Presence Of Norepinephrine Research Articles

Non-canonical Role of CaVα2δ1 in Cardiac Hypertrophy

Excessive neurohormonal activity contributes to the progression of pathological hypertrophy and subsequent transition to heart failure. Although increased diastolic Ca2+ represents a hallmark feature of heart failure, the role of L-type CaV1.2 channels in this process remains controversial. CaV1.2 is an oligomeric protein complex formed by the distinctive pore-forming subunit CaVα1 with auxiliary subunits (CaVβ, calmodulin, and CaVα2δ1). CaVα2δ1 is a large extracellular GPI-anchored protein that facilitates channel activation at physiological voltages and stimulates (≈ 5-fold) peak current density. The present study examined the time-dependent changes in the expression of CaV auxiliary subunits in an in vitro model of cardiac hypertrophy. Cultured 1-day old neonatal rat ventricular cardiomyocytes (NRVMs) were treated for 24-48 h with 1 μM norepinephrine or 50 nM endothelin-1. In the presence of norepinephrine or endothelin-1, cardiac troponin-T protein levels and cell surface area (confocal imaging) were increased 1.8 ± 0.3-fold (n=3) and 1.5 ± 0.3-fold (n=4), respectively. Analysis of the CaV1.2 subunits revealed that CaVα2δ1 protein levels were 1.4 ± 0.15-fold higher (n=3) in hypertrophic cardiomyocytes compared to untreated NRVMs without a concomitant change in the expression of CaVα1 and CaVβ subunits. CaVα2δ1 was identified at the plasma membrane and unexpectedly detected at the perinuclear region co-localized with calreticulin. The increased expression of CaVα2δ1 protein levels persisted in hypertrophic NRVMs treated with 1 μM nifedipine. Lastly, confocal imaging identified modest CaV1.2 and CaVα2δ1 immunoreactivity in ventricular fibroblasts. Experiments are currently examining the identity of CaVα2δ1 protein partners in NRVMs. Collectively, these data demonstrate that the heavily glycosylated CaVα2δ1 protein is upregulated in hypertrophic NRVMs and localization at the perinuclear region may contribute in part to the progression of pathological cardiac hypertrophy via a potential non-canonical paradigm.

LIM Kinase Inhibition Diminishes Hypertension and Vasoconstriction‐Induced Inward Remodeling in Mouse and Human Resistance Arteries

Vascular remodeling is an early biomarker of cardiovascular disease (CVD) related to structural changes in resistance arteries (RA). Arteriolar inward remodeling, characterized by a reduced luminal diameter that is associated with changes in the homeostasis of actin polymerization/depolymerization pathways, is posited to precede the development of hypertension. We hypothesize that inward remodeling involves changes in the actin cytoskeleton that facilitate cell motility and the rearrangement of vascular smooth muscle cells (VSMCs) around the vascular lumen. Moreover, we predict that inhibition of actin polymerization with a Lim kinase (LIMK) inhibitor would block the inward remodeling process associated with prolonged vasoconstriction and hypertension. We have previously reported in ex vivo experiments in rats that long‐term exposure of RA to vasoconstrictors induces vascular inward remodeling via activation of the Rho pathway, which in part promotes actin polymerization. We now demonstrate that VSMCs increase the inward remodeling ratio of filamentous to globular actin (F/G‐actin) in response to GPCR‐activation by a norepinephrine (NE) and angiotensin‐II (AngII). GPCR‐mediated Rho activates (LIMK), which subsequently phosphorylates and inactivates the actin severing enzyme, cofilin, thus effecting an increase in F/G‐actin ratios. In isolated rat cremasteric RA we have demonstrated that LIMK inhibition blocks the inward remodeling induced by of GPCR agonists and that this is coincident with a reduction in cofilin phosphorylation. To study the arterial remodeling process in vivo, we imaged the outer ear of a transgenic mouse line that expresses smooth muscle actin‐GFP in VSMCs by multiphoton microscopy. We treated these mice with AngII (1000ng/kg/min) for 14 days to induce vasoconstriction and hypertension. We imaged the same resistance artery within each ear before (Day 0) and after (Day 14) AngII infusion to study the progression of remodeling over time. Starting at day 0, one ear was injected every four days with a LIMK inhibitor (LIMKi), while the other ear received vehicle (control). We observed a significant inward remodeling in vessels of the control ear compared to those of the ear treated with LIMKi (p<0.05). We then assessed the effect of LIMKi in the inward remodeling process associated with ex vivo prolonged vasoconstriction in isolated human RA from omental tissues and studied by pressure myography under passive conditions before and after a 4h incubation in the presence of NE + AngII (control) or NE + AngII + LIMKi. Exposure to NE + AngII induced vascular inward remodeling that was completely ablated by presence of LIMKi and resulted in outward remodeling (p<0.05). These data indicate that LIMK plays a necessary role in the vasoconstriction‐hypertension vascular remodeling process that makes it a potential target for the treatment of CVDs associated with resistance artery inward remodeling.Support or Funding InformationNational Institutes of Health grants: R01 HL137769 (JP), R01 HL088105 (LM‐L).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Влияние рН среды на нейрогенную реактивность внутренней сонной артерии крысы на фоне действия норадреналина

The effect of pH on the neurogenic contractile response to electrical field stimulation (at frequencies of 1, 3, 10, and 40 Hz) of the rat internal carotid artery segment in the absence and in the presence of 0.01-10.0 μM noradrenaline was studied. In the absence of noradrenaline, acidosis (рН 6.6) was shown to inhibit this response most significantly at high frequencies of stimulation, while in the presence of noradrenaline, acidosis rather potentiated and alkalosis (рН 7.6) inhibited the response most significantly at low frequencies of stimulation used. Acidosis tended to increase the artery contraction evoked by noradrenaline in all concentrations except for 10.0 μM. Noradrenaline potentiated the neurogenic vasoconstriction, which was more pronounced at noradrenaline concentrations from 0.5 μM to 10.0 μM, increased at acidosis and decreased at alkalosis. Increase in the contractility of the internal carotid artery under the condition of the pH shift from alkaline to acidic may be of importance for the prevention of the increase in the cerebral perfusion pressure during exercise characterized by elevated blood pressure, acidosis, and increased levels of noradrenaline.

In vitro pharmacological characterization of SPN-810M (molindone).

BackgroundImpulsive aggression (IA) is considered a maladaptive form of aggression that is reactive and overt and occurs outside of the acceptable social context. Many children and adolescents with attention-deficit/hyperactivity disorder (ADHD) display clinically significant aggression, with the predominant subtype being IA. However, there is currently no Food and Drug Administration-approved medication specifically to treat IA. The pathophysiology of IA is not fully understood, although it has been suggested to include the dopamine, norepinephrine, and serotonin systems.MethodsSPN-810 (extended-release molindone) is being developed for the novel indication of IA and is currently being studied in patients treated for ADHD. Molindone is an indole derivative and a dopamine D2 receptor antagonist.ResultsThe in vitro pharmacological studies described in the current manuscript demonstrate that the active substance molindone (SPN-810M) is a potent antagonist for the dopamine receptors, D2S and D2L, and the serotonin receptor, 5-HT2B, at therapeutic concentrations. The in vitro studies further demonstrate that the antagonist effect of SPN-810M is due to the parent drug and not the metabolites, and that the antagonism is not affected by the presence of norepinephrine or dopamine neurotransmitters. In addition, studies investigating the potential differential effects of the enantiomers of SPN-810M have demonstrated that the R(−) enantiomer is more potent than S(+), showing greater regulatory effect on D2S and D2L receptors.ConclusionOverall, the results of the in vitro SPN-810M pharmacological studies provide some insight into how SPN-810M modulates the serotonin and dopamine pathways that play a role in IA.

Follicular Development and Secretion of Ovarian Hormones during the Juvenile and Adult Reproductive Lives of the Myelin Mutant taiep Rat: An Animal Model of Demyelinating Diseases.

Infertility and reproductive problems have been reported in women with several neurological disorders, for example, demyelination. However, the physiology of such problems has remained unknown so far. The taiep rats are an animal neurological model that initially shows a hypomyelination followed by a progressive demyelination of the central nervous system. This animal has reproductive problems, and the aim of this work is to characterize the follicular development, secretion of ovarian hormones, and presence of noradrenaline in the ovaries of the female taiep rats in the juvenile and adult stages. The taiep rats have low body weight (approximately 19% less than that of SD rats), a delay of 4 days in the age of vaginal opening, and an irregularity in the estrous cycle by the absence or prolongation of some estral cycle stage. In the juvenile stage, we observed a decrease of approximately 44% in the total number of follicles with a 15% increase of atresia and an 80% decrease in the fluorescence intensity of catecholamines in the ovaries, with a 21% increment in plasma concentrations of testosterone. In the adult stage, we observed follicular cysts and a 50% decrease in fluorescence intensity of catecholamines in the ovaries, with changes in the secretion of ovarian hormones, an increase of 20 times in progesterone, and a decrement of a half in estradiol. The demyelination in taiep rats affects follicular development and steroidogenesis in the early stages of the animal's life, and this is maintained until adulthood.

Cold-induced stress impairs the production of Th1 protective cytokines during co-culturing of dendritic cells and naïve T helper cells of Chlamydia muridarum infected mice.

Abstract The purpose of this study was to determine how cold-induced stress affects the maturing of mouse bone marrow-derived dendritic cells (BMDCs) and to see the dynamics of cytokines production associated with T helper subtypes. Quantitative real time PCR and ELISA were used to measure gene expression and production of cytokines in co-culture of BMDC and Naïve CD4+ T cells. Culturing BMDC in vitro stimulated with LPS in the presence of norepinephrine (NE) or Feroterol, β-AR agonist resulted in a marked increase (75 pg/ml) of IL-10 compared to that of stressed mice. Treatment with β-AR antagonist, ICI 118,551 resulted in lowering production of IL-10 in BMDC of nonstressed (control) mice. In contrast, IL-12 production in stressed group was significantly low (132 vs 239 pg/ml) compared to that of control mice. Naïve T cells co-cultured with BMDC pretreated with NE or Feroterol showed decreased IL-12 production compared to controls. In contrast IL-4 production was increased with pretreatment of NE or Feroterol (64 vs 28 pg/ml) compared to control group. Increased IL-4 production in the genital tracks of mice compared to control (114 vs 0 pg/ml) was observed. The relative expression of GTAT 3 in T cells co-cultured with BMDC of stressed mice was high (2.8 vs 0.49) compared to control mice. The same gene expression trend of IL-4 in stressed mice group (1.8 versus 0.12) compared to control group was observed. In control mice, T-bet expression was (1.78 vs 1.0) and interferon gamma (2.12 versus 1.0) compared to that of stressed mice group. Our results demonstrate that stress influences the activation and differentiation of DCs stimulated by LPS shifting the Th1/Th2 ratio and impairing the production of Th1 protective cytokines during Chlamydia muridarum infection.

Norepinephrine Increases Cytosolic Ca2+ in Neurons of the Paraventricular Nucleus of the Hypothalamus

The paraventricular nucleus (PVN), an important nucleus of the hypothalamus, is involved in cardiorespiratory and central autonomic responses to a variety of stimuli or stressors. The PVN receives dense innervation from catecholaminergic neurons in the nucleus tractus solitarii, ventrolateral medulla, and locus coeruleus. Stressors, including hypoxia, activate these projections to the PVN, releasing neurotransmitters and modulators including norepinephrine (NE). Within the PVN, adrenergic receptor activation by NE increases excitatory postsynaptic currents, and induces depolarization and elevated action potential discharge. However, NE's role in modulating cytosolic Ca2+ in PVN neurons remains to be determined. In this study, we determined the effects of NE on PVN neuron cytosolic Ca2+ using fura‐2 ratiometric fluorescent Ca2+ imaging. PVN neurons were dissociated from 3–4 wk old male Sprague‐Dawley rats and used within 12 hours of isolation. Neurons were exposed to aCSF baseline (Bsl, 5 min), followed by vehicle (5 min) or NE (100 mM, 5 min). Compared to aCSF Bsl, PVN neurons showed stable basal Ca2+ during vehicle controls (n=34). In contrast, addition of 100 mM NE elevated basal Ca2+ (n=69). To examine the contribution of Ca2+ from internal sarco/endoplasmic reticulum Ca2+‐ATPase (SERCA) stores in this elevation, neurons were exposed to the SERCA blocker Thapsigargin (TG, 1 mM, 30 min). When NE was added in the presence of TG the increase in basal Ca2+ persisted, although slightly attenuated (n=11). To examine if NE elevates cytosolic Ca2+ via extracellular Ca2+ entry, cells were exposed to aCSF with zero Ca2+. In the absence of extracellular Ca2+, NE did not elevate basal Ca2+ and rather showed a significant decrease (n=63). Spontaneous Ca2+ peaks in PVN neurons were also recorded. While NE did not affect the number of spontaneous peaks or the number of cells that exhibited spontaneous peaks, NE did increase spontaneous peak amplitude (n=69). NE in presence of TG replicated NE's increase in spontaneous peak amplitude (n=11). NE in the absence of extracellular Ca2+ significantly decreased spontaneous peak amplitude (n=63). To study the potential influence of NE on use (voltage)‐dependent Ca2+ channels, cells were repeatedly depolarized with 55 mM K+ (5×, 20 sec, 5 min intervals). NE or vehicle was applied between the 3rd and 4th depolarization and the amplitude of Ca2+ peaks and basal Ca2+ was evaluated. Across the 5 peaks, there was a slight decrease in amplitude over time that was not altered by vehicle (n=34). Application of 100 mM NE induced a small, non‐significant increase in Ca2+ peak amplitude, and a significant increase in basal Ca2+ (n=34). In contrast to the NE response that occurred in neurons studied immediately after isolation, PVN neurons incubated for 2–5 days saw the effects of NE greatly diminished, if not completely abolished, and 24 hour incubation in 10 mM NE did not restore NE sensitivity. These preliminary findings demonstrate NE increases cytosolic Ca2+, likely from an influx of extracellular Ca2+, and support previous work that NE is associated with depolarization of PVN neurons.Support or Funding InformationHL 098602This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Oxidative burst and Dectin-1-triggered phagocytosis affected by norepinephrine and endocannabinoids: implications for fungal clearance under stress.

A prolonged stress burden is known to hamper the efficiency of both the innate and the adaptive immune systems and to attenuate the stress responses by the catecholaminergic and endocannabinoid (EC) systems. Key mechanisms of innate immunity are the eradication of pathogens through phagocytosis and the respiratory burst. We tested the concentration-dependent, spontaneous and stimulated (via TNFα and N-formylmethionine-leucyl-phenylalanine) release of reactive oxygen species (ROS) by human polymorphonuclear leukocytes (PMNs) in vitro in response to norepinephrine (NE) and AM1241, a pharmacological ligand for the EC receptor CB2. We evaluated phagocytosis of Dectin-1 ligating zymosan particles and tested the cytokine response against Candida antigen in an in vitro cytokine release assay. Increasing concentrations of NE did not affect phagocytosis, yet stimulated ROS release was attenuated gradually reaching maximum suppression at 500 nM. Adrenergic receptor (AR) mechanisms using non-AR-selective (labetalol) as well as specific α-(prazosin) and β-(propranolol) receptor antagonists were tested. Results show that only labetalol and propranolol were able to recuperate cytotoxicity in the presence of NE, evidencing a β-receptor-mediated effect. The CB2 agonist, AM1241, inhibited phagocytosis at 10 µM and spontaneous peroxide release by PMNs. Use of the inverse CB2 receptor agonist SR144528 led to partial recuperation of ROS production, confirming the functional role of CB2. Additionally, AM1241 delayed early activation of monocytes and induced suppression of IL-2 and IL-6 levels in response to Candida via lower activity of mammalian target of rapamycin (mTOR). These findings provide new insights into key mechanisms of innate immunity under stressful conditions where ligands to the sympatho-adrenergic and EC system are released.

Effect of Coscinium Fenestratum stem extracts on hypertension in animal model

This study was done to evaluate the anti-hypertensive effects of stem extract of Coscinium fenestratum (CF). The stems were air dried for 2 days, grounded and then soaked in organic solvent for 2 days. Thereafter the filtered extract was concentrated using rotary evaporator at 450C to obtain the crude extract. Partial purification was done using silica gel column chromatography to separate the crude extract into five fractions A-E. The effect on hypertension was tested by intravenous injection of each extractinto a normotensive anaesthetized rat andperfused frog heart. The effect Mean Arterial Blood Pressure (MABP), Heart rate (beats per minute) (HR) and force of contraction (FOC) was recorded via the BIOPAC LAB PRO SOFTWARE. The phytochemical compound present in the extract was identified using a simple colorimetric biochemical method. The extract showed a reduction of theMABP, HR and FOC in the presence of norepinephrine and without norepinephrine. The effect of the extract was more than that of commercial drug atenolol. Colorimetric phytochemistry testing revealed the presence of flavonoids, terpenoids, saponins, tanins, cardiac glycoside and alkaloids. The effect maybe because of the type of bioactive compounds present in the extract which has previously shown antioxidant property.

Glucose autoregulation is the dominant component of the hormone-independent counterregulatory response to hypoglycemia in the conscious dog.

The contribution of hormone-independent counterregulatory signals in defense of insulin-induced hypoglycemia was determined in adrenalectomized, overnight-fasted conscious dogs receiving hepatic portal vein insulin infusions at a rate 20-fold basal. Either euglycemia was maintained (group 1) or hypoglycemia (≈45 mg/dl) was allowed to occur. There were three hypoglycemic groups: one in which hepatic autoregulation against hypoglycemia occurred in the absence of sympathetic nervous system input (group 2), one in which autoregulation occurred in the presence of norepinephrine (NE) signaling to fat and muscle (group 3), and one in which autoregulation occurred in the presence of NE signaling to fat, muscle, and liver (group 4). Average net hepatic glucose balance (NHGB) during the last hour for groups 1-4 was -0.7 ± 0.1, 0.3 ± 0.1 (P < 0.01 vs. group 1), 0.7 ± 0.1 (P = 0.01 vs. group 2), and 0.8 ± 0.1 (P = 0.7 vs. group 3) mg·kg-1·min-1, respectively. Hypoglycemia per se (group 2) increased NHGB by causing an inhibition of net hepatic glycogen synthesis. NE signaling to fat and muscle (group 3) increased NHGB further by mobilizing gluconeogenic precursors resulting in a rise in gluconeogenesis. Lowering glucose per se decreased nonhepatic glucose uptake by 8.9 mg·kg-1·min-1, and the addition of increased neural efferent signaling to muscle and fat blocked glucose uptake further by 3.2 mg·kg-1·min-1 The addition of increased neural efferent input to liver did not affect NHGB or nonhepatic glucose uptake significantly. In conclusion, even in the absence of increases in counterregulatory hormones, the body can defend itself against hypoglycemia using glucose autoregulation and increased neural efferent signaling, both of which stimulate hepatic glucose production and limit glucose utilization.

Sympathetic nervous system activity and anti-lipolytic response to iv-glucose load in subcutaneous adipose tissue of obese and obese type 2 diabetic subjects.

The study aim was to investigate the effect of endogenous insulin release on lipolysis in subcutaneous adipose tissue after adrenergic stimulation in obese subjects diagnosed with type 2 diabetes (T2D). In 14 obese female T2D subjects, or 14 obese non-T2D controls, glycerol concentration was measured in response to the α1,2,ß-agonist norepinephrine, the α1-agonist norfenefrine and the ß2-agonist terbutaline (each 10−4 M), using the microdialysis technique. After 60 minutes of stimulation, an intravenous glucose load (0.5 g/kg lean body mass) was given. Local blood flow was monitored by means of the ethanol technique. Norepinephrine and norfenefrine induced a four and three fold rise in glycerol dialysate concentration (p<0.001, each), with a similar pattern in adipose tissue. Following agonist stimulation and glucose infusion, endogenous insulin release inhibited lipolysis in the presence of norepinephrine, which was more rapid and pronounced in healthy obese controls than in T2D subjects (p = 0.024 obese vs T2D subjects). Insulin-induced inhibition of lipolysis in the presence of norfenefrine was similar in all study participants. In the presence of terbutaline the lipolysis rate increased two fold until the effect of endogenous insulin (p<0.001). A similar insulin-induced decrease in lipolysis was observed for each of the norfenefrine groups and the terbutaline groups, respectively. Adipose tissue blood flow remained unchanged after the iv-glucose load. Both norepinephrine and norfenefrine diminished blood flow slightly, but insulin reversed this response (p<0.001 over the entire time). Terbutaline alone and terbutaline plus increased endogenous insulin augmented local blood flow (p<0.001 over the entire time). In conclusion, a difference in insulin-induced inhibition of lipolysis was observed in obese T2D subjects compared to obese healthy controls following modulation of sympathetic nervous system activity and is assumed to be due to ß1-adrenoceptor mediated stimulation by norepinephrine.

Heterogeneous effects of norepinephrine on spontaneous and stimulus-driven activity in the male accessory olfactory bulb.

Norepinephrine (NE) release has been linked to experience-dependent plasticity in many model systems and brain regions. Among these is the rodent accessory olfactory system (AOS), which is crucial for detecting and processing socially relevant environmental cues. The accessory olfactory bulb (AOB), the first site of chemosensory information processing in the AOS, receives dense centrifugal innervation by noradrenergic fibers originating in the locus coeruleus. Although NE release has been linked to behavioral plasticity through its actions in the AOB, the impacts of noradrenergic modulation on AOB information processing have not been thoroughly studied. We made extracellular single-unit recordings of AOB principal neurons in ex vivo preparations of the early AOS taken from adult male mice. We analyzed the impacts of bath-applied NE (10 μM) on spontaneous and stimulus-driven activity. In the presence of NE, we observed overall suppression of stimulus-driven neuronal activity with limited impact on spontaneous activity. NE-associated response suppression in the AOB came in two forms: one that was strong and immediate (21%) and one other that involved gradual, stimulus-dependent monotonic response suppression (47%). NE-associated changes in spontaneous activity were more modest, with an overall increase in spontaneous spike frequency observed in 25% of neurons. Neurons with increased spontaneous activity demonstrated a net decrease in chemosensory discriminability. These results reveal that noradrenergic signaling in the AOB causes cell-specific changes in chemosensory tuning, even among similar projection neurons.NEW & NOTEWORTHY Norepinephrine (NE) is released throughout the brain in many behavioral contexts, but its impacts on information processing are not well understood. We studied the impact of NE on chemosensory tuning in the mouse accessory olfactory bulb (AOB). Electrophysiological recordings from AOB neurons in ex vivo preparations revealed that NE, on balance, inhibited mitral cell responses to chemosensory cues. However, NE's effects were heterogeneous, indicating that NE signaling reshapes AOB output in a cell- and stimulus-specific manner.

Adrenergic control of pinealocyte chondriome - an in vitro study.

Norepinephrine released from sympathetic innervation plays the main role in the regulation of melatonin secretion in mammalian pinealocytes. The present study was conducted for the following reasons: 1) to establish whether the pinealocyte chondriome is controlled by norepinephrine, 2) to determine the effect of adrenergic stimulation on mitochondria, and 3) to characterize adrenoceptors involved in the regulation of the chondriome. The static organ culture of the pineal gland was used. The explants were incubated for 5 consecutive days in control medium and between 20:00 and 08:00 in medium with the presence of 10 μM norepinephrine - adrenergic agonist; isoproterenol - beta-adrenoceptor agonist; cirazoline, methoxamine, M-6364 - alfa1 - adrenoceptors agonists or PMA - activator of PKC. The explants were then subjected to ultrastructural examination and morphometric analysis. The incubation of explants in the presence of norepinephrine or isoproterenol caused a decrease in the relative volume and the numerical density of mitochondria and induced an increase in the percentage of free mitochondria in pinealocytes. Significant changes in these parameters were not observed after treatment with methoxamine, cirazoline, M-6463 and PMA. The results obtained show that the chondriome of pig pinealocytes is controlled by norepinephrine acting via beta-adrenoceptors. Adrenergic stimulation, repeated for five consecutive days of organ culture, causes a decrease in the number of mitochondria and a shift in the distribution of mitochondria from the form of networks and filaments into the form of single particles. This indicates the intensive remodeling of the mitochondria network, which is closely linked to the metabolic status of the cell.

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.

Abstract 088: Chronic Efferent Vagal Stimulation Enhances Norepinephrine-mediated Inhibition of Splenic Pro-inflammatory Cytokine Release in Lupus Hypertension

Hypertension is prevalent in the autoimmune disease systemic lupus erythematosus (SLE), occurring with alarming frequency in reproductive-age women. Recent studies implicate the adaptive immune system in the development and maintenance of hypertension, and neuroimmune pathways may regulate this source of inflammation. One example is the cholinergic anti-inflammatory pathway (CAP), an endogenous nerve-to-spleen mechanism that regulates splenic pro-inflammatory cytokine release. We hypothesized that this pathway is impaired in SLE and that chronic stimulation of the CAP at the level of the efferent vagus nerve would attenuate hypertension in SLE. Starting at 30 and 32 weeks of age, female NZBWF1 SLE mice and NZW control mice were treated with the pharmacologic efferent vagal stimulators CNI-1493 (CNI; 8mg/kg; twice weekly; i.p.) or galantamine (GAL; 4mg/kg; daily; i.p.), or saline. At 34 weeks of age, we measured mean arterial pressure (MAP), finding that MAP (mmHg) in SLE mice was elevated compared to controls (139.83 ± 4.56 vs. 120.70 ± 2.96; n=4-6/group, p = 0.002), while the rise in MAP was prevented by CNI (134.45 ± 3.07)and GAL (129.25 ± 3.97) in SLE mice. We further hypothesized that splenocytes isolated from SLE mice conditioned by efferent vagal stimulation would release fewer pro-inflammatory cytokines in the presence of norepinephrine, which stimulates splenic β2 adrenergic receptors. We incubated isolated splenocytes for 24 hours at 37°C with and without norepinephrine (100 μM), then measured pro-inflammatory cytokines in the supernatant via ELISA. Compared to control mice, splenocytes from SLE mice secreted 70.7% and 146.5% higher concentrations of IL-6 and TNF-α (8.24 vs. 4.83 and 2.79 vs. 1.13 pg/mL, respectively; n=2/group) in the presence of norepinephrine. Compared to saline-treated SLE mice, splenocytes from CNI and GAL-treated SLE mice released fewer cytokines when incubated with norepinephrine (8.24 vs. 5.31 and 5.79 pg/mL IL-6; 2.79 vs. 2.18 and 0.81 pg/mL TNF-α; n=2/group). These in vivo and in vitro data suggest that stimulation of the CAP at the level of the efferent vagus may promote anti-inflammatory splenocyte activity, which may be protective against hypertension in the setting of chronic inflammation.

Quantification of neurotransmitters and metabolically related compounds at glassy carbon electrodes modified with bamboo-like carbon nanotubes dispersed in double stranded DNA

This work reports the application of glassy carbon electrodes (GCE) modified with bamboo-like carbon nanotubes (bCNT) dispersed in calf-thymus double-stranded DNA (dsDNA) (GCE/bCNT-dsDNA) for the highly sensitive and selective quantification of neurotransmitters and metabolically related compounds by Adsorptive Stripping. Dopamine (Do) was quantified in the presence of its most important metabolite, 3,4-dihydroxyphenylacetic acid (dopac), while normetanephrine (NM) was determined in the presence of its precursor norepinephrine (NE). The accumulation of the analytes was performed at open circuit potential and the quantification was carried out, after medium exchange, in a 0.200M acetate buffer solution pH5.0 using Differential Pulse Voltammetry. The sensitivity for Do in the presence of 1.0×10−5M dopac was (7.1±0.2)×105μAM−1, (just 7.0% higher than the one obtained for Do alone), the detection limit was 68nM, and the linear range 5.0×10−7M-7.0×10−6M. The sensitivities for NM in the absence and presence of NE were (2.8±0.1)×104μAM−1 and (2.6±0.2)×104μAM−1, with a linear range between 1.0×10−6 and 2.0×10−5M, and a detection limit of 0.5μM. The platform was successfully used for the quantification of Do and NM in enriched human urine samples.

Enhanced activity of hormone sensitive lipase (HSL) in mesenteric but not epididymal fat correlates with higher production of epinephrine in mesenteric adipocytes in rat model of cachectic rheumatoid arthritis

Cachectic rheumatoid arthritis, the less frequent form of the disease, is associated with loss of fat mass and often more severe course of the disease. Its experimental model represents rat adjuvant arthritis (AA) characterized by edema, lack of appetite, sharp body weight and fat loss. As individual fat depots display functional differences, here we studied lipolytic activity and sensitivity to lipolytic stimuli of nodeless epididymal fat (eWAT) and perinodal mesenteric fat (mWAT) depots at the peak of AA. We also examined changes in catecholamine and cytokine levels involved in lipolysis in plasma and/or isolated adipocytes from both WATs to identify the contribution of local, adipocyte-based processes and/or systemic events to adiposity loss in cachectic rheumatoid arthritis. AA was induced to male Lewis rats by complete Freund’s adjuvant. Groups of ad libitum-fed and pair-fed controls were used to distinguish the effects of food restriction from inflammation-induced cachexia. Adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL) and its phosphorylated form (pHSL) were analyzed by western blot. CRP and catecholamine levels in plasma or adipocyte lysates were determined using ELISA kits. Cytokine-induced neutrophil chemoattractant-1 (CINC-1/CXCL1), monocyte chemoattractant protein-1 (MCP-1/CCL2), IL-1β, IL-6, IL-10 and leptin in adipocyte lysate were analyzed by quantitative protein microarray. Plasma glycerol and FFA were measured spectrophotometrically. AA rats developed severe cachexia, with lower adiposity in mWAT compared to normal and pair-fed controls, whereas in eWAT the adiposity was similarly reduced in AA and pair-fed groups. ATGL levels in both WATs were not affected by AA or pair feeding. AA upregulated levels of HSL, pHSL and pHSL/HSL ratio in mWAT, whereas none of these parameters has changed in eWAT of AA rats or in either WATs of pair-fed rats. In AA rats plasma glycerol was elevated, whereas FFA concentration was reduced. Plasma norepinephrine and epinephrine were increased in AA compared with both groups of controls. In eWAT adipocytes, AA but not pair feeding, upregulated norepinephrine levels. In mWAT adipocytes, AA rats showed higher epinephrine levels than pair-fed controls. Leptin levels in both WATs were depleted in AA animals in accordance with body weight loss. None of the measured cytokines in eWAT and mWAT was enhanced. Our results demonstrate augmented lipolytic activity in mWAT and not eWAT during cachectic arthritis. The adipocyte-derived cytokines do not seem to contribute to activated lipolysis. We first demonstrated enhanced presence of norepinephrine in perinodal adipocytes that may contribute to the regulation of local lipolytic activity by auto/paracrine fashion and thus provide independent fuel supply to activated lymph nodes.

Adrenergic Effect on Cytokine Release After Ex Vivo Healthy Volunteers' Whole Blood LPS Stimulation.

Catecholamines are molecules with immunomodulatory properties in health and disease. Several studies showed the effect of catecholamines when administered to restore hemodynamic stability in septic patients. This study investigates the effect of norepinephrine and dobutamine on whole blood cytokine release after ex vivo lipopolysaccharide (LPS) stimulation. Whole blood collected from healthy individuals was stimulated with LPS, in the presence of norepinephrine or dobutamine at different concentrations, with or without metoprolol, a β1 receptor antagonist. Cytokine measurement was performed in isolated cell culture supernatants with ELISA. Results are expressed as mean ± SEM and compared with Mann-Whitney rank-sum test. Both norepinephrine and dobutamine significantly reduced TNF-α and IL-6 production after ex vivo LPS stimulation of whole blood in a dose-dependent manner, and this effect was partially reversed by the presence of metoprolol. Norepinephrine and dobutamine reduce the LPS-induced production of pro-inflammatory cytokines, thus possibly contributing to altered balance between the inflammatory and anti-inflammatory responses, which are vital for a successful host response to severe disease, shock, and sepsis.

Down Regulation of L-Type Calcium Current in Rat Atrial Myocytes during Heart Failure

Heart failure causes down-regulation of the atrial L-type Ca2+ current (ICaL) that has consequences on contractile function and susceptibility to atrial fibrillation. The reduction in atrial ICaL density has been suggested to involve changes in both the expression and phosphorylation of channel protein. The objective of this project was to examine the mechanism of the decrease in atrial ICaL in a surgical model of heart failure. Heart failure was induced in adult male Wistar rats by surgical ligation of the left anterior descending coronary artery (CAL) under general anesthesia. Control animals were subject to equivalent sham operation (Sham). Left atrial myocytes were isolated ∼18 weeks after surgery and superfused with a Tyrode's solution (37 °C). Whole-cell L-type Ca currents (ICaL) were recorded during depolarisation to 0 mV from a holding potential of −80 mV and normalised to whole-cell capacitance, which was also used as an index of cell size. CAL cells were hypertrophied (94.7±5.2 pF, n=63) compared to Sham (56.6±2.1 pF, n=47; P≤0.0001). ICaL density at 0 mV was reduced in CAL compared to Sham (3.3±0.4 pA/pF; n=31 vs 7.3±0.7 pA/pF; n=33; P≤0.001), but the response of ICaL to 1 μmol/L norepinephrine was greater in CAL (x6.8) than in Sham (x3.2) so that there was no difference in ICaL density between the two groups in the presence of norepinephrine. These data indicate a change in channel protein phosphorylation without a change in channel density. The regulation of L-type Ca2+ channel phosphorylation may be a potential therapeutic target for atrial arrhythmias in heart failure.This work was supported by the British Heart Foundation

Role of Various Potassium Channels in Caffeine-induced Aortic Relaxation in Rats.

Background:Studies done on caffeine-induced changes in aortic rings have demonstrated inconclusive results. Moreover, the role of various potassium channels in caffeine-induced effects has not been explored so far. The present in vitro study was designed to explore the direct effects of caffeine on rat aortic rings and the role of various potassium channels in those changes/effects.Materials and Methods:This study was carried out in College of Medicine, University of Dammam. Aortic rings obtained from Sprague Dawley rats were mounted in the organ bath. Tension in the aortic rings was measured with an isometric force transducer and recorded with a PowerLab data-acquisition system. Aortic rings in relaxed and contractile state were exposed to caffeine and various potassium channel blockers (glyburide, 4-aminopyridine, or tetraethylammonium).Results:Caffeine produced significant relaxation of isolated aortic rings (baseline tension: 1.26 ± 0.30 g, tension after adding cumulative concentrations of caffeine: 1.12 ± 0.31 g, P < 0.05) in the absence or presence of norepinephrine (NE) (tension induced by NE: 1.06 ± 0.37 g, tension after adding cumulative concentrations of caffeine: 1.01 ± 0.36 g, P < 0.05). Caffeine's vasodilatory effects were, however, blocked in aortic rings pretreated with different types of potassium channel blockers such as 4-aminopyridine (tension induced by NE: 1.52 ± 0.41 g, tension after adding cumulative concentrations of caffeine: 1.50 ± 0.37 g, P > 0.05), glyburide (tension induced by NE: 0.82 ± 0.35 g, tension after adding cumulative concentrations of caffeine: 0.79 ± 0.42 g, P > 0.05), and tetraethylammonium (tension induced by NE: 0.68 ± 0.34 g, tension after adding cumulative concentrations of caffeine: 0.67 ± 0.33 g, P > 0.05).Conclusion:Caffeine causes significant dilation of aortic rings, and this vasodilatory effect may involve ATP-dependent, calcium-mediated, or voltage-dependent potassium channels.