Enzyme Phenylethanolamine N-methyltransferase Research Articles

Exercise-stimulated Resolvin Biosynthesis in Adipose Tissue is Abrogated by High Fat Diet-induced Adrenergic Deficiency.

Diet-induced white adipose tissue inflammation is associated with insulin resistance and metabolic perturbations. Conversely, exercise (Exe) protects against the development of chronic inflammation and insulin resistance independent of changes in weight; however, the mechanisms remain largely unknown. We have recently shown that, through adrenergic stimulation of macrophages, exercise promotes resolution of acute peritoneal inflammation by enhancing the biosynthesis of specialized pro-resolving lipid mediators (SPMs). In this study, we sought to determine if exercise stimulates pro-resolving pathways in adipose tissue and whether this response is modified by diet. Specifically, we hypothesized that high fat diet feeding disrupts exercise-stimulated resolution by inhibiting adrenergic signaling, priming the development of chronic inflammation in adipose tissue (AT). To explore the dietary dependence of the pro-resolving effects of Exe, mice were fed either a control or high-fat diet (HFD) for 2 weeks prior to, and throughout, a 4 wk period of daily treadmill running. Glucose handling, body weight and composition, and exercise performance were evaluated at the end of the feeding and exercise interventions. Likewise, catecholamines and their biosynthetic enzymes were measured along with AT SPM biosynthesis and macrophage phenotype and abundance. When compared with sedentary controls (Sed), macrophages isolated from mice exposed to 4 wk of exercise display elevated expression of the SPM biosynthetic enzyme Alox15, while whole AT SPM levels and anti-inflammatory CD301+ M2 macrophages increased. These changes were dependent upon diet as 6 wk of feeding with HFD abrogated the pro-resolving effect of exercise when compared with control diet-fed animals. Interestingly, exercise-induced epinephrine production was inhibited by HFD, which diminished expression of the epinephrine biosynthetic enzyme phenylethanolamine N-methyltransferase (PNMT) in adrenal glands. Taken together, these results suggest that a diet high in fat diminishes the pro-resolving effects of exercise in adipose tissue via decreasing the biosynthesis of catecholamines.

Abstract 13651: Adipose Tissue Resolvin Biosynthesis is Abrogated by High Fat Diet-Induced Adrenergic Deficiency

Diet-induced chronic inflammation in adipose tissue (AT) is associated with systemic insulin resistance. Conversely, exercise (Exe) protects against the development of chronic inflammation and insulin resistance. Our lab recently showed that exercise promotes inflammation resolution through adrenergic stimulation of specialized proresolving mediators (SPMs). In this study, we sought to determine if the proresolving benefits of exercise were dietary dependent. When compared with sedentary controls (Sed), mice exposed to 4 wk of Exe display elevated AT expression of SPM biosynthetic enzyme Alox15 (Sed 1.12±0.18 vs. Exe 1.68±0.13 relative expression, n =5-7), SPM levels (Sed vs. Exe; RvD1 156.7±12.02 vs. 223.0±24.71; 17 R -RvD1 354.0±35.86 vs. 534.4±98.92; RvD4 31.31±6.93 vs. 123.1±30.69; and 17 R -RvD3 2.92±1.08 vs. 8.20±1.70 pg/g, n =5-7), and anti-inflammatory macrophages (F4/80 + CD301 + ; Sed 17.5±3.2 vs. Exe 40.9±4.5 %F4/80 + , n =4-6). These changes were dependent upon diet as short duration feeding with high fat diet (60% kcal from fat; HFD) abrogated the proresolving effect of exercise when compared with low fat diet (10% kcal from fat; LFD) controls. Interestingly, exercise-induced epinephrine (Epi) production in LFD-fed mice (Sed 147.2±24.4 vs Exe 312.5±60.9 ng/mL) was inhibited by HFD (Sed 121.1±22.7 vs. Exe 140.0±16.8 ng/mL). HFD feeding resulted in diminished expression of the Epi biosynthetic enzyme phenylethanolamine N-methyltransferase (PNMT) in adrenal glands when compared with LFD fed animals (protein expression: LFD 1.0±0.25 vs. HFD 0.36±0.15, n =4). These results suggest that the proresolving effects of exercise in adipose tissue are dietary and adrenergic dependent.

Identification of a novel off-target of paroxetine: Possible role in sexual dysfunction induced by this SSRI antidepressant drug

Paroxetine is a widely used antidepressant drug, usually prescribed to treat major depression disorder, anxiety, but is also common in managing chronic pain, eating disorders, some forms of headache and sleep disturbances. Antidepressants, and paroxetine in particular, are linked to side effects, and sexual dysfunction is one of the most prevalent. To explore possible mechanisms leading to this symptomatology, an unbiased approach was applied to retrieve potential paroxetine off-target proteins. A 3D proteome-wide scale in silico screening of a human and murine protein database using SPILLO-PBSS software indicated that the enzyme phenylethanolamine N-methyltransferase (PNMT), the limiting enzyme in the formation of epinephrine, also potentially interacts with paroxetine. Then, a multidisciplinary approach was applied to confirm this finding. Indeed, docking and molecular dynamics analysis and an in vitro assay indicated that paroxetine is able to inhibit PNMT, a result also confirmed in an animal model. The catecholamines norepinephrine and epinephrine are involved in sexual function, and their altered levels have been associated with erectile dysfunction. Thus, based on the fact that the PNMT enzyme is involved in epinephrine production, and that the data here reported indicate that paroxetine inhibits PNMT, a possible role of this enzyme in the sexual dysfunction reported by antidepressant users can be suggested.

Structure-Based Drug Design of Bisubstrate Inhibitors of Phenylethanolamine N-Methyltransferase Possessing Low Nanomolar Affinity at Both Substrate Binding Domains1.

The enzyme phenylethanolamine N-methyltransferase (PNMT, EC 2.1.1.28) catalyzes the final step in the biosynthesis of epinephrine and is a potential drug target, primarily for the control of hypertension. Unfortunately, many potent PNMT inhibitors also possess significant affinity for the a2-adrenoceptor, which complicates the interpretation of their pharmacology. A bisubstrate analogue approach offers the potential for development of highly selective inhibitors of PNMT. This paper documents the design, synthesis, and evaluation of such analogues, several of which were found to possess human PNMT (hPNMT) inhibitory potency <5 nM versus AdoMet. Site-directed mutagenesis studies were consistent with bisubstrate binding. Two of these compounds (19 and 29) were co-crystallized with hPNMT and the resulting structures revealed both compounds bound as predicted, simultaneously occupying both substrate binding domains. This bisubstrate inhibitor approach has resulted in one of the most potent (20) and selective (vs the a2-adrenoceptor) inhibitors of hPNMT yet reported.

Neurotransmitter Switching Coupled to β-Adrenergic Signaling in Sympathetic Neurons in Prehypertensive States.

Single or combinatorial administration of β-blockers is a mainstay treatment strategy for conditions caused by sympathetic overactivity. Conventional wisdom suggests that the main beneficial effect of β-blockers includes resensitization and restoration of β1-adrenergic signaling pathways in the myocardium, improvements in cardiomyocyte contractility, and reversal of ventricular sensitization. However, emerging evidence indicates that another beneficial effect of β-blockers in disease may reside in sympathetic neurons. We investigated whether β-adrenoceptors are present on postganglionic sympathetic neurons and facilitate neurotransmission in a feed-forward manner. Using a combination of immunocytochemistry, RNA sequencing, Förster resonance energy transfer, and intracellular Ca2+ imaging, we demonstrate the presence of β-adrenoceptors on presynaptic sympathetic neurons in both human and rat stellate ganglia. In diseased neurons from the prehypertensive rat, there was enhanced β-adrenoceptor-mediated signaling predominantly via β2-adrenoceptor activation. Moreover, in human and rat neurons, we identified the presence of the epinephrine-synthesizing enzyme PNMT (phenylethanolamine-N-methyltransferase). Using high-pressure liquid chromatography with electrochemical detection, we measured greater epinephrine content and evoked release from the prehypertensive rat cardiac-stellate ganglia. We conclude that neurotransmitter switching resulting in enhanced epinephrine release, may provide presynaptic positive feedback on β-adrenoceptors to promote further release, that leads to greater postsynaptic excitability in disease, before increases in arterial blood pressure. Targeting neuronal β-adrenoceptor downstream signaling could provide therapeutic opportunity to minimize end-organ damage caused by sympathetic overactivity.

The Beta 2 Adrenergic Receptor Antagonist Timolol Improves Healing of Combined Burn and Radiation Wounds.

In a scenario involving a nuclear detonation during war or a terrorist attack, acute radiation exposure combined with thermal and blast effects results in severe skin injury. Although the cutaneous injury in such a scenario may not be lethal, it may lead to inflammation, delayed wound healing and loss of the skin barrier, resulting in an increased risk of infection. In this study, we tested the potential use of timolol, a beta-adrenergic receptor antagonist, to improve epidermal wound closure after combined burn and radiation injury using an ex vivo human skin culture model. Daily application of 10 μ M timolol after combined injury (burn and 10 Gy ex vivo irradiation) increased wound epithelialization by 5-20%. In addition, exposure to 10 Gy significantly suppressed epidermal keratinocyte proliferation by 46% at 48 h postirradiation. Similar to what has been observed in a thermal burn injury, the enzyme phenylethanolamine N-methyltransferase (PNMT), which generates epinephrine, was elevated in the combined thermal burn and radiation wounds. This likely resulted in elevated tissue levels of this catecholamine, which has been shown to delay healing. Thus, with the addition of timolol to the wound to block the binding of locally generated epinephrine to the beta-adrenergic receptor, healing is improved. This work suggests that by antagonizing local epinephrine action within the wound, a beta-adrenergic receptor antagonist such as timolol may be a useful adjunctive treatment to improve healing in the combined burn and radiation injury.

Acetylcholine nicotinic receptor subtypes in chromaffin cells.

In the adrenal gland, acetylcholine released on stimulation of the sympathetic splanchnic nerve activates neuronal-type nicotinic receptors (nAChRs) in chromaffin cells and triggers catecholamine secretion. At least two subtypes of nAChRs have been described in bovine chromaffin cells. The main subtype, a heteromeric assembly of α3, β4 and perhaps α5 subunits, is involved in the activation step of the catecholamine secretion process and is not blocked by the snake toxin α-bungarotoxin. The other is α-bungarotoxin-sensitive, and its functional role has not yet been well defined. The α7 subunit conforms the homomeric structure of this subtype. All nAChR subunits share the same molecular organization and structural data at atomic resolution level are now available for some homomeric and heteromeric ensembles. The α3, β4 and α5 subunits are clustered in genomes of different species, with the transcription factor Sp1 playing a co-ordinating role in the transcriptional regulation of these three subunits. The transcription factor Egr-1 controls the differential expression of α7 nAChR in adrenergic chromaffin cells, as happens with the enzyme phenylethanolamine N-methyl transferase. For unknown reasons, whole cell currents observed in bovine chromaffin cells clearly differ of the ones observed when different combinations of subunit RNAs are injected in oocytes. In addition to the typical nicotinic ligands, a variety of unrelated substances with clinical relevance can target nAChRs in chromaffin cells and, therefore, affect catecholamine secretion. They can act as agonists, antagonists or allosteric modulators.

Suppression of adrenal gland‐derived epinephrine enhances the corticosterone‐induced antinociceptive effect in the mouse formalin test

There is both clinical and experimental evidence to support the application of corticosterone in the management of inflammation and pain. Corticosterone has been used to treat painful inflammatory diseases and can produce antinociceptive effects. Epinephrine is synthesized from norepinephrine by the enzyme phenylethanolamine N-methyltransferase (PNMT) and works as an endogenous adrenoceptor ligand secreted peripherally by the adrenal medulla. It is currently unclear whether corticosterone's antinociceptive effect is associated with the modulation of peripheral epinephrine. We first determined whether exogenous corticosterone treatment actually produced an antinociceptive effect in a formalin-induced pain model, and then examined whether this corticosterone-induced antinociceptive effect was altered by suppression of adrenal-derived epinephrine, using the following three suppression methods: (1) inhibition of the PNMT enzyme; (2) blocking peripheral epinephrine receptors; and (3) adrenalectomy. Exogenous treatment with corticosterone at a high dose (50 mg/kg), but not at lower doses (5, 25 mg/kg), significantly reduced pain responses in the late phase. Moreover, injection of 2,3-dichloro-a-methylbenzylamine, a PNMT enzyme inhibitor, (10 mg/kg) before corticosterone treatment caused a leftward shift in the dose-response curve for corticosterone and injection of propranolol (5 mg/kg), but not phentolamine, also shifted the dose-response curve to the left during the late phase. Chemical sympathectomy with 6-hydroxydopamine had no effect on corticosterone-induced antinociceptive effect, but injection of a low dose of corticosterone produced an antinociceptive effect in adrenalectomized animals. These results demonstrate that suppression of epinephrine, derived from adrenal gland, enhances the antinociceptive effect of exogenous corticosterone treatment in an inflammatory pain model.

Amperometric sensing of norepinephrine at picomolar concentrations using screen printed, high surface area mesoporous carbon

Norepinephrine (NE) is detected amperometrically using the enzyme Phenylethanolamine N-methyl transferase and cofactor S-(5′-Adenosyl)-l-methionine chloride dihydrochloride with disposable screen printed mesoporous carbon electrodes. The role of internal surface area and pore size of the mesoporous carbon is systematically examined using soft-templated, mesoporous silica–carbon powders with highly microporous walls obtained from etching of the silica to produce powders with surface areas ranging from 671–2339m2g−1. As the surface area increases, the sensitivity of the biosensor at very low NE concentrations (0–500pgmL−1) in phosphate buffered saline (PBS) increases just as the current signal increases with respect to the NE concentration of 81–1581μAmLng−1cm−2 for the mesoporous carbons. The best performing electrode provides similar sensitivity in whole rabbit blood in comparison to PBS despite no membrane layer to filter the non-desired reactants; the small (<5nm) pore size and large internal surface area acts to minimize non-specific events that decrease sensitivity.

Chronic physical stress changes gene expression of catecholamine biosynthetic enzymes in the adrenal medulla of adult rats

In this study we examined how chronic forced running (CFR) affects the expression of catecholamine biosynthetic enzymes and cAMP response element-binding (CREB) in the adrenal medulla and the weight of adrenal glands of rats. Also, we examined how CFR and additional acute immobilization stress affect the expression of catecholamine biosynthetic enzymes in the adrenal medulla and the concentration of catecholamines and corticosterone (CORT) in the blood plasma. In this experiment we used as a model forced exercise in rats (treadmill running). We used the most advanced method for determining the level of gene expression, Real-time PCR with TaqMan probes, as well as Western blot analysis (ECL). We found that CFR decreases tyrosine hydroxylase (TH), and dopamine-β-hydroxylase (DBH) mRNA and protein levels in the adrenal medulla. The decreased TH and DBH mRNA levels coincide with the reduced expression of CREB in the adrenal medulla and with the reduced plasma CORT level. Additionally, CFR reduces the level of phenylethanolamine N-methyltransferase (PNMT) mRNA, but elevates its protein level in the adrenal medulla and increases the concentration of adrenaline (A) in the plasma. Reduced level of PNMT mRNA in the adrenal medulla coincides with reduced plasma CORT level. The additional acute immobilization stress increases gene expression of catecholamine biosynthetic enzymes in the adrenal medulla, as well as catecholamines and CORT levels in the plasma. The increased synthesis of PNMT enzyme in the adrenal medulla may result in an increased biosynthesis of A under chronic stress conditions. Additionally, increased level of catecholamines in the plasma after chronic physical stress is the allostatic load that may induce numerous diseases and pathological conditions.

Modulation of catecholamine-synthesizing enzymes in adrenal medulla and stellate ganglia by treadmill exercise of stressed rats

The sympatho-adrenal system represents one of the main systems involved in the response to stressful events because its stress-induced activation results in an increased release of catecholamines. Exercise training acts as an important modulator of sympatho-adrenal system, adrenal medulla and stellate ganglia being two components of this system. This study aimed at investigating physical exercise-related changes in gene expression of catecholamine biosynthetic enzymes tyrosine hydroxylase (TH), dopamine-β-hydroxylase (DBH) and phenylethanolamine N-methyltransferase in the adrenal medulla and stellate ganglia of chronically psychosocially stressed adult rats exposed daily to 20-min treadmill exercise for 12 weeks, using TaqMan RT-PCR assay. Chronic psychosocial stress decreased gene expression of the examined enzymes in the adrenal medulla and treadmill exercise did not lead to further modulation of the corresponding gene expression. On the other hand, chronic psychosocial stress produced a significant increase of TH (about 51%) and DBH (about 103%) gene expression in stellate ganglia, while treadmill exercise decreased gene expression of these enzymes to control levels in psychosocially stressed rats. Our data indicate that treadmill exercise leads to a decreased gene transcription of catecholamine biosynthetic enzymes in stellate ganglia and attenuation of cardiac noradrenaline production in stressful situations. Reduction of catecholamine synthesis in stellate ganglia may be linked to the beneficial effects of treadmill exercise on cardiovascular system in stressed animals.

Role of Reactive Oxygen Species in the Neural and Hormonal Regulation of the PNMT Gene in PC12 Cells

The stress hormone, epinephrine, is produced predominantly by adrenal chromaffin cells and its biosynthesis is regulated by the enzyme phenylethanolamine N-methyltransferase (PNMT). Studies have demonstrated that PNMT may be regulated hormonally via the hypothalamic-pituitary-adrenal axis and neurally via the stimulation of the splanchnic nerve. Additionally, hypoxia has been shown to play a key role in the regulation of PNMT. The purpose of this study was to examine the impact of reactive oxygen species (ROS) produced by the hypoxia mimetic agent CoCl2, on the hormonal and neural stimulation of PNMT in an in vitro cell culture model, utilizing the rat pheochromocytoma (PC12) cell line. RT-PCR analyses show inductions of the PNMT intron-retaining and intronless mRNA splice variants by CoCl2 (3.0- and 1.76-fold, respectively). Transient transfection assays of cells treated simultaneously with CoCl2 and the synthetic glucocorticoid, dexamethasone, show increased promoter activity (18.5-fold), while mRNA levels of both splice variants do not demonstrate synergistic effects. Similar results were observed when investigating the effects of CoCl2-induced ROS on the neural stimulation of PNMT via forskolin. Our findings demonstrate that CoCl2-induced ROS have synergistic effects on hormonal and neural activation of the PNMT promoter.

Fragment-based screening by X-ray crystallography, MS and isothermal titration calorimetry to identify PNMT (phenylethanolamine N-methyltransferase) inhibitors

CNS (central nervous system) adrenaline (epinephrine) is implicated in a wide range of physiological and pathological conditions. PNMT (phenylethanolamine N-methyltransferase) catalyses the final step in the biosynthesis of adrenaline, the conversion of noradrenaline (norepinephrine) to adrenaline by methylation. To help elucidate the role of CNS adrenaline, and to develop potential drug leads, potent, selective and CNS-active inhibitors are required. The fragment screening approach has advantages over other lead discovery methods including high hit rates, more efficient hits and the ability to sample chemical diversity more easily. In the present study we applied fragment-based screening approaches to the enzyme PNMT. We used crystallography as the primary screen and identified 12 hits from a small commercial library of 384 drug-like fragments. The hits include nine chemicals with two fused rings and three single-ring chemical systems. Eight of the hits come from three chemical classes: benzimidazoles (a known class of PNMT inhibitor), purines and quinolines. Nine of the hits have measurable binding affinities (~5-700 μM) as determined by isothermal titration calorimetry and all nine have ligand efficiencies of 0.39 kcal/mol per heavy atom or better (1 kcal≈4.184 kJ). We synthesized five elaborated benzimidazole compounds and characterized their binding to PNMT, showing for the first time how this class of inhibitors interact with the noradrenaline-binding site. Finally, we performed a pilot study with PNMT for fragment-based screening by MS showing that this approach could be used as a fast and efficient first-pass screening method prior to characterization of binding mode and affinity of hits.

Conserved regulatory motifs at phenylethanolamine N-methyltransferase (PNMT) are disrupted by common functional genetic variation: an integrated computational/experimental approach

The adrenomedullary hormone epinephrine transduces environmental stressors into cardiovascular events (tachycardia and hypertension). Although the epinephrine biosynthetic enzyme PNMT genetic locus displays both linkage and association to such traits, genetic variation underlying these quantitative phenotypes is not established. Using an integrated suite of computational and experimental approaches, we elucidate a functional mechanism for common (minor allele frequencies > 30%) genetic variants at PNMT. Transcription factor binding motif prediction on mammalian PNMT promoter alignments identified two variant regulatory motifs, SP1 and EGR1, disrupted by G-367A (rs3764351), and SOX17 motif created by G-161A (rs876493). Electrophoretic mobility shifts of approximately 30-bp oligonucleotides containing ancestral versus variant alleles validated the computational hypothesis. Queried against chromaffin cell nuclear protein extracts, only the G-367 and -161A alleles shifted. Specific antibodies applied in electrophoretic gel shift experiments confirmed binding of SP1 and EGR1 to G-367 and SOX17 to -161A. The in vitro allele-specific binding was verified in cella through promoter reporter assays: lower activity for -367A haplotypes cotransfected by SP1 (p = 0.002) and EGR1 (p = 0.034); and enhanced inhibition of -161A haplotypes (p = 0.0003) cotransfected with SP1 + SOX17. Finally, we probed cis/trans regulation with endogenous factors by chromatin immunoprecipitation using SP1/EGR1/SOX17 antibodies. We describe the systematic application of complementary computational and experimental techniques to detect and document functional genetic variation in a trait-associated regulatory region. The results provide insight into cis and trans transcriptional mechanisms whereby common variation at PNMT can give rise to quantitative changes in human physiological and disease traits. Thus, PNMT variants in cis may interact with nuclear factors in trans to govern adrenergic activity.Electronic supplementary materialThe online version of this article (doi: 10.1007/s00335-010-9253-y) contains supplementary material which is available to authorized users.

Molecular recognition of physiological substrate noradrenaline by the adrenaline-synthesizing enzyme PNMT and factors influencing its methyltransferase activity

Substrate specificity is critically important for enzyme catalysis. In the adrenaline-synthesizing enzyme PNMT (phenylethanolamine N-methyltransferase), minor changes in substituents can convert substrates into inhibitors. Here we report the crystal structures of six human PNMT complexes, including the first structure of the enzyme in complex with its physiological ligand R-noradrenaline. Determining this structure required rapid soak methods because of the tendency for noradrenaline to oxidize. Comparison of the PNMT-noradrenaline complex with the previously determined PNMT-p-octopamine complex demonstrates that these two substrates form almost equivalent interactions with the enzyme and show that p-octopamine is a valid model substrate for PNMT. The crystal structures illustrate the adaptability of the PNMT substrate binding site in accepting multi-fused ring systems, such as substituted norbornene, as well as noradrenochrome, the oxidation product of noradrenaline. These results explain why only a subset of ligands recognized by PNMT are methylated by the enzyme; bulky substituents dictate the binding orientation of the ligand and can thereby place the acceptor amine too far from the donor methyl group for methylation to occur. We also show how the critical Glu(185) catalytic residue can be replaced by aspartic acid with a loss of only 10-fold in catalytic efficiency. This is because protein backbone movements place the Asp(185) carboxylate almost coincident with the carboxylate of Glu(185). Conversely, replacement of Glu(185) by glutamine reduces catalytic efficiency almost 300-fold, not only because of the loss of charge, but also because the variant residue does not adopt the same conformation as Glu(185).

Adrenergic Responses to Stress

Stress effects on adrenergic responses in rats were examined in adrenal medulla, the primary source of circulating epinephrine (Epi). Irrespective of duration, immobilization (IMMO) increased adrenal corticosterone to the same extent. In contrast, Epi changed little, suggesting that Epi synthesis replenishes adrenal pools and sustains circulating levels for the heightened alertness and physiological changes required of the "flight or fight" response. IMMO also induced the Epi-synthesizing enzyme, phenylethanolamine N-methyltransferase (PNMT). The rise in its mRNA and protein was preceded by increases in Egr-1 and Sp1 mRNA, protein, and protein-DNA binding complex formation. With repeated and prolonged stress, PNMT protein did not reflect the magnitude of change in mRNA. The latter suggests that post-transcriptional, in addition to transcriptional mechanisms, regulate PNMT responses to stress. To further reveal molecular mechanisms underlying stress-induced changes in adrenergic function, the effects of hypoxia on PNMT promoter-driven gene expression are being examined in adrenal medulla-derived PC12 cells. Hypoxia activates the PNMT promoter to increase PNMT promoter-driven luciferase reporter gene expression and endogenous PNMT in PC12 cells. Induction of both appear mediated via activation of multiple signaling pathways and downstream activation of hypoxia inducible factor and PNMT transcriptional activators, Egr-1 and Sp1. Hypoxia generates both partially and fully processed forms of PNMT mRNA. The former reportedly is translated into a truncated, nonfunctional protein, and the latter into enzymatically active PNMT. Together, findings suggest that stress increases PNMT gene transcriptional activity but post-transcriptional regulatory mechanisms limit the biological end-point of functional PNMT enzyme and, thereby, Epi.

Epinephrine Is Required for Normal Cardiovascular Responses to Stress in the Phenylethanolamine N-Methyltransferase Knockout Mouse

Epinephrine (EPI) is an important neurotransmitter and hormone. Its role in regulating cardiovascular function at rest and with stress is unclear, however. An epinephrine-deficient mouse model was generated in which the epinephrine-synthesizing enzyme phenylethanolamine N-methyltransferase was knocked out (KO). Blood pressure and heart rate were monitored by telemetry at rest and during graded treadmill exercise. Cardiac structure and function were evaluated by echocardiography in mice under 1 of 2 conditions: unstressed and lightly anesthetized or restrained and awake. In KO mice, resting cardiovascular function, including blood pressure, heart rate, and cardiac output, was the same as that in wild-type mice, and the basal norepinephrine plasma level was normal. However, inhibition of sympathetic innervation with the ganglion blocker hexamethonium caused a 54% smaller decrease in blood pressure in KO mice, and treadmill exercise caused an 11% higher increase in blood pressure, both suggesting impaired vasodilation in KO mice. Interestingly, phenylethanolamine N-methyltransferase KO did not change the heart rate response to ganglionic blockade and exercise. By echocardiography, KO mice had an increased ratio of left ventricular posterior wall thickness to internal dimensions but did not have cardiac hypertrophy, suggesting concentric remodeling in the KO heart. Finally, in restrained, awake KO mice, heart rate and ejection fraction remained normal, but cardiac output was significantly reduced because of diminished end-diastolic volume. Our data suggest that epinephrine is required for normal blood pressure and cardiac filling responses to stress but is not required for tachycardia during stress or normal cardiovascular function at rest.

Morphological Changes in the Atlantic Bottlenose Dolphin (Tursiops truncatus) Adrenal Gland Associated with Chronic Stress

Beach-stranded Atlantic bottlenose dolphins (n=68) were categorized as either "acutely stressed" (if they died from net entanglement, boat strike, or acute infection; 31 animals) or "chronically stressed" (if they suffered from or died as a result of long-term disease or debilitating injury; 37 animals). No significant differences in mass between the right and left adrenal glands were found within each category. However, the average gland mass (AGM), based on the right and left glands together, was 5.2g for acutely stressed animals and 11.01 g for chronically stressed animals (P<0.001). Significant differences were also found, in terms of the ratio of cross-sectional areas of the cortex to medulla, between acutely stressed (ratio 1.22) and chronically stressed (ratio 1.63) animals (P=0.027). Adrenal glands of acutely stressed animals consisted of 48% cortex, 41% medulla, and 11% other tissue elements (connective tissue, blood vessels and gland capsule), whereas the corresponding figures for chronically stressed animals were 53%, 36%, and 11%. The mean estimated mass values for cortex, medulla and other tissue were, for acutely stressed animals, 2.36, 1.9, and 0.54, respectively, whereas for chronically stressed animals the corresponding figures were 6.06, 4.04, and 1.29 (P<0.001 for each of the three comparisons). Overstaining with haematoxylin (HEM) and immunohistochemical labelling (IHC) of the enzyme phenylethanolamine N-methyl transferase (which converts norepinephrine to epinephrine) were used to determine the percentage of epinephrine-producing cells in relation to the overall cross-sectional area of the adrenal gland. The percentage values in acutely as compared with chronically stressed dolphins were 6.7% and 15.93%, respectively (P=0.021). The results therefore suggest that in bottlenose dolphins chronic stress results in increases in (1) adrenal mass, (2) cortex to medulla ratio, and (3) epinephrine-producing cells within the medulla, giving rise to an increase in the thickness of the medullary band.

Gene Expression of Phenylethanolamine N-Methyltransferase in Corticotropin-Releasing Hormone Knockout Mice During Stress Exposure

Epinephrine (EPI) synthesizing enzyme phenylethanolamine N-methyltransferase (PNMT, EC 2.1.1.28) is primarily localized in the adrenal medulla (AM). We have recently described existence of the PNMT gene expression in cardiac atria and ventricles and in sympathetic ganglia of adult rats and mice. The aim of the present work was to study regulation of the PNMT gene expression in corticotropin-releasing hormone knockout mice (CRH KO) and matched control wild-type mice (WT) under normal and stress conditions. Levels of the PNMT mRNA were determined by RT-PCR; PNMT immunoprotein and protein of transcription factor EGR-1 by Western Blot. Plasma EPI and corticosterone (CORT) levels were determined by radioenzymatic and RIA methods. Immobilization (IMMO) was used as a stressor. Stress-induced increases in the PNMT mRNA and protein levels observed in WT mice were almost completely absent in CRH KO mouse adrenal medulla, stellate ganglia, and cardiac atria, while ventricular PNMT mRNA elevation was not CRH-dependent. Plasma EPI and CORT levels were markedly reduced in CRH KO compared to WT mice both before and after the stress. Levels of EGR-1, crucial transcription factor for regulation of the PNMT were highly increased in stressed WT and CRH KO mice in cardiac areas, but not in the adrenal medulla. Data show that the CRH deficiency can markedly prevent immobilization-triggered induction of the PNMT mRNA and protein levels in the adrenal medulla and stellate ganglia. Reduced plasma epinephrine and corticosterone levels and adrenal medullary EGR-1 protein levels in CRH knockout versus WT mice during stress indicate that the HPA axis plays a crucial role in regulation of the PNMT gene expression in these organs. Cardiac atrial PNMT gene expression with stress is also dependent on intact HPA axis. However, in cardiac ventricles, especially after the single stress exposure, its expression is not impaired by CRH deficiency. Since cardiac EGR-1 protein levels in CRH KO mice are also not affected by the single stress exposure, we propose existence of different regulation of the PNMT gene expression, especially in the cardiac ventricles.Overall, our findings reveal that the PNMT gene expression is regulated through the HPA in both sympathoadrenal system and the heart and also via EGR-1 in the adrenal medulla, but apparently not in the heart. Regulation of the PNMT gene expression in various compartments of heart includes both corticosterone-dependent and independent mechanisms.

Chronic nicotine effects on adrenergic function

Smokers suggest that smoking relaxes them but after smoking, the two stress hormones epinephrine (EPI) and cortisol rapidly rise. Smoking is highly associated with cardiovascular pathology and morbidity, and EPI, a major vasopressor, may be an underlying factor. We propose that nicotine (NIC), the major addictive component in cigarettes, acts directly via nicotinic receptors and indirectly via the hypothalamic-pituitary-adrenal axis to elevate EPI biosynthesis by activating the EPI-synthesizing enzyme phenylethanolamine N-methyltransferase (PNMT). In PC12 cells transfected with a PNMT promoter-luciferase reporter gene construct, NIC (100 μM) stimulates luciferase activity in a dose- and time-dependent manner, with activity highest at 6 hr (2.5-fold), back to basal levels by 24 hr and increasing again with continued exposure. In combination with DEX (1 μM), synergistic activation is apparent at 24 hr. In addition, NIC and DEX induce endogenous PNMT mRNA and protein in untransfected PC12 cells. Whether the combination leads to cooperative stimulation remains to be determined. When NIC is administered chronically to rats (1.6 mg/kg i.p. twice daily for 7 days), PNMT mRNA and protein rise 2.0 to 2.5-fold in the adrenal medulla. Finally, in both NIC-treated PC12 cells (24 hr) or adrenal medulla of NIC-treated rats (7 days), mRNA for the immediate early gene transcription factor Egr-1 markedly rises. Together these results suggest that NIC and glucocorticoids, while independently activating the PNMT gene, may synergistically stimulate PNMT with chronic exposure, Egr-1 being one factor mediating induction.