Phenylethanolamine N-methyltransferase Research Articles

Establishing an Artificial Pathway for the Biosynthesis of Octopamine and Synephrine.

In this study, we designed an artificial pathway composed of tyramine β-hydroxylase (TBH) and phenylethanolamine N-methyltransferase (PNMT) for the biosynthesis of both octopamine and synephrine. As most TBH and PNMT originate from eukaryotic animals and plants, the heterologous expression and identification of functional TBH and PNMT are critical for establishing the pathway in mode microorganisms like Escherichia coli. Here, three TBHs were evaluated, and only TBH from Drosophila melanogaster was successfully expressed in the soluble form in E. coli. Its expression was promoted by evaluating the effects of different expression strategies. The specific enzyme activity of TBH was optimized up to 229.50 U·g-1, and the first step in the biosynthetic pathway was successfully established and converted tyramine to synthesize 0.10 g/L of octopamine. Furthermore, the second step to produce synephrine from octopamine was developed by screening PNMT, enhancing enzyme activity, and optimizing reaction conditions, with a maximum synephrine production of 2.02 g/L. Finally, based on the optimization of the reaction conditions for each individual reaction, the one-pot cascade reaction for synthesizing synephrine from tyramine was constructed by combining the TBH and PNMT. The synthetic synephrine reached 30.05 mg/L with tyramine as substrate in the two-step enzyme cascade system. With further optimization and amplification, the titers of octopamine and synephrine were increased to 0.45 and 0.20 g/L, respectively, with tyramine as substrate. This work was the first achievement of the biosynthesis of octopamine and synephrine to date.

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

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 β2-adrenoceptor of parasympathetic nerve terminal, has been reported. As NE is a weak β2-adrenoceptor agonist, there is a possibility that synaptic NE is converted to epinephrine by phenylethanolamine-N-methyltransferase (PNMT) and then acts on the β2-adrenoceptors to induce neurogenic vasodilation. Blood vessel myography technique was used to measure relaxation and contraction responses of isolated basilar arterial rings of rats. 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 β1-adrenoceptor antagonist) combined with ICI-118,551 (a β2-adrenoceptor antagonist). 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.

The 3D in vitro Adrenoid cell model recapitulates the complexity of the adrenal gland

The crosstalk between the chromaffin and adrenocortical cells is essential for the endocrine activity of the adrenal glands. This interaction is also likely important for tumorigenesis and progression of adrenocortical cancer and pheochromocytoma. We developed a unique in vitro 3D model of the whole adrenal gland called Adrenoid consisting in adrenocortical carcinoma H295R and pheochromocytoma MTT cell lines. Adrenoids showed a round compact morphology with a growth rate significantly higher compared to MTT-spheroids. Confocal analysis of differential fluorescence staining of H295R and MTT cells demonstrated that H295R organized into small clusters inside Adrenoids dispersed in a core of MTT cells. Transmission electron microscopy confirmed the strict cell–cell interaction occurring between H295R and MTT cells in Adrenoids, which displayed ultrastructural features of more functional cells compared to the single cell type monolayer cultures. Adrenoid maintenance of the dual endocrine activity was demonstrated by the expression not only of cortical and chromaffin markers (steroidogenic factor 1, and chromogranin) but also by protein detection of the main enzymes involved in steroidogenesis (steroidogenic acute regulatory protein, and CYP11B1) and in catecholamine production (tyrosine hydroxylase and phenylethanolamine N-methyltransferase). Mass spectrometry detection of steroid hormones and liquid chromatography measurement of catecholamines confirmed Adrenoid functional activity. In conclusion, Adrenoids represent an innovative in vitro 3D-model that mimics the spatial and functional complexity of the adrenal gland, thus being a useful tool to investigate the crosstalk between the two endocrine components in the pathophysiology of this endocrine organ.

Pharmacometabolomics and mass spectrometry imaging approach to reveal the neurochemical mechanisms of Polygala tenuifolia

Polygala tenuifolia, commonly known as Yuanzhi (YZ) in Chinese, has been shown to possess anti-insomnia properties. However, the material basis and the mechanism underlying its sedative-hypnotic effects remain unclear. Herein, we investigated the active components and neurochemical mechanism of YZ extracts using liquid chromatography tandem mass spectrometry (LC-MS/MS)-based pharmacometabolomics and mass spectrometry imaging (MSI)-based spatial resolved metabolomics. According to the results, 17 prototypes out of 101 ingredients in the YZ extract were detected in both the plasma and brain, which might be the major components contributing to the sedative-hypnotic effects. Network pharmacology analysis revealed that these prototypes may exert their effects through neuroactive ligand-receptor interaction, serotonergic synapse, dopaminergic synapse, and dopaminergic synapse, among other pathways. LC-MS/MS-based targeted metabolomics and Western blot (WB) revealed that tryptophan-serotonin-melatonin (Trp-5-HT-Mel) and tyrosine-norepinephrine-adrenaline (Tyr-Ne-Ad) are the key regulated pathways. Dopa decarboxylase (DDC) upregulation and phenylethanolamine N-methyltransferase (PNMT) downregulation further confirmed these pathways. Furthermore, MSI-based spatially resolved metabolomics revealed notable alterations in 5-HT in the pineal gland (PG), and Ad in the brainstem, including the middle brain (MB), pons (PN), and hypothalamus (HY). In summary, this study illustrates the efficacy of an integrated multidimensional metabolomics approach in unraveling the sedative-hypnotic effects and neurochemical mechanisms of a Chinese herbal medicine, YZ.

Differential molecular mechanisms of substrate recognition by selenium methyltransferases, INMT and TPMT, in selenium detoxification and excretion

It is known that the recommended dietary allowance of selenium (Se) is dangerously close to its tolerable upper intake level. Se is detoxified and excreted in urine as trimethylselenonium ion (TMSe) when the amount ingested exceeds the nutritional level. Recently, we demonstrated that the production of TMSe requires two methyltransferases: thiopurine S-methyltransferase (TPMT) and indolethylamine N-methyltransferase (INMT). In this study, we investigated the substrate recognition mechanisms of INMT and TPMT in the Se-methylation reaction. Examination of the Se-methyltransferase activities of two paralogs of INMT, namely, nicotinamide N-methyltransferase (NNMT) and phenylethanolamine N-methyltransferase (PNMT), revealed that only INMT exhibited Se-methyltransferase activity. Consistently, molecular dynamics simulations demonstrated that dimethylselenide (DMSe) was preferentially associated with the active center of INMT. Using the fragment molecular orbital method, we identified hydrophobic residues involved in the binding of DMSe to the active center of INMT. The INMT-L164R mutation resulted in a deficiency in Se- and N-methyltransferase activities. Similarly, TPMT-R152, which occupies the same position as INMT-L164, played a crucial role in the Se-methyltransferase activity of TPMT. Our findings suggest that TPMT recognizes negatively charged substrates, whereas INMT recognizes electrically neutral substrates in the hydrophobic active center embedded within the protein. These observations explain the sequential requirement of the two methyltransferases in producing TMSe.

Post-Finasteride Syndrome And Post-Ssri Sexual Dysfunction: Two Clinical Conditions Apparently Distant, But Very Close

Post-finasteride syndrome and post-SSRI sexual dysfunction, are two poorly explored clinical conditions in which men treated for androgenetic alopecia with finasteride or for depression with SSRI antidepressants show persistent side effects despite drug suspension (e.g., sexual dysfunction, psychological complaints, sleep disorders). Because of some similarities in the symptoms, common pathological mechanisms are proposed here. Indeed, as discussed, clinical studies and preclinical data obtained so far suggest an important role for brain modulators (i.e., neuroactive steroids), neurotransmitters (i.e., serotonin, and cathecolamines), and gut microbiota in the context of the gut-brain axis. In particular, the observed interconnections of these signals in these two clinical conditions may suggest similar etiopathogenetic mechanisms, such as the involvement of the enzyme converting norepinephrine into epinephrine (i.e., phenylethanolamine N-methyltransferase). However, despite the current efforts, more work is still needed to advance the understanding of these clinical conditions in terms of diagnostic markers and therapeutic strategies.

Regulation of epinephrine biosynthesis in HRAS-mutant paragangliomas.

The biochemical phenotype of paragangliomas (PGLs) is highly dependent on the underlying genetic background and tumor location. PGLs at extra-adrenal locations usually do not express phenylethanolamine N-methyltransferase (PNMT), the enzyme required for epinephrine production, which was explained by the absence of glucocorticoids. PGLs with pathogenic variants (PVs) in Harvey rat sarcoma viral oncogene homolog (HRAS) can occur in or outside of the adrenal, but always synthesize epinephrine independently of the localization. Here, we characterize the signaling pathways through which PVs in HRAS influence PNMT expression. Catecholamines, cortisol, and transcriptional features of PGL tissues with known genetic background were analyzed. Genetically modified rat pheochromocytoma cells carrying PVs in Hras were generated and analyzed for regulation of Pnmt expression. Elevated epinephrine contents in PGLs with PVs in HRAS were accompanied by enrichment in mitogen-activated protein kinase (MAPK) signaling compared to PGLs with PVs in genes that activate hypoxia pathways. In vitro, Hras PVs increased Pnmt expression and epinephrine biosynthesis through increased phosphorylation of stimulatory protein 1 via MAPK signaling. Here, we provide a molecular mechanism that explains the PV-dependent epinephrine production of PGLs.

Catecholamines and Parkinson's disease: tyrosine hydroxylase (TH) over tetrahydrobiopterin (BH4) and GTP cyclohydrolase I (GCH1) to cytokines, neuromelanin, and gene therapy: a historical overview.

The author identified the genes and proteins of human enzymes involved in the biosynthesis of catecholamines (dopamine, norepinephrine, epinephrine) and tetrahydrobiopterin (BH4): tyrosine hydroxylase (TH), aromatic L-amino acid decarboxylase (AADC), dopamine β-hydroxylase (DBH), phenylethanolamine N-methyltransferase (PNMT), and GTP cyclohydrolase I (GCH1). In Parkinson's disease (PD), the activities and levels of mRNA and protein of all catecholamine-synthesizing enzymes are decreased, especially in dopamine neurons in the substantia nigra. Hereditary GCH1 deficiency results in reductions in the levels of BH4 and the activities of TH, causing decreases in dopamine levels. Severe deficiencies in GCH1 or TH cause severe decreases in dopamine levels leading to severe neurological symptoms, whereas mild decreases in TH activity in mild GCH1 deficiency or in mild TH deficiency result in only modest reductions in dopamine levels and symptoms of DOPA-responsive dystonia (DRD, Segawa disease) or juvenile Parkinsonism. DRD is a treatable disease and small doses of L-DOPA can halt progression. The death of dopamine neurons in PD in the substantia nigra may be related to (i) inflammatory effect of extra neuronal neuromelanin, (ii) inflammatory cytokines which are produced by activated microglia, (iii) decreased levels of BDNF, and/or (iv) increased levels of apoptosis-related factors. This review also discusses progress in gene therapies for the treatment of PD, and of GCH1, TH and AADC deficiencies, by transfection of TH, AADC, and GCH1 via adeno-associated virus (AAV) vectors.

Abstract P2007: Novel Regulatory Axis In Heart-adrenal Cross Talk Mediated By Mtor-eprs

Adrenal catecholamines, including norepinephrine and epinephrine play an important role in cardiovascular physiology. Serum catecholamine level is tightly regulated in response to metabolic and cardiac stress. The biosynthesis of catecholamine is driven by a series of enzymatic reaction carried out by the key rate-limiting enzymes, including tyrosine hydroxylase (TH), DOPA decarboxylase (DDC) and phenylethanolamine-N-methyltransferase (PNMT). Although the transcription regulation of these enzymes has been described before, the regulatory mechanism at post-transcription or protein translation level has never been explored. Using a system genetics approach, we explored genes associated with the adrenal gland traits in HMDP at basal and post isoproterenol treatment. We have identified EPRS (Glutamyl-Prolyl-tRNA synthetase) to be significantly associated with adrenal gland mass post ISO treatment. EPRS is a tRNA synthetase responsible for charging tRNA during protein translation, EPRS also regulates mRNA translation as an RNA binding protein. To explore the functional impact of EPRS in adrenal gland physiology, we generated an EPRS adrenal gland specific knockout mouse model (EPRS-aKO). Inactivation of EPRS in adrenal gland led to a decrease of adrenal gland mass associated with reduced systemic catecholamine level. Interestingly, we further observed a moderate yet significantly decreased heart rate and ejection fraction in EPRS-aKO mice. Through both in vivo and in vitro analysis, we confirmed inactivation of EPRS led to a reduced protein, but not mRNA expression for both TH and DDC. In adipose tissue, activation of EPRS through mTOR signaling pathway is critical for its function as RNA binding protein. To further explore the mechanism through EPRS mediated catecholamine synthesis, we treated PC12 cells with rapamycin to inhibit mTOR activity. Treatment with Rapamycin diminished EPRS activity and expression, reduced TH protein expression along with a decrease of catecholamine level. Our results identified a novel regulatory scheme of catecholamine synthesis through mTOR-EPRS axis, adding a novel layer of gene regulation in catecholamine production and providing new mechanistic insights for heart-adrenal cross talk.

Cell-Effective Transition-State Analogue of Phenylethanolamine N-Methyltransferase.

Phenylethanolamine N-methyltransferase (PNMT) catalyzes the S-adenosyl-l-methionine (SAM)-dependent methylation of norepinephrine to form epinephrine. Epinephrine is implicated in the regulation of blood pressure, respiration, Alzheimer's disease, and post-traumatic stress disorder (PTSD). Transition-state (TS) analogues bind their target enzymes orders of magnitude more tightly than their substrates. A synthetic strategy for first-generation TS analogues of human PNMT (hPNMT) permitted structural analysis of hPNMT and revealed potential for second-generation inhibitors [Mahmoodi, N.; J. Am. Chem. Soc. 2020, 142, 14222-14233]. A second-generation TS analogue inhibitor of PNMT was designed, synthesized, and characterized to yield a Ki value of 1.2 nM. PNMT isothermal titration calorimetry (ITC) measurements of inhibitor 4 indicated a negative cooperative binding mechanism driven by large favorable entropic contributions and smaller enthalpic contributions. Cell-based assays with HEK293T cells expressing PNMT revealed a cell permeable, intracellular PNMT inhibitor with an IC50 value of 81 nM. Structural analysis demonstrated inhibitor 4 filling catalytic site regions to recapitulate both norepinephrine and SAM interactions. Conformation of the second-generation inhibitor in the catalytic site of PNMT improves contacts relative to those from the first-generation inhibitors. Inhibitor 4 demonstrates up to 51,000-fold specificity for PNMT relative to DNA and protein methyltransferases. Inhibitor 4 also exhibits a 12,000-fold specificity for PNMT over the α2-adrenoceptor.

Target Class Profiling of Small-Molecule Methyltransferases.

Target class profiling (TCP) is a chemical biology approach to investigate understudied biological target classes. TCP is achieved by developing a generalizable assay platform and screening curated compound libraries to interrogate the chemical biological space of members of an enzyme family. In this work, we took a TCP approach to investigate inhibitory activity across a set of small-molecule methyltransferases (SMMTases), a subclass of methyltransferase enzymes, with the goal of creating a launchpad to explore this largely understudied target class. Using the representative enzymes nicotinamide N-methyltransferase (NNMT), phenylethanolamine N-methyltransferase (PNMT), histamine N-methyltransferase (HNMT), glycine N-methyltransferase (GNMT), catechol O-methyltransferase (COMT), and guanidinoacetate N-methyltransferase (GAMT), we optimized high-throughput screening (HTS)-amenable assays to screen 27,574 unique small molecules against all targets. From this data set, we identified a novel inhibitor which selectively inhibits the SMMTase HNMT and demonstrated how this platform approach can be leveraged for a targeted drug discovery campaign using the example of HNMT.

Components of the sympathetic nervous system as targets to modulate inflammation – rheumatoid arthritis synovial fibroblasts as neuron-like cells?

BackgroundCatecholamines are major neurotransmitters of the sympathetic nervous system (SNS) and they are of pivotal importance in regulating numerous physiological and pathological processes. Rheumatoid arthritis (RA) is influenced by the activity of the SNS and its neurotransmitters norepinephrine (NE) and dopamine (DA) and early sympathectomy alleviates experimental arthritis in mice. In contrast, late sympathectomy aggravates RA, since this procedure eliminates anti-inflammatory, tyrosine hydroxylase (TH) positive cells that appear in the course of RA. While it has been shown that B cells can take up, degrade and synthesize catecholamines it is still unclear whether this also applies to synovial fibroblasts, a mesenchymal cell that is actively engaged in propagating inflammation and cartilage destruction in RA. Therefore, this study aims to present a detailed description of the catecholamine pathway and its influence on human RA synovial fibroblasts (RASFs).ResultsRASFs express all catecholamine-related targets including the synthesizing enzymes TH, DOPA decarboxylase, dopamine beta-hydroxylase, and phenylethanolamine N-methyltransferase. Furthermore, vesicular monoamine transporters 1/2 (VMAT1/2), dopamine transporter (DAT) and norepinephrine transporter (NET) were detected. RASFs are also able to degrade catecholamines as they express monoaminoxidase A and B (MAO-A/MAO-B) and catechol-O-methyltransferase (COMT). TNF upregulated VMAT2, MAO-B and NET levels in RASFs. DA, NE and epinephrine (EPI) were produced by RASFs and extracellular levels were augmented by either MAO, COMT, VMAT or DAT/NET inhibition but also by tumor necrosis factor (TNF) stimulation. While exogenous DA decreased interleukin-6 (IL-6) production and cell viability at the highest concentration (100 μM), NE above 1 μM increased IL-6 levels with a concomitant decrease in cell viability. MAO-A and MAO-B inhibition had differential effects on unstimulated and TNF treated RASFs. The MAO-A inhibitor clorgyline fostered IL-6 production in unstimulated but not TNF stimulated RASFs (10 nM-1 μM) while reducing IL-6 at 100 μM with a dose-dependent decrease in cell viability in both groups. The MAO-B inhibitor lazabemide hydrochloride did only modestly decrease cell viability at 100 μM while enhancing IL-6 production in unstimulated RASFs and decreasing IL-6 in TNF stimulated cells.ConclusionsRASFs possess a complete and functional catecholamine machinery whose function is altered under inflammatory conditions. Results from this study shed further light on the involvement of sympathetic neurotransmitters in RA pathology and might open therapeutic avenues to counteract inflammation with the MAO enzymes being key candidates.

Circuit-Specific Control of Blood Pressure by PNMT-Expressing Nucleus Tractus Solitarii Neurons

The nucleus tractus solitarii (NTS) is one of the morphologically and functionally defined centers that engage in the autonomic regulation of cardiovascular activity. Phenotypically-characterized NTS neurons have been implicated in the differential regulation of blood pressure (BP). Here, we investigated whether phenylethanolamine N-methyltransferase (PNMT)-expressing NTS (NTSPNMT) neurons contribute to the control of BP. We demonstrate that photostimulation of NTSPNMT neurons has variable effects on BP. A depressor response was produced during optogenetic stimulation of NTSPNMT neurons projecting to the paraventricular nucleus of the hypothalamus, lateral parabrachial nucleus, and caudal ventrolateral medulla. Conversely, photostimulation of NTSPNMT neurons projecting to the rostral ventrolateral medulla produced a robust pressor response and bradycardia. In addition, genetic ablation of both NTSPNMT neurons and those projecting to the rostral ventrolateral medulla impaired the arterial baroreflex. Overall, we revealed the neuronal phenotype- and circuit-specific mechanisms underlying the contribution of NTSPNMT neurons to the regulation of BP.

PS-BPR01-5: DOPAMINE-SECRETING PHEOCHROMOCYTOMA: AN OVERLOOKED CAUSE OF INCIDENTALLY DISCOVERED ADRENAL TUMOR AND NORMAL BLOOD PRESSURE

A 64-year-old woman with a history of chronic thyroiditis and no family history of endocrine disease was incidentally determined to have a left retroperitoneal mass on abdominal ultrasonography. A subsequent abdominal computed tomography (CT) scan revealed a 5.6 × 5.1 × 10.6 cm left adrenal tumor (10–50 Hounsfield Units of CT attenuation value), which showed marginal irregularity and an internal cystic structure [Figure 1A]. She was admitted to our hospital for further investigation of a suspected adrenocortical carcinoma. On admission, she was normotensive and had no symptoms of catecholamine excess. The 24-hour urine catecholamine level showed normal norepinephrine (122.9 ug/day), epinephrine (24.3 ug/day), whereas markedly elevated dopamine (148,212.4 ug/day). 123I-MIBG scintigraphy revealed tumor uptake. After alpha-blockade as preoperative management, she successfully underwent laparoscopic left adrenalectomy and was finally diagnosed with an exclusively dopamine-secreting pheochromocytoma. The tumor was histologically comprised of small polygonal cells with high cellularity and was immunohistochemically positive for all three catecholamine synthesizing enzymes: tyrosine hydroxylase (very weak), dopamine beta-hydroxylase (heterogeneous), and phenylethanolamine N-methyltransferase (very weak). Electron microscopy revealed very few catecholamine-containing small vesicles with a few organelles, which reflected immature cells. No biochemical or imaging evidence of recurrence or metastasis was evident 1 year after the surgery. To our knowledge, this is the first case of an exclusively dopamine-secreting pheochromocytoma in which immunostaining of catecholamine synthetic enzymes and electron microscopy of tumor tissues were performed together to identify the mechanism underlying a dopamine-secreting PPGL. The purpose of this report was to identify the mechanism underlying a dopamine-secreting pheochromocytoma from the histopathological findings. We also discuss the reasons for the lack of catecholamine excess symptoms, whether preoperative management of alpha-blockade is needed, and the association between the prognosis and genetic mutation with an extensive literature review. JOURNAL/jhype/04.03/00004872-202301001-00862/figure1/v/2023-10-24T163949Z/r/image-jpeg

Clinical identification of expressed proteins in adrenal medullary hyperplasia detected with hypertension.

Hypertension remains a challenging public health problem worldwide, and adrenal gland-related diseases are one class of the major causes for secondary hypertension. Among them, one relatively rare pattern is adrenal hyperplastic hypertension caused by adrenal medullary hyperplasia (AMH), leading to excessive secretion of autonomic catecholamine. Given that the pathological changes of adrenal medulla are not well correlated to the onset and even severity of secondary hypertension, the molecular basis why some AMH patients are accompanied with hypertension remains unclear and is worth exploring. For this reason, this study aims at investigating differentially expressed proteins in clinical AMH tissue, with special focus on the potential contribution of these differentially expressed proteins to AMH development, in order to have a better understanding of mechanisms how AMH leads to secondary hypertension to some extent. To this end, AMH specimens were successfully obtained and verified through computed tomography (CT) and haematoxylin-eosin (HE) staining. Proteomic analyses of AMH and control tissues revealed 782 kinds of differentially expressed proteins. Compared with the control tissue, there were 357 types of upregulated proteins and 425 types of downregulated proteins detected in AMH tissue. Of interest, these differentially expressed proteins were significantly enriched in 60 gene ontology terms (P < 0.05), including 28 biological process terms, 14 molecular function terms, and 18 cellular component terms. Pathway analysis further indicated that 306 proteins exert their functions in at least one Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. Western blotting showed enhanced expression of phenylethanolamine N- methyltransferase (PNMT), myelin protein zero (MPZ), and Ras-related protein Rab-3C (RAB3C), and reduced expression of cluster of differentiation 36 (CD36) observed in AMH tissue in comparison with controls. Clinical AMH specimens display a different proteomic profile compared to control tissue. Of note, PNMT, MPZ, RAB3C, and CD36 are found to differentially expressed and can be potential targets for AMH, providing a theoretical basis for mechanistic exploration of AMH along with hypertension.

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.

In-silico analysis of deleterious single nucleotide polymorphisms of PNMT gene

ABSTRACT PNMT is a protein-coding gene that codes for the Phenylethanolamine N-methyltransferase enzyme. It is primarily associated with neurodegenerative disorders such as Parkinson’s disease and Alzheimer’s disease. In the present study, over 17 different sequence-based and structure-based tools as well as 4 MSA tools and a ConSurf server were used to screen the highly deleterious single nucleotide polymorphisms (SNPs) of PNMT. Out of 286 missense SNPs, five were found to be deleterious (Y35D, I78N, I78S, G79D and L217Q) after the screening process. This was followed by performing oncogenic and phenotypic analysis, modelling of mutant proteins, structural analysis, STRING analysis, and post-translational modification (PTM) analysis. Finally, molecular dynamics simulations analysis was performed in duplicates to understand the impact of deleterious mutations on the stability of PNMT. All five mutations were predicted to be oncogenic and PTM analysis revealed that mutation Y35D occurs at a tyrosine kinase phosphorylation site.

1317-P: Intermittent Hypoxia Increased the Expression of DBH and PNMT in Neuronal Cells via MicroRNA-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 have reported correlation between IH and insulin resistance. However, why hypertension is induced by IH has been elusive. Here, we investigated the effect of IH on the expression of catecholamine metabolizing enzymes using in vitro IH system. Human and mouse neuronal cells, NB-1 and Neuro-2a, were exposed to IH (70 cycles of 5 min hypoxia [1% O2] and min normoxia [21% O2]) or normoxia for 24 hours. 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 (P=0.0271 and P&amp;lt;0.0001, respectively) and Neuro-2a (P=0.0135 and P=0.0341, respectively) . Western blot showed that the expression of DBH and PNMT in the NB-1 cells significantly increased by IH (P=0.0296 and P=0.0007, respectively) . We next prepared reporter plasmids containing human DBH (-1018˜+10) and PNMT (-600˜+67) upstream of luciferase reporter gene and transfected them into NB-1 cells. The promoter activities of these genes were not increased by IH. Target mRNA search of microRNA (miR) s using MicroRNA.org program revealed that the mRNAs have a potential target sequence for miR-375. The miR-375 level of IH-treated cells was significantly decreased than that of normoxia-treated cells (P=0.0222) . To clarify the role of miR-375, miR-375 mimic and non-specific control RNA (miR-375 mimic NC) were introduced into NB-1 cells. The IH-induced up-regulation of mRNAs of DBH and PNMT was abolished by introduction of miR-375 mimic but not by miR-375 mimic NC. Theses results strongly suggest that IH stress down-regulates the miR-375 in neuronal cells, resulting in the increased levels of DBH and PNMT via the inhibition of the miR-375-mediated mRNA degradation, leading SAS patients to hypertension. Disclosure S.Takasawa: None. R.Shobatake: None. Y.Takeda: None. T.Uchiyama: None. A.Yamauchi: None. M.Makino: None. K.Asai: None. H.Ota: None.

Differences in the Functional Activity and Redox Homeostasis Between the Left and Right Adrenal Gland of Rats Exposed to Chronic Isolation Stress

Abstract The aim of this study was to examine whether there are differences in adrenomedullary function in respect to the left and right sides in chronic stress conditions. We investigated how chronic stress isolation (CSI 12 weeks) affected the protein levels of key enzymes involved in adrenaline (A) synthesis (phenyl ethanolamine N-methyltransferase -PNMT), storage (vesicular monoamine transporters 2 - VMAT2) and degradation (catechol-O-methyltransferase - COMT), as well as the concentrations of A as an index for adrenomedullary function in the left and right adrenal medulla. Also, we examined the concentrations of malondialdehyde (MDA), protein levels of nuclear factor κB (NF-κB), and activity of catalase (CAT) in the left and right adrenal medulla. The investigated parameters were quantified by Western blot analysis, assay of enzymatic activity, and CAT Research ELISA kits. We found that CSI pro duced significantly increased levels of PNMT protein, and VMAT2 protein, as well as increased concentrations of A in the right adrenal medulla. However, we recorded that CSI increased protein levels of COMT and NF-κB, as well as the concentrations of MDA in the left adrenal medulla. Also, CSI decreased the activity of CAT only in the left adrenal medulla. Based on these results, it may be concluded that adrenomedullary function is different in respect to the left and right sides in chronic stress conditions.