Absence Of Norepinephrine Research Articles

Chronic stress promotes non-small cell lung cancer (NSCLC) progression through circMBOAT2 upregulation mediated by CTCF.

Circular RNA (circRNA) has been demonstrated to play a pivotal role in tumor development. This study aimed to investigate the regulatory mechanism of circMBOAT2 in non-small cell lung cancer (NSCLC) and its association with tumor growth induced by chronic stress. We constructed stably transfected A549 and H1299 cell lines with circMBOAT2 overexpression and knockdown. Colony formation, scratch healing, Transwell and CCK-8 assays were conducted to evaluate the effects of circMBOAT2 in the presence or absence of norepinephrine (NE) treatment on the proliferation, migration, and invasion of NSCLC cells, respectively. Additionally, A chronic unpredictable mild stress (CUMS)-induced depression with heterotopic transplantation LLC and injection of antisense oligonucleotides (ASOs) targeting circMBOAT2 mouse model was established to evaluate the effect of chronic stress on tumorigenesis via circMBOAT2. Moreover, we investigated the regulatory effect of CCCTC binding factor (CTCF) on circMBOAT2 expression through in vivo and in vitro silencing of CTCF. Our results revealed a significant upregulation of circMBOAT2 in NSCLC cell lines and tumor tissues. circMBOAT2 knockdown inhibited the proliferation, migration, and invasion of NSCLC cells, while NE treatment reversed the cell suppression effect caused by circMBOAT2 knockdown. Notably, CUMS promoted tumor growth, while silencing circMBOAT2 inhibited tumor growth in vivo. Furthermore, we identified CTCF as the upstream regulator of circMBOAT2, which exhibited upregulation in NSCLC cells and tissues. Knockdown of CTCF reversed the promotional effect of CUMS on circMBOAT2 expression and tumor growth. Our findings provide evidence that CTCF mediates chronic stress in promoting of NSCLC progression through circMBOAT2. circMBOAT2 may serve as a potential biomarker and therapeutic target for NSCLC as well as the treatment of comorbid depression in NSCLC patients.

Norepinephrine Drives Sleep Fragmentation Activation of Asparagine Endopeptidase, Locus Ceruleus Degeneration, and Hippocampal Amyloid-β42 Accumulation.

Chronic sleep disruption (CSD), from insufficient or fragmented sleep and is an important risk factor for Alzheimer's disease (AD). Underlying mechanisms are not understood. CSD in mice results in degeneration of locus ceruleus neurons (LCn) and CA1 hippocampal neurons and increases hippocampal amyloid-β42 (Aβ42), entorhinal cortex (EC) tau phosphorylation (p-tau), and glial reactivity. LCn injury is increasingly implicated in AD pathogenesis. CSD increases NE turnover in LCn, and LCn norepinephrine (NE) metabolism activates asparagine endopeptidase (AEP), an enzyme known to cleave amyloid precursor protein (APP) and tau into neurotoxic fragments. We hypothesized that CSD would activate LCn AEP in an NE-dependent manner to induce LCn and hippocampal injury. Here, we studied LCn, hippocampal, and EC responses to CSD in mice deficient in NE [dopamine β-hydroxylase (Dbh)-/-] and control male and female mice, using a model of chronic fragmentation of sleep (CFS). Sleep was equally fragmented in Dbh -/- and control male and female mice, yet only Dbh -/- mice conferred resistance to CFS loss of LCn, LCn p-tau, and LCn AEP upregulation and activation as evidenced by an increase in AEP-cleaved APP and tau fragments. Absence of NE also prevented a CFS increase in hippocampal AEP-APP and Aβ42 but did not prevent CFS-increased AEP-tau and p-tau in the EC. Collectively, this work demonstrates AEP activation by CFS, establishes key roles for NE in both CFS degeneration of LCn neurons and CFS promotion of forebrain Aβ accumulation, and, thereby, identifies a key molecular link between CSD and specific AD neural injuries.

START domain-mediated nuclear translocation of Them1 regulates the expression of metabolic genes relative to thermogenesis in brown adipocytes

Thioesterase superfamily member 1 (Them1) is a long-chain fatty acyl-CoA thioesterase that is highly expressed in brown adipose tissue (BAT), where it limits energy expenditure. Them1 is comprised of tandem enzymatic domains and a C-terminal lipid-binding steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domain. Fatty acid binding to the START domain stabilizes the enzymatic domains and increases activity, whereas lysophosphatidylcholine (LPC) acts in the opposite manner. In brown adipocytes, Them1 localizes to puncta within the cytoplasm, but diffuses in response to β-adrenergic stimulation after which it is also found in the nucleus. The aim of this study was to determine the mechanism of Them1 nuclear trafficking and the metabolic consequences. METHODS: Identification of a putative nuclear localization signal (NLS) was determined by computational analysis. Full-length, NLS-deleted, or truncated Them1 cDNA constructs were cloned and linked to EGFP. Plasmid or adenoviral vectors were transfected into immortalized brown adipose cells (iBAs) that do not express Them1. Them1 was visualized by live cell confocal imaging via EGFP and with NucSpot nuclear staining. Signal quantification was performed with Imaris software. Them1 nuclear localization was evaluated in the presence or absence of norepinephrine (NE), leptomycin B, cycloheximide, or lipids that bind the START domain. Them1 nuclear localization was also quantified in BAT from mice treated with a β3-adrenergic receptor agonist, CL316,243. mRNA expression in iBAs expressing full-length or NLS-mutant Them1 in response to NE was determined by RNA-Seq. RESULTS: In the diffuse state, Them1 localized to the nucleus in a time-dependent manner. The NLS resides in a hinge region between the thioesterase domains and the START domain. Deletion of either the NLS or the START domain blocked nuclear targeting. Conversely, truncated Them1 containing only the START domain was targeted to the nucleus. Entry into the nucleus was balanced by trafficking out of the nucleus and new protein synthesis was not required, as evidenced by using leptomycin B to block nuclear efflux and cycloheximide to block protein synthesis, respectively. Incubation of NE-stimulated iBAs with LPC promoted nuclear Them1 translocation. Them1 protein was also detected in the nucleus of BAT from wild-type mice in response to CL administration. Nuclear Them1 localization in iBAs was associated with down-regulated mRNA expression of genes that control lipid and glucose metabolism during cold adaptation. CONCLUSIONS: Our data suggest that LPC generated during thermogenesis binds and destabilizes the START domain, leading to exposure of the NLS and nuclear trafficking of Them1 in response to β-adrenergic stimulation. Once inside the nucleus, Them1 regulates the transcription of lipid and glucose metabolic enzymes that are required for chronic cold adaptation. This work was supported by NIH DK103046 to DEC, SJH, and EAO This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

QseBC regulates in vitro and in vivo virulence of Aeromonas hydrophila in response to norepinephrine.

The inter-kingdom communication between host and pathogenic bacteria mediated by the host hormones epinephrine (Epi)/norepinephrine (NE)/autoinducer-3 (AI-3) and transduced by the bacterial two-component signal transduction system QseBC has been well demonstrated in mammalian pathogens. Aeromonas hydrophila, a common opportunistic pathogen in freshwater aquaculture, responds to NE by increased bacterial growth and enhanced virulence. However, the underlying mechanisms remain poorly understood. Our study demonstrated that deletion of qseB and qseC significantly inhibited NE-promoted growth, biofilm formation, and hemolytic activity of A. hydrophila. The adhesion ability of ΔqseB and ΔqseC to J774a.1cells was significantly decreased compared with the wild-type strain in the presence and absence of NE, whereas NE still enhanced the adhesion ability of the mutant and wild-type strains with a similar effect, suggesting that NE-enhanced cell adhesion was independent of QseBC. Moreover, QseBC did not affect the swimming and swarming motility of A. hydrophila with or without NE. Quantitative real-time PCR analyses revealed the down-regulated expression of some virulence-related genes (hly, ast, act, aerA) in each mutant compared with the wild-type strain in the presence of NE. Tilapia infection experiments indicated that deletion of qseB or qseC weakened NE-promoted virulence of A. hydrophila. In conclusion, our study suggests that NE stimulates the growth, biofilm formation, and hemolytic activity of A. hydrophila and enhances the virulence of the pathogen in fish via the QseBC system.

Cross-interactions between Norepinephrine, Methicillin-Resistant Staphylococcus aureus and Human Osteoblast Cells in Culture Conditions

Objective: The role of norepinephrine (NE) on growth, adhesion and invasion of methicillin-resistant Staphylococcus aureus (MRSA) ATTC 43300 was examined in human osteoblast (HOB) cells. The effects of NE and/or MRSA on the viability and cell death pathways of HOB cells were also investigated. Furthermore, the alterations of bacterial response to oxidative stress (H2O2) were analyzed in the presence/ absence of NE. Materials and Methods: Bacterial growth was detected spectrophotometrically. The colony counting method was examined for adhesion-invasion. The alteration of HOB cell viability was determined by methyl thiazolyl diphenyl-tetrazolium bromide (MTT) assay. The death pathways of HOB cells were examined microscopically using acridine orange-ethidium bromide dual staining and dichlorofluorescein-diacetate (DCF-DA) dye. The bacterial response to H2O2 was investigated by agar dilution. Results: The growth of bacterium was not affected in the presence of NE. Bacterial adhesion was decreased by NE (p<0.0001) while high-level NE induced invasion (p=0.013). HOB cell viability was reduced by MRSA and/or NE (p<0.001). MRSA and co-existence of MRSA and NE caused necrosis more than apoptosis in HOB cells (p<0.05). NE did not alter the bacterial response to oxidative stress. Conclusion: Norepinephrine has different effects on the biological properties of both MRSA and HOB cells.

Arterial Load and Norepinephrine Are Associated With the Response of the Cardiovascular System to Fluid Expansion.

BackgroundFluid responsiveness has been extensively studied by using the preload prism. The arterial load might be a factor modulating the fluid responsiveness. The norepinephrine (NE) administration increases the arterial load and modifies the vascular properties. The objective of the present study was to determine the relationship between fluid responsiveness, preload, arterial load, and NE use. We hypothesized that as a preload/arterial load, NE use may affect fluid responsiveness.MethodsThe retrospective multicentered analysis of the pooled data from 446 patients monitored using the transpulmonary thermodilution before and after fluid expansion (FE) was performed. FE was standardized between intensive care units (ICUs). The comparison of patients with and without NE at the time of fluid infusion was performed. Stroke volume (SV) responsiveness was defined as an increase of more than 15% of SV following the FE. Pressure responsiveness was defined as an increase of more than 15% of mean arterial pressure (MAP) following the FE. Arterial elastance was used as a surrogate for the arterial load.ResultsA total of 244 patients were treated with NE and 202 were not treated with NE. By using the univariate analysis, arterial elastance was correlated to SV variations with FE. However, the SV variations were not associated with NE administration (26 [15; 46]% vs. 23 [10; 37]%, p = 0.12). By using the multivariate analysis, high arterial load and NE administration were associated with fluid responsiveness. The association between arterial elastance and fluid responsiveness was less important in patients treated with NE. Arterial compliance increased in the absence of NE, but it did not change in patients treated with NE (6 [−8; 19]% vs. 0 [−13; 15]%, p = 0.03). The changes in total peripheral and arterial elastance were less important in patients treated with NE (−8 [−17; 1]% vs. −11 [−20; 0]%, p < 0.05 and −10 [−19; 0]% vs. −16 [−24; 0]%, p = 0.01).ConclusionThe arterial load and NE administration were associated with fluid responsiveness. A high arterial load was associated with fluid responsiveness. In patients treated with NE, this association was lower, and the changes of arterial load following FE seemed to be driven mainly by its resistive component.

3-LB: Glutamate Metabolism Is Coupled to Lactate Production from Astrocytes under Low-Glucose Conditions

Recurrent hypoglycemia (RH) results in elevated lactate levels in the ventromedial hypothalamus that impair the counter-regulatory responses to hypoglycemia. While the mechanisms that enhance lactate production are not clear, our previous data suggests that astrocytic glycogen is the major source of lactate resulting from RH. A number of different inputs regulate glycogen turnover in astrocytes and the interaction between metabolic and neurotransmitter signals may alter glycogenolytic capacity and ultimately, lactate production. The current study investigates how the interaction between norepinephrine and glutamate influence lactate production from astrocytes in the setting of hypoglycemia. We cultured primary rat astrocytes in normal (2.5mM) and low (0.1mM) glucose medium in the presence and absence of norepinephrine and/or glutamate for 3 hours and quantified lactate in the culture media. We also examined the contribution of glutamate oxidation using the glutamate dehydrogenase inhibitor, hexachlorophene. Our data indicates that under low glucose conditions, glutamate alone dramatically enhances lactate production in astrocytes compared to controls (P&amp;lt;0.0001). Of note, the inhibition of glutamate oxidation also increased lactate secretion, but not to the same extent as glutamate alone (P&amp;lt;0.01). In contrast, the addition of norepinephrine and glutamate significantly reduced lactate secretion (P&amp;lt;0.05). Our data suggests that glutamate uptake during acute hypoglycemia likely plays a large role in driving astrocytic lactate production, a metabolic deficit that is offset by the oxidation of glutamate and by norepinephrine. In conclusion, our data suggests that under low glucose conditions, the metabolism of glutamate is tightly coupled to lactate production in astrocytes. Disclosure D. A. Appadurai: None. O. Chan: None. Funding National Institutes of Health (DK099315)

Norepinephrine Dysregulates the Immune Response and Compromises Host Defense during Sepsis.

Rationale: Sepsis is characterized by a dysregulated immune response to infection. Norepinephrine, the cornerstone vasopressor used in septic shock, may contribute to immune dysregulation and impact host defense.Objectives: To investigate effects of norepinephrine and the alternative vasopressor vasopressin on the immune response and host defense.Methods: Leukocytes from six to nine donors were stimulated in the presence or absence of norepinephrine and vasopressin. A total of 190 C57BL/6J mice received a continuous infusion of norepinephrine or vasopressin via microosmotic pumps and were challenged with LPS or underwent cecal ligation and puncture. Thirty healthy volunteers were randomized to a 5-hour infusion of norepinephrine, vasopressin, or saline and intravenously challenged with LPS. The relationship between the norepinephrine infusion rate and the use of β-blockers and plasma cytokines was assessed in 195 patients with septic shock.Measurements and Main Results: Norepinephrine attenuated the production of proinflammatory mediators and reactive oxygen species and augmented antiinflammatory IL-10 production both in vitro and in LPS-challenged mice. Norepinephrine infusion during cecal ligation and puncture resulted in increased bacterial dissemination to the spleen, liver, and blood. In LPS-challenged volunteers, norepinephrine enhanced plasma IL-10 concentrations and attenuated the release of the proinflammatory cytokine IFN-γ-induced protein 10. Vasopressin exerted no immunomodulatory effects across these experimental setups. In patients, higher norepinephrine infusion rates were correlated with a more antiinflammatory cytokine balance, whereas β-blocker use was associated with a more proinflammatory cytokine balance.Conclusions: Norepinephrine dysregulates the immune response in mice and humans and compromises host defense. Therefore, it may significantly contribute to sepsis-induced immunoparalysis, whereas vasopressin does not have untoward immunologic effects.

Thioesterase Superfamily Member 1 Undergoes Stimulus‐coupled Reorganization to Regulate Metabolic Activity in Brown Adipose Tissue

Thioesterase superfamily member 1 (Them1) is a long chain fatty acyl‐CoA thioesterase that is enriched in brown adipose tissue. Them1 is markedly upregulated by cold ambient temperatures, but functions to suppress energy expenditure. Consequently Them1−/− mice are highly resistant to diet‐induced obesity‐related disorders, most notably hepatic steatosis and insulin resistance. Although Them1 reduces fatty acid availability for b‐oxidation in mitochondria, how it limits thermogenesis remains undefined. Here we show that Them1 undergoes marked intracellular conformational changes after stimulation to regulate metabolic activity.MethodsCultured brown adipocytes, which do not constitutively express Them1, were transfected with full‐length Them1 fused to EGFP as well as an engineered ascorbate peroxidase (APEX2) fusion protein. EGFP was visualized by confocal microscopy in the presence or absence of norepinephrine (NE) stimulation to determine the 3‐D localization of Them1. APEX2 was used for high resolution correlative light and electron microscopy (CLEM). Cells were further transfected with Them1 phosphomimetic mutants at S15, 18, and 25 to evaluate the role of phosphorylation in Them1 organization. Rates of NE‐stimulated fatty acid oxidation were determined by using a Seahorse XFe96. The localization of Them1 in brown adipose tissue of mice was determined after injection of CL316,243, a β3 adrenoceptor agonist. The primary amino acid sequence of Them1 was subjected to multiple analyses to predict disordered regions, aggregation, and hydrophobicity.ResultsThem1 formed puncta that were localized near lipid droplets and mitochondria in cultured brown adipose cells. In response to NE, Them1 was phosphorylated, which resulted in a diffuse intracellular localization. This change in Them1 localization also occurred in brown adipose tissue after CL316,243 administration to mice. Puncta formation activated Them1, as evidenced by suppression of oxygen consumption. CLEM revealed that puncta are highly organized membraneless organelles, which typically form after a phase separation in cells. To support this possibility, Them1 contains one intrinsically disordered region at the N‐terminus that contains multiple interacting motifs, as is frequently found in phase‐separating proteins. Similarly, phosphorylation is known to disrupt aggregation after a phase transition, resulting in a diffuse localization.ConclusionsOur data establish that Them1 forms membraneless organelles (puncta) that function to suppress thermogenesis. Additionally, puncta are dispersed after phosphorylation at S15 to maximize fatty acid oxidation during a period of energy demand and to allow for the rapid suppression of thermogenesis once the demand no longer exists. We speculate that Them1 is an attractive molecular target in the management of non‐alcoholic fatty liver disease and other obesity‐related disorders.

Analysis of &lt;i&gt;Coscinium fenestratum&lt;/i&gt; Colebr stem extract and effect of the extract on mean arterial blood pressure, heart rate and force of contraction in rats

Purpose : To evaluate the effect of Coscinium fenestratum (CF) crude extract on hypertension in animal models. Methods : Dichloromethane (DCM), ethyl acetate (EA), and butanol (BuOH) extracts were obtained by fractionation from the crude methanol extract. The dried extracts were then administered to Sprague- Dawley (250 – 300 mg) rats (n = 20) and frogs (n = 20) of either sex. CF effect on mean arterial blood pressure (MABP), heart rate (HR) and force of contraction (FOC) were assessed with the aid of Biopac Lab Pro Software. Atenolol (5 ug/ml) and acetylcholine (5 ug/ml) were used as controls. High performance liquid chromatography (HPLC) analysis and phytochemical studies were carried out on the extract. Results : The methanol extract (M - E) reduced MABP by 45.60 %, HR by 48.50 %, FOC .by 58.40 %; Fraction - E of the methanol extract reduced MABP by 60.60 %, HR by 58.60 %, FOC by 65.80 %; compared to atenolol which lowered MABP by 52.80 %, HR by 50.60 %, and FOC by 58.80 %; acetylcholine which reduced MABP by 30.50 % HR by 33.60 %, and FOC by 10.50 % in the presence and absence of norepinephrine. Preliminary phytochemical analysis by HPLC indicate the presence of flavonoids, terpenoids, saponins, tannins, cardiac glycoside, alkaloids, columbamine, berberine and naringin. Conclusion : The results show that Coscinium fenestratum extract lowers MABP, HR and FOC in rats. These effects may be due to the bioactive components present in the extract. Keywords : Coscinium fenestratum , Hypertension, Norepinephrine, Acetylcholine, Atenolol

In vitro reduction of antibacterial activity of tigecycline against multidrug-resistant Acinetobacter baumannii with host stress hormone norepinephrine

The host stress hormone norepinephrine (NE), also called noradrenaline, is reported to augment bacterial growth and pathogenicity, but few studies have focused on the effect of NE on the activity of antimicrobials. The aim of this study was to clarify whether NE affects antimicrobial activity against multidrug-resistant Acinetobacter baumannii (MDR-AB). Time–kill studies of tigecycline (TIG) and colistin (COL) against MDR-AB as well as assays for factors contributing to antibiotic resistance were performed using MDR-AB clinical strains both in the presence and absence of 10 µM NE. In addition, expression of three efflux pump genes (adeB, adeJ and adeG) in the presence and absence of NE was analysed by quantitative reverse transcription PCR. Viable bacterial cell counts in TIG-supplemented medium containing NE were significantly increased compared with those in medium without NE. In contrast, NE had little influence on viable bacterial cell counts in the presence of COL. NE-supplemented medium resulted in an ca. 2 log increase in growth and in bacterial cell numbers adhering on polyurethane, silicone and polyvinylchloride surfaces. Amounts of biofilm in the presence of NE were ca. 3-fold higher than without NE. Expression of the adeG gene was upregulated 4–6-fold in the presence of NE. In conclusion, NE augmented factors contributing to antibiotic resistance and markedly reduced the in vitro antibacterial activity of TIG against MDR-AB. These findings suggest that NE treatment may contribute to the failure of TIG therapy in patients with MDR-AB infections.

Green tea catechins enhance norepinephrine-induced lipolysis via a protein kinase A-dependent pathway in adipocytes

Green tea catechins have been shown to attenuate obesity in animals and humans. The catechins activate adenosine monophosphate-activated protein kinase (AMPK), and thereby increase fatty acid oxidation in liver and skeletal muscles. Green tea catechins have also been shown to reduce body fat in humans. However, the effect of the catechins on lipolysis in adipose tissue has not been fully understood. The aim of this study was to clarify the effect of green tea catechins on lipolysis in adipocytes and to elucidate the underlying mechanism. Differentiated mouse adipocyte cell line (3T3-L1) was stimulated with green tea catechins in the presence or absence of norepinephrine. Glycerol and free fatty acids in the media were measured. Phosphorylation of hormone-sensitive lipase (HSL) was determined by Western blotting, and the mRNA expression levels of HSL, adipose triglyceride lipase (ATGL), and perilipin were determined by quantitative RT-PCR. The cells were treated with inhibitors of protein kinase A (PKA), protein kinase C (PKC), protein kinase G (PKG), or mitogen-activated protein kinase (MAPK) to determine the responsible pathway. Treatment of 3T3-L1 adipocytes with green tea catechins increased the level of glycerol and free fatty acids released into the media in the presence, but not absence, of norepinephrine, and increased the level of phosphorylated HSL in the cells. The catechins also increased mRNA and protein levels of HSL and ATGL. PKA inhibitor (H89) attenuated the catechin-induced increase in glycerol release and HSL phosphorylation. The results demonstrate that green tea catechins enhance lipolysis in the presence of norepinephrine via a PKA-dependent pathway in 3T3-L1 adipocytes, providing a potential mechanism by which green tea catechins could reduce body fat.

Escherichia coli O157:H7 Lacking the qseBC -Encoded Quorum-Sensing System Outcompetes the Parental Strain in Colonization of Cattle Intestines

The qseBC-encoded quorum-sensing system regulates the motility of Escherichia coli O157:H7 in response to bacterial autoinducer 3 (AI-3) and the mammalian stress hormones epinephrine (E) and norepinephrine (NE). The qseC gene encodes a sensory kinase that autophosphorylates in response to AI-3, E, or NE and subsequently phosphorylates its cognate response regulator QseB. In the absence of QseC, QseB downregulates bacterial motility and virulence in animal models. In this study, we found that 8- to 10-month-old calves orally inoculated with a mixture of E. coli O157:H7 and its isogenic qseBC mutant showed significantly higher fecal shedding of the qseBC mutant. In vitro analysis revealed similar growth profiles and motilities of the qseBC mutant and the parental strain in the presence or absence of NE. The magnitudes of the response to NE and expression of flagellar genes flhD and fliC were also similar for the qseBC mutant and the parental strain. The expression of ler (a positive regulator of the locus of enterocyte effacement [LEE]), the ler-regulated espA gene, and the csgA gene (encoding curli fimbriae) was increased in the qseBC mutant compared to the parental strain. On the other hand, growth, motility, and transcription of flhD, fliC, ler, espA, and csgA were significantly reduced in the qseBC mutant complemented with a plasmid-cloned copy of the qseBC genes. Thus, in vitro motility and gene expression data indicate that the near-parental level of motility, ability to respond to NE, and enhanced expression of LEE and curli genes might in part be responsible for increased colonization and fecal shedding of the qseBC mutant in calves.

The foetal pig pineal gland is richly innervated by nerve fibres containing catecholamine-synthesizing enzymes, neuropeptide Y (NPY) and C-terminal flanking peptide of NPY, but it does not secrete melatonin.

Innervation of the mammalian pineal gland during prenatal development is poorly recognized. Therefore, immunofluorescence studies of the pineals of 70- and 90-day-old foetuses of the domestic pig were performed using antibodies against tyrosine hydroxylase (TH), dopamine-β-hydroxylase (DβH), neuropeptide Y (NPY) and C-terminal flanking peptide of NPY (CPON). The investigated glands were supplied by numerous nerve fibres containing TH and DβH. The density of these fibres was higher in the distal and middle parts of the gland than in the proximal one. NPY and CPON were identified in the majority of DβH-positive fibres as well as in a small population of DβH-negative fibres localized mainly in the proximal part of the pineal. The immunoreactive fibres were more numerous in 90-day-old foetuses than in 70-day-old ones. The effect of norepinephrine on melatonin secretion by the foetal pineals in the short-term organ culture was studied to determine the role of DβH-positive fibres during prenatal life. For the same purpose melatonin was measured in the blood in the umbilical cords and in the jugular vein of the mother. The pineals of both groups of foetuses did not secrete melatonin in the organ culture, independently of the presence or absence of norepinephrine in the medium. Melatonin concentrations in the blood in the umbilical cords of foetuses from the same litter and in the jugular vein of their mother were similar. The presence of adrenergic nerve fibres in the pig pineal during gestation does not seem to be associated with the control of melatonin secretion.

Abstract 24: Autonomic Function and Obesity in the Coupling Between Interleukin-6 and C-Reactive Protein

C-reactive protein (CRP) is a marker of inflammation in obesity, induced in part by interleukin-6 (IL-6) derived from adipocytes. To test the hypothesis that sympathetic overactivity contributes to the increase in IL-6 and CRP, we measured these inflammatory markers in female subjects: 16 obese (42±2 yrs., BMI 37.5±1.3), 30 healthy controls (32±3 yrs., BMI 23.2±0.4), and 54 with postural tachycardia syndrome (POTS, 31±1 yrs., BMI 22.2±0.4), a condition characterized by increased sympathetic tone. We also included 3 patients with congenital absence of norepinephrine (dopamine ß-hydroxylase deficiency, DBH) as a comparison group for isolated sympathetic failure. Sympathetic activation, measured by low-frequency variability of blood pressure (LFSBP), was greater in obese and POTS compared to healthy controls (8.5±0.8 and 6.1±0.6 vs. 3.4±0.4 mmHg^2, P&lt;0.01. Figure A). High-frequency heart rate variability (HFRRI), a marker of parasympathetic tone, was lower in POTS and in obese subjects compared to healthy controls (357±56, 402±146 and 1556±394 ms^2, respectively, P&lt;0.01 for POTS vs. controls). IL-6 was higher in the obese and POTS groups compared to healthy controls (4.4±0.4 and 4.2±0.5 vs. 2.2±0.4 pg/mL, P&lt;0.01. Figure B), but hsCRP was increased only in obese subjects (4.5±1.2 vs. 1.1±0.2 and 0.9±0.2 mg/L for POTS and healthy controls, P&lt;0.01). We conclude that sympathetic activation and parasympathetic withdrawal are associated with increased serum IL-6 levels even in lean patients, but not with increased CRP. The coupling between IL-6 and CRP requires increased adiposity.

Effects of Adrenergic Agents on Intracellular Ca2+ Homeostasis and Metabolism of Glucose in Astrocytes with an Emphasis on Pyruvate Carboxylation, Oxidative Decarboxylation and Recycling: Implications for Glutamate Neurotransmission and Excitotoxicity

Glucose and glycogen are essential sources of energy for maintaining glutamate homeostasis as well as glutamatergic neurotransmission. The metabolism of glycogen, the location of which is confined to astrocytes, is affected by norepinephrine (NE), and hence, adrenergic signaling in the astrocyte might affect glutamate homeostasis with implications for excitatory neurotransmission and possibly excitotoxic neurodegeneration. In order to study this putative correlation, cultured astrocytes were incubated with 2.5 mM [U-(13)C]glucose in the presence and absence of NE as a time course for 1 h. Employing mass spectrometry, labeling in intracellular metabolites was determined. Moreover, the involvement of Ca(2+) in the noradrenergic response was studied. In unstimulated astrocytes, the labeling pattern of glutamate, aspartate, malate and citrate confirmed important roles for pyruvate carboxylation and oxidative decarboxylation in astrocytic glucose metabolism. Importantly, pyruvate carboxylation was best visualized at 10 min of incubation. The abundance and pattern of labeling in lactate and alanine indicated not only an extensive activity of malic enzyme (initial step for pyruvate recycling) but also a high degree of compartmentalization of the pyruvate pool. Stimulating with 1 μM NE had no effect on labeling patterns and glycogen metabolism, whereas 100 μM NE increased glutamate labeling and decreased labeling in alanine, the latter supposedly due to dilution from degradation of non-labeled glycogen. It is suggested that further experiments uncovering the correlation between adrenergic and glutamatergic pathways should be performed in order to gain further insight into the role of astrocytes in brain function and dysfunction, the latter including excitotoxicity.

Thermogenically competent nonadrenergic recruitment in brown preadipocytes by a PPARγ agonist

Most physiologically induced examples of recruitment of brown adipose tissue (BAT) occur as a consequence of chronic sympathetic stimulation (norepinephrine release within the tissue). However, in some physiological contexts (e.g., prenatal and prehibernation recruitment), this pathway is functionally contraindicated. Thus a nonsympathetically mediated mechanism of BAT recruitment must exist. Here we have tested whether a PPARgamma activation pathway could competently recruit BAT, independently of sympathetic stimulation. We continuously treated primary cultures of mouse brown (pre)adipocytes with the potent peroxisome proliferator-activated receptor-gamma (PPARgamma) agonist rosiglitazone. In rosiglitazone-treated cultures, morphological signs of adipose differentiation and expression levels of the general adipogenic marker aP2 were manifested much earlier than in control cultures. Importantly, in the presence of the PPARgamma agonist the brown adipocyte phenotype was significantly enhanced: UCP1 was expressed even in the absence of norepinephrine, and PPARalpha expression and norepinephrine-induced PGC-1alpha mRNA levels were significantly increased. However, the augmented levels of PPARalpha could not explain the brown-fat promoting effect of rosiglitazone, as this effect was still evident in PPARalpha-null cells. In continuously rosiglitazone-treated brown adipocytes, mitochondriogenesis, an essential part of BAT recruitment, was significantly enhanced. Most importantly, these mitochondria were capable of thermogenesis, as rosiglitazone-treated brown adipocytes responded to the addition of norepinephrine with a large increase in oxygen consumption. This thermogenic response was not observable in rosiglitazone-treated brown adipocytes originating from UCP1-ablated mice; hence, it was UCP1 dependent. Thus the PPARgamma pathway represents an alternative, potent, and fully competent mechanism for BAT recruitment, which may be the cellular explanation for the enigmatic recruitment in prehibernation and prenatal states.

Catecholamine activation of p38 MAPK in S49 T lymphosarcoma cells is dependent on Protein Kinase A

The catecholamine norepinephrine (NE) functions through a β2AR/cAMP/PKA pathway in T cells leading to altered immune function. p38 MAPK is an important intracellular signaling mediator for environmental stressors and plays a role in cell survival, apoptosis, and inflammation. To determine a relationship between NE‐mediated immune dysfunction and the p38 MAPK pathway, S49 wild type cells were incubated in the presence and absence of NE or a cAMP analogue, and the levels of activated (dually phosphophorylated, pTpY180/182) p38 MAPK were detected by ELISA. Activated p38 MAPK increased upon NE or cAMP treatment within 5 min and was sustained for 30 min. When the cells were treated with nadolol, a β2AR antagonist, the activation of p38 MAPK by NE was inhibited. Treatment of an S49 PKA mutant cell line (kin‐) with NE or cAMP, unlike wt, showed no change in activated p38 MAPK levels. These results suggest that NE can signal through the β2AR/cAMP/PKA pathway to activate p38 MAPK and reveal a new signaling pathway for engagement of p38 MAPK through a G protein‐coupled receptor/PKA dependent mechanism. Supported by NIH.

Insulin modulates norepinephrine-mediated melatonin synthesis in cultured rat pineal gland

The mammalian pineal gland synthesizes melatonin in a circadian manner, peaking during the dark phase. This synthesis is primarily regulated by sympathetic innervations via noradrenergic fibers, but is also modulated by many peptidergic and hormonal systems. A growing number of studies reveal a complex role for melatonin in influencing various physiological processes, including modulation of insulin secretion and action. In contrast, a role for insulin as a modulator of melatonin synthesis has not been investigated previously. The aim of the current study was to determine whether insulin modulates norepinephrine (NE)-mediated melatonin synthesis. The results demonstrate that insulin (10 − 8M) potentiated norepinephrine-mediated melatonin synthesis and tryptophan hydroxylase (TPOH) activity in ex vivo incubated pineal glands. When ex vivo incubated pineal glands were synchronized (12h NE-stimulation, followed by 12h incubation in the absence of NE), insulin potentiated NE-mediated melatonin synthesis and arylalkylamine- N-acetyltransferase (AANAT) activity. Insulin did not affect the activity of hydroxyindole- O-methyltranferase (HIOMT), nor the gene expression of tpoh, aanat, or hiomt, under any of the conditions investigated. We conclude that insulin potentiates NE-mediated melatonin synthesis in cultured rat pineal gland, potentially through post-transcriptional events.

Abstract 1295: The Vasomotor Effects of Apelin in vivo in Man

BACKGROUND In preclinical models, apelin, the endogenous ligand for the G protein-coupled APJ receptor, lowers systemic venous tone, causes nitric oxide (NO)-mediated arterial vasodilatation and is the most potent inotrope yet studied. We aimed to establish the in vivo vasomotor effects of apelin in man. METHODS Dorsal hand vein diameter was measured by the Aellig technique in 6 subjects during local intravenous infusions (0.1–3 nmol/min) of two apelin agonists, apelin-36 and (Pyr1)apelin-13, and sodium nitroprusside (SNP; 0.6 nmol/min) in the presence and absence of norepinephrine preconstriction. Forearm blood flow (FBF) responses to intrabrachial infusions of apelin-36 and (Pyr1)apelin-13 (0.1–30 nmol/min) were measured by venous occlusion plethysmography in 8 subjects on 4 occasions. In a 2×2 factorial design, a further 8 subjects received an intrabrachial infusion of (Pyr1)apelin-13 (0.3–3 nmol/min) in the presence or absence of the NO clamp (NO synthase inhibitor, L-N-monomethylarginine (8 micromol/min), co-infused with SNP (1–128 ng/min) to maintain basal flow and clamp endogenous NO production), and following a single oral dose of aspirin (600 mg) or matched placebo. RESULTS Although SNP caused venodilatation (P&lt;0.0001), apelin-36 and (Pyr1)apelin-13 had no effect on dorsal hand vein diameter in the presence or absence of norepinephrine (P=0.2). Both apelin-36 and (Pyr1)apelin-13 caused reproducible dose-dependent vasodilatation in forearm resistance vessels (P&lt;0.0001): maximal increase of 198+/−27% and 90+/−7% respectively. Vasodilatation had a rapid onset (within 6 min) and slow offset that remained 36 min post-infusion (P&lt;0.01). Plasma apelin concentrations and non-infused FBF (P&lt;0.05) were increased at doses &gt;3 nmol/min, consistent with systemic spillover. (Pyr1)apelin-13-mediated vasodilatation was attenuated by the NO clamp (AUC reduced by 64%; P=0.004) but was unaffected by aspirin (P=0.7). CONCLUSION Although having no apparent effect on venous tone, apelin causes a rapid onset and sustained NO-dependent arterial vasodilatation in vivo in man. The apelin-APJ system merits further clinical investigation to determine its role in cardiovascular homeostasis and its therapeutic potential in heart failure.