detecting cooperative binding in fragment-based drug design.

The aim of fragment-based drug design (FBDD) is to identify molecular fragments that bind to alternate subsites within a given binding pocket leading to cooperative binding when linked. In this study, the binding of fragments to human phenylethanolamine N-methyltransferase is used to illustrate how (a) current protocols may fail to detect fragments that bind cooperatively, (b) theoretical approaches can be used to validate potential hits, and (c) apparent false positives obtained when screening against cocktails of fragments may in fact indicate promising leads.

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-â-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 percent) compared to the corresponding control. Expression of the DBH gene was markedly decreased both in the right (about 62 percent) and left (about 81 percent) 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 percent), DBH (about 37 percent) and PNMT (about 60 percent) 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.