bluuebunny/crossref_metadata_2025 huggingface dataset:
Obesity and Chagas Disease: Cardiovascular, Oxidative and Metabolic Aspects of this Relationship
Authors: Martins‐Pinge Marli Cardoso , Lucchetti Bruno Fernando Cruz , Lopes Fernanda Novi Cortegoso , Pinge‐Filho Phileno
Published: June 2021

Chagas disease (CD) is one of the leading neglected tropical infectious diseases, and people living in endemic regions are at potential risk for developing CD. The incidence of obesity has been increasing lately in countries endemic to DC, leading to high blood pressure, insulin resistance, increased levels of inflammation and oxidative stress. This picture promoted by obesity may have effects on the development of CD. In the present study, we evaluated the influence of obesity on acute infection by Trypanosoma cruzi, on cardiovascular, inflammatory parameters and insulin resistance. Obese swiss mice, on the 70th day of life were submitted to intraperitoneal infection with 5×102 trypomastigote forms of the Y strain. The cardiovascular parameters were evaluated before and during infection. The parasitemia and the survival up to 30º dpi were analyzed. In the 13th dpi the tissue was collected. Cytokines were determined using commercial kit CBA. Nitric oxide (NO) was determined by the cadmium and Griess technique. The antioxidant capacity and levels of oxidative stress were determined by the ABTS, FRAP, NBT and TBARS assays. Obese animals had higher blood pressure. We observed a higher parasitemia in the infected obese group (OI) compared to the infected control (CI) on days 13 and 15 post infection. All animals in the OI group died until the 19th day after infection while 87.5% of the IC survived to 30º. We observed a drop in mean arterial pressure in OI from the 9th dpi remaining until the end of the evaluation. No changes in blood pressure were found in the IC group. An increase in plasma NO in adipose tissue and aorta in OI was observed. Higher concentrations of INF‐Y and MCP‐1, and a lower concentration of IL‐10 was observed in OI vs CI. It was found a lower insulin sensitivity in obese animals and accentuated after infection. Higher parasitic load was found in adipose and hepatic tissue, and increased levels of oxidative stress in cardiac, hepatic and adipose tissue in the OI group. Our results suggest that the association of obesity and CD during the acute phase promote greater damage to infected animals.

Support or Funding Information

CAPES (felowship)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Extracellular Superoxide Dismutase (SOD3) Links Tumor Necrosis Factor‐alpha Receptor 1 to Integrin Signaling
Authors: Choi Hyehun , Raja Benjamin S , Miller Michael , Stark Ryan J , Lamb Fred S
Published: June 2021

Tumor necrosis factor‐α (TNFα) signaling requires superoxide (O2•−) to be produced extracellularly by NADPH oxidase 1 (Nox1). Extracellular superoxide dismutase (ecSOD) can convert this O2•− to hydrogen peroxide (H2O2). ecSOD is anchored to the cell surface by interaction with either heparin sulfate proteoglycan via a heparin binding domain, or by binding to fibulin‐5 (Fib5), an extracellular matrix protein which interacts with integrin receptors. Fib5 binding does not activate integrins, but blocks interaction with other components of the extracellular matrix. The physiologic significance of ecSOD‐Fib5‐integrin interaction has not been well defined. We hypothesized that ecSOD regulates extracellular redox signaling of TNFα/Nox1 by modulating secondary effects on integrin signaling. We analyzed TNFα‐induced interleukin‐6 (IL‐6) production and NF‐κB activation in cultured vascular smooth muscle cells (VSMC). Knockdown of ecSOD by siRNA blocked TNFα‐mediated IL‐6 (siControl+TNFα 2922±354, si ecSOD+TNFα 952.5±99 pg/mL, p<0.05, n=8–9) and NF‐κB activation (siControl+TNFα 10.8±1.0, si ecSOD+TNFα 4.0±0.3 fold change, p<0.05, n=7). Similar inhibitory effects were observed following knockdown of Fib5 (IL‐6; siControl+TNFα 2372±123, siFib5+TNFα 1686±124 pg/mL, p<0.05, n=4. NF‐κB activation; siControl+TNFα 10.8±1.0, siFib5+TNFα 5.4±0.3 fold change, p<0.05, n=7). We next sought to determine how ecSOD affected integrin signaling. Focal adhesion kinase (FAK) and integrin‐linked kinase (ILK) are critical downstream effectors of integrin activation. TNFα induced phosphorylation of both FAK and Akt, which was used as a surrogate for ILK activation. This approach was validated by the ability of ILK inhibition (Cpd22; 3 μM) to prevent Akt phosphorylation. Both FAK and Akt phosphorylation in response to TNFα were inhibited by an anti‐integrin β1 antibody. Knockdown of ecSOD significantly enhanced TNFα‐induced phosphorylation of both FAK and Akt. FAK inhibition (FAK inhibitor 14; 2 μM) blocked while ILK inhibition (Cpd22; 3 μM) markedly potentiated NF‐κB activation. These results suggest that TNFα transactivates β1 integrins and therefore FAK and ILK in VSMCs. We speculate that ecSOD localizes H2O2 production to the vicinity of integrin receptors via physical association with Fib5 and provides tonic suppression of β1integrin receptor activation. Knockdown of either ecSOD or Fib5 would promote ILK‐dependent inhibition of NF‐κB activation and IL‐6 secretion which occurs via direct TNFR1‐dependent signaling.

Support or Funding Information

American Heart Association (16SDG30610002)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of living at moderate altitude on pulmonary vascular function and exercise capacity in mice with sickle cell anemia
Authors: Ferguson Scott Kohman , Stenmark Kurt R , Buehler Paul , Irwin David C.
Published: June 2021

Exposure to high altitude worsens symptoms and crises in patients with sickle cell disease (SCD). However, it remains unclear if prolonged exposure to low barometric pressures exacerbates SCD etiologies or impairs quality of life. We tested the hypothesis that, relative to wild‐type (WT) mice, Berkley sickle cell mice (BERK‐SS) residing at sea level, mild (1609 m) and moderate (2438 m) altitude would have a higher rate of hemolysis, impaired cardiac function, and reduced exercise tolerance and that the level of altitude would worsen these decrements. Following 3 months of altitude exposure, right ventricular systolic pressures (RVSP) were measured (solid‐state transducer). In addition, the adaptive balance between pulmonary vascular endothelial nitric oxide synthase and endothelin was assessed in lung tissue to determine differences in pulmonary vascular adaptation and the speed/duration relationship (critical speed, CS) was used to evaluate treadmill exercise tolerance. At all altitudes, BERK‐SS mice had a significantly lower %Hct and higher total bilirubin and free hemoglobin concentration (P<0.05 for all). RVSPs in BERK‐SS were higher than WT at moderate altitude and when compared to BERK‐SS at sea level (P<0.05, for both). CS was significantly lower in BERK‐SS at mild and moderate altitude (P<0.05). BERK‐SS demonstrated exacerbated SCD complications and reduced exercise capacity associated with an increase in altitude. These results suggest that exposure to mild and moderate altitude enhances the progression of SCD in BERK‐SS mice compared to healthy WT cohorts and BERK‐SS mice at sea level and provides crucial information for the clinical counseling of SCD patients.

Support or Funding Information

This study was supported by the National Heart, Lung and Blood Institute Grants 1R01HL125642‐01A1 (Irwin DC), 1R01HL086680‐07 (Nozik‐Grayck E), 5P01HL014985‐38 (Stenmark KR), and T32‐HL007171 (Stenmark KR), Colorado Sickle Cell Treatment and Research Center (Hassell K and Nuss R).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
ROS‐NO Signaling is Altered in Exercising Swine with Multiple Comorbidities
Authors: Sorop Oana , Van De Wouw Jens , Van Drie Ruben , Merkus Daphne , Duncker Dirk J.
Published: June 2021

Introduction

Multiple comorbidities, such as diabetes mellitus (DM), hypercholesterolemia and chronic kidney disease (CKD) induce sustained systemic inflammation and coronary endothelial dysfunction and increased oxidative stress. The resulting alterations in microvascular tone may contribute to impaired myocardial perfusion.

Hypothesis

Increased production of reactive oxygen species (ROS) due to DM, CKD and high fat diet (HFD) results in loss of NO bioavailability in the coronary microvasculature of exercising swine leading finally to altered myocardial perfusion.

Methods and Results

In 6 female swine, DM (streptozotocin 3×50mg/kg iv), CKD (renal embolization), and hypercholesterolemia (via high fat diet, HFD) were induced for 6 months (DM+HFD+CKD). 8 female healthy swine on normal pig chow served as controls (CON). The role of ROS‐NO pathways in the regulation of coronary flow regulation was studied at rest and during graded treadmill exercise with/without eNOS inhibition (nitro‐L‐arginine), and ROS scavengers (MPG+Tempol). 6 months after the induction of comorbidities, DM+HFD+CKD animals had hyperglycemia (18.7±1.9 vs 7.5±0.6mmol/L), renal dysfunction (glomerular filtration rate: 123±12 vs 202±8 ml/min), hypercholesterolemia (7.3±0.7 vs 1.7±0.1mmol/l) and sustained systemic inflammation (TNF 51.6±5.1 vs 24.6±5.5pg/ml, all P<0.05). Myocardial superoxide production was increased in DM+HFD+CKD animals due to NADPH oxidase activation and eNOS uncoupling and this was associated with a lower myocardial NO production, (P<0.05). Myocardial oxygen delivery was impaired in DM+HFD+CKD swine at rest and during exercise, resulting in reduced coronary venous oxygen saturation (cvSaO2) compared to CON (P<0.05). eNOS‐inhibition resulted in coronary microvascular constriction in CON, reflected by reduced cvSaO2, however, it had no effect in DM+HFD+CKD (Fig. A, B, *P<0.05). Interestingly, scavenging of ROS resulted in decreased cvSaO2 in DM+HFD+CKD and had no effect in CON (Fig. C, D), suggesting that the increased oxidative stress resulted in increased production of the vasodilator hydrogen peroxide.

Conclusion

In swine, 6 months exposure to multiple comorbidities resulted in impaired myocardial oxygen delivery mediated by altered NO bioavailability, which was associated with increased oxidative stress. This was partially compensated by increased hydrogen peroxide‐mediated vasodilation.

Support or Funding Information

This study was supported by a grant from the Netherlands CardioVascular Research Initiative: an initiative with support of the Dutch Heart Foundation [CVON2014‐11 (RECONNECT)]

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Knowledge, Attitudes, and Practices on the use of Energy Drinks among Division I Athletes
Authors: Raynes Edilberto Alcantara , Waller Lauren , Glymph Kelly , Jones Timothy , Michaels Natalie , Barredo Ronald , Heimdal James , Johnson Owen , Pounders Vance , Bradley William , Hubbert Douglas
Published: June 2021

The consumption of energy drinks has recently become a popular practice among athletes and college students. Research has shown the inappropriate use of energy drinks can increase the risk for cardiovascular complications. The objective of this study was to explore on the knowledge, attitudes and practices on the use of energy drinks among 137 Division I athletes. A cross sectional study design was utilized with an online survey questionnaire. In terms of attitudes, 77% of the participants did not use energy drinks with alcohol and did not mix with other substances and 19% consumed energy drinks to enhance sports performance. In terms of practices, more than 80% of the participants did not use energy drinks during sports activity and rarely consumed it monthly. Based on the results, the participants appear knowledgeable on the appropriate use of energy drinks.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dissection is the future. Or is it?
Authors: Pickering James
Published: June 2021

The use of dissection as an effective modality to teaching anatomy to healthcare professions is longstanding, with the debate about its necessity and role sometimes appearing to have been going on for even longer. Recently, this debate has returned to the educational landscape due to the increasing level of anatomy education scholarship underway that is morphing the field of anatomy education from one of pure delivery to evidence‐based curriculum design. However, how do we make decisions on our curriculum delivery, what informs our decision making processes? Traditionally, the approach would be to use the established evidence‐base to justify decisions and implement changes that are supported by literature, but is this really the best way? Although this approach may work within other scientific fields, education delivery is complex with multiple factors, such as cohort mix, technology, logistical arrangements, and timetable availability. Moreover, there is also the societal context in which our students will be entering and the changes in healthcare systems that await them as healthcare professionals. Within this context is teaching anatomy primarily through dissection, to undifferentiated healthcare students, the most effective and efficient approach?

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Menthol‐induced hyperthermia and its implication for a pharmacological approach to obesity prevention and treatment
Authors: Armentano Giovana Marchini , Vizin Robson Cristiano Lillo , Carrettiero Daniel Carneiro , Almeida Maria Camila
Published: June 2021

We have previously shown that short‐term repeated treatment with topical menthol results in less body mass gain in lean Wistar rats compared to controls due to a persistent increase in energy expenditure, with limited compensatory thermoregulatory adaptations and, most unexpectedly, without affecting food intake (Vizin et al, J Appl Physiol. 2018 124(3):672–683). Menthol seems thus to be a promising drug for the prevention of obesity. Whether repeated topical application of menthol affects body weight gain in obese animals is not known. We tested the effects of topical menthol (5%) treatment on body weight gain and body temperature on both genetic obese ob/ob mice and in high‐fat diet (HFD)‐induced obesity in Wistar rats. In control mice (WT) repeated application of menthol (9 days) resulted in a reduction of body mass gain, as a consequence of increased energy expenditure, as measured by the persistent hyperthermic response after menthol. However, in obese ob/ob mice repeated application of menthol for 9 days was not sufficient to cause a significant reduction in body mass gain. The hyperthermic response of ob/ob mice to menthol was reduced as compared to WT thermoregulatory response. In HFD‐obese rats, topical menthol treatment for 9 days resulted in a decrease in ~20g in mean body weight, which is correspondent to a 5% reduction in body mass. Menthol treatment was accompanied by a hyperthermic response in obese rats, which most probably accounted for the increased energy expenditure that allowed body mass reduction. Our data support the suggestion that menthol is a promising drug not only for obesity prevention (as it reduces body mass gain in lean animals) but also for the treatment of HFD‐induced obesity as well.

Support or Funding Information

Fapesp (grants 2017/13062‐5 and 2015/02991‐0)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Determining Changes in Thermal Patterns in Canines based on Coat Type using Infrared Thermographic Imaging
Authors: Kwon Claire , Brundage Cord
Published: June 2021

Infrared thermography detects variations in surface temperatures. This imaging modality has been used to approximate body temperature, inflammation, and wellness. In mammals, the insulatory effect of hair coat type is likely to have disparate effects on surface temperature and patterning. To measure the difference in surface temperatures based on coat type in dogs (Canis lupus familiaris), an infrared thermographic imaging camera (i.e. FLIR B400) was used. Thermographic images were taken of the right and left sides of privately‐owned dogs (n = 50) Each animal acclimated in an indoor environment (23 ± 2.0 °C) for 15 minutes; images were taken at a minimum distance of 0.4 m. Mean surface temperatures were analyzed across the lateral surface of each animal. No difference in surface temperature was identified based on the laterality (P = 0.983). Mean surface temperatures were then categorized based on each dog's hair coat type: short coat (SC), long coat (LC), curly coat (CC), and double coat (DC). SC animals had a significantly higher surface temperature (31.57 ± 0.32 °C) than LC or DC animals (28.11 ± 0.95 °C for LC and 28.29 ± 2.00 °C for DC respectively; P < 0.001). Dogs characterized with a CC had an intermediate surface temperature of 29.96 ± 1.94 °C. Variability in surface temperature was independent of rectal temperature (P > 0.05). This evidence suggests that coat type should be considered when evaluating the surface temperature of canids and elevations in surface temperature should be interpreted within the context of an animal's coat characteristics.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Specific Role of Adiponectin in Pulmonary Artery Smooth Muscle Cells Proliferation and Inflammatory Cytokines Production during Rat Lung Development
Authors: Ivanovska Julijana , Kang Cindy , Tamir‐Hostovsky Liran , Belik Jaques , Gauda Estelle B.
Published: June 2021

Adiponectin (APN), an abundant circulating plasma protein mostly generated from adipose tissue, is implicated in lung vascular, oxidative and anti‐inflammatory protection. Circulating APN levels are low in premature and small for gestational age neonates, who are at the greatest risk for developing lung oxidative and inflammatory injury leading to bronchopulmonary (BPD) and pulmonary artery hypertension (PAH). We previously characterized the expression pattern of APN and its receptors (Adipor1 and Adipor2) during lung development in the newborn rat and localized APN and Adipor1 and Adipor2 protein expression in pulmonary artery smooth muscle cells (PASMC). Here we describe the effect of recombinant APN (rAPN) on PASMC proliferation and pro‐ and anti‐inflammatory cytokine production in PASMC during early lung development.

METHODS

Postnatal day (PND) 4 and 7 PASMC were treated with recombinant APN (rAPN) (2 and 5 μg/ml), in the presence of water‐soluble tetrazolium salt 8 (WST8). The formation of formazan in viable cells was detected spectrophotometrically and used as an indicator of cell proliferation. The effect of rAPN on lipopolysaccharide (LPS) (3 μg/ml) ‐ induced protein expression of IL6, TNFα, and IL10 was also examined. Western blotting technique were used to measure cytokine protein expression.

RESULTS

rAPN decreased the proliferation of PASMC from both age groups, p<0.05 vs only culture medium control cells. rAPN (2μg/ml): 1) decreased IL6 protein levels in PND 4 PASMC at baseline and in presence of LPS with no effect on IL‐6 expression in PASMC from PND 7 animals, 2) decreased TNFα protein levels in PND 4 and PND 7 PASMC in response to LPS and at baseline only in PND 4 PASMC, and 3) increased the anti‐inflammatory cytokine IL10 protein levels from PASMC at baseline in PND4 and in response to LPS in PND 7 (p<0.05 vs control)

CONCLUSIONS

For the first time, we show that rAPN inhibits PASMC proliferation and pro‐inflammatory cytokine production in PASMC during early postnatal lung development. Overall rAPN decreased pro‐inflammatory and increased anti‐inflammatory cytokine production in these cells at PND 4 and 7. These novel findings are relevant to the premature infant at high risk for developing BPD and associated PAH. We speculate that increasing lung APN levels could potentially decrease lung injury and vascular remodeling from inflammatory stimuli and reduce the development of BPD. The role of rAPN delivered to the lung, in an in vivo model of LPS‐induced lung injury in newborn rats is currently being investigated

Support or Funding Information

Women's Auxilary of The Hospital for Sick Children

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Contribution of Mitochondria‐Derived Reactive Oxygen Species to Augmented Pulmonary Vasoconstriction Following Chronic Hypoxia
Authors: Yan Simin , Sheak Joshua R. , Jernigan Nikki L. , Walker Benjimen R. , Resta Thomas C.
Published: June 2021

Chronic hypoxia (CH) augments pulmonary arterial tone through O2−‐dependent stimulation of RhoA, a response that may contribute to the vasoconstrictor component of pulmonary hypertension. Although mitochondria are an important source of reactive oxygen species (ROS) in the pulmonary vasculature, the role of mitochondria‐derived ROS (mitoROS) in enhanced vasoconstrictor reactivity following CH is unknown. We hypothesized that mitoROS contribute to elevated basal pulmonary arterial tone and endothelin‐1 (ET‐1) induced vasoconstriction following CH. To test our hypothesis, we measured basal tone by videomicroscopy in isolated, pressurized small pulmonary arteries [~150 μm inner diameter (i.d.)] from normoxic and CH (4 wk, 0.5 atm) rats following administration of the mitochondria‐targeted antioxidants, MitoQ (1 μM) or MitoTEMPO (200 μM), or their respective vehicles. Parallel studies evaluated effects of mitoROS inhibition on vasoconstrictor responses to increasing concentrations of ET‐1. Experiments were performed either in the presence of the nitric oxide (NO) synthase inhibitor Nω‐nitro‐L‐arginine (300 μM) or after endothelial disruption to limit the influence of endogenous NO. Pulmonary arterial tone was calculated as the percent difference in i.d. between Ca2+‐free and Ca2+‐containing conditions, whereas vasoconstriction to ET‐1 was expressed as a percentage of baseline i.d. Exposure to CH significantly (P < 0.05) increased basal pulmonary arterial tone and vasoconstrictor responsiveness to ET‐1 in both NOS‐inhibited and endothelium‐disrupted arteries. Treatment with either MitoQ or MitoTEMPO prevented these responses to CH and normalized values between groups. We conclude that mitoROS provide a major contribution to CH‐induced increases in pulmonary vasoconstrictor reactivity.

Support or Funding Information

This work was supported by NIH grant R01 HL132883 (to T.C. Resta).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Implementation of LabVIEW as a Virtual Instrument in a Cost‐Effective Isolated Lung Setup
Authors: Cleveland William J. , Hees Josephine E. , Balzer Claudius , Lotze Felicia P. , Pille Johannes A. , Salzman Michele M. , Riess Matthias L.
Published: June 2021

Introduction

Isolated lung experiments allow for ex vivo characterization of lung function and prove to be a valuable model for various fields such as cancer, lung transplantation, and ischemia/reperfusion injury. Unlike other models that require damaging the lung, the isolated lung allows for collection of physiological changes including airway pressure, pulmonary artery and vein pressure, airway flow, and, in our model, ventilated gas concentrations. We describe the use of LabVIEW combined with the free statistical software R for highly flexible, effective, and automated data collection in an isolated lung system.

Methods

Pressure transducers connected to bioamplifiers allow for collection of dynamic pulmonary artery and vein pressures, while airway pressure and flow are collected from the ventilator with the former used to visualize positive end expiratory pressure. Signals from these devices are connected to an analog‐digital converter from National InstrumentsTM that collects data into LabVIEW software, which is then automatically collected and displayed at 200Hz. Use of a roller pump with analog input/output allows for fully automated control of the perfusion speed. Output from a gas mixer for use in ventilated gas studies is collected and used to generate gas composition data. Additional experimental data that do not have signal output capability can be recorded manually in LabVIEW.

Results

LabVIEW is a reliable alternative to other commercially available data acquisition systems. The creation of a virtual instrument requires significant programming knowledge but allows for completely customizable data collection in a modular isolated lung system. Data become available in an Excel‐compatible file which can then be imported and analysed in R as another affordable alternative to commercial programs.

Conclusion

We describe the design and implementation of a LabVIEW virtual instrument as an adjustable alternative to conventional data acquisition systems.

Support or Funding Information

This work was supported, in part, by institutional funds, NIH grant (5R01 HL123227), and a Merit Review Award (I01 BX003482) from the U.S. Department of Veterans Affairs Biomedical Laboratory R&D Service.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Microanalytical Mass Spectrometry Detects RAS Metabolites in Small Identified Mouse Brain Samples
Authors: Lombard‐Banek Camille , Yu Zhe , Swiercz Adam P. , Marvar Paul J , Nemes Peter
Published: June 2021

Background

There is increasing evidence for cardiovascular disease (CVD) development in post‐traumatic stress disorder (PTSD). The renin‐angiotensin system (RAS) critical for cardiovascular homeostasis, has been identified as potential therapeutic target and link to comorbid PTSD‐CVD development. However, the mechanisms are unclear. To better understand how the RAS contributes to CVD risk in PTSD, measurements of the RAS metabolites, the angiotensin peptides, in targeted brain areas are necessary. The low concentrations of angiotensin peptides in the brain however, challenges modern analytical approaches. This challenge is further compounded when studying small identified areas such as brain tissue punches. As a response to this challenge, we developed a microanalytical mass spectrometry approach to enable the detection of angiotensin peptides from brain tissue punches.

Methods

Using 6 angiotensin‐like peptide standards, we reconfigured a laboratory‐built capillary electrophoresis electrospray ionization platform (CE‐ESI) platform and a high‐resolution mass spectrometer (HRMS) for targeted peptide detection, essentially serving as a highly confident peptide assay. We benchmarked our approach to nano‐liquid chromatography (LC) ESI‐HRMS, the traditional technology for peptide analysis in HRMS.

Results

We first developed a targeted approach on a quadrupole‐orbitrap mass spectrometer for the detection and quantification of the 6 selected angiotensin‐like peptides. Comparison of our CE‐ESI‐HRMS approach to the traditional nanoLC‐ESI‐HRMS revealed comparable peptide signals despite a 5‐fold reduction in sample loading. This demonstrated that our platform was suited for the measurement of angiotensin peptides from small sample volumes. The lower‐limit of quantification was established in the pM–nM range for all the selected RAS metabolites. After revising the extraction method to enhance peptide recovery, we applied our methodology to measure different levels of RAS metabolites in brain punches from the subfornical organ (SFO) and the paraventricular nucleus of the hypothalamus (PVN) of control and water deprived (WD) mice. Differences in the level of all RAS metabolites in the SFO concurred with the effect of WD in increasing angiotensin levels. On the other hand, levels for most of the RAS metabolites in the PVN did not change noticeably.

Conclusions

These results demonstrate that our CE‐ESI‐HRMS based analytical approach enabled the sensitive detection of RAS metabolites in small brain tissue samples, raising a potential to help study the chemistry of the brain RAS during PTSD and CVD.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Combined V1a and V1b Receptor Activation Within the Paraventricular Nucleus Contributes to Sympathoexcitation and Hypertension in Rats Exposed to Chronic Mild Unpredictable Stress
Authors: Komnenov Dragana , Quaal Harrison , Rossi Noreen Felice
Published: June 2021

The paraventricular nucleus (PVN) in the brain has been recognized as a major integrative center for neurohumoral regulation which receives a broad array of afferent inputs. As such, the function of the PVN becomes even more important in times of physical or physiological stress. Two distinct neuronal populations we are interested in include the magnocellular and parvocellular neurons. Specifically, we are interested in deciphering how intranuclearly released vasopressin (AVP) from the magnocellular neurons acts upon the parvocellular pre‐sympathetic neurons and glia that are richly endowed with V1a and V1b receptors in a rat model of depression. It has been demonstrated that V1a receptor activation depolarizes PVN pre‐sympathetic neurons and initiates an action potential, which puts forth a signaling cascade that increases sympathetic nerve activity, arterial pressure and heart rate. Additionally, we previously published that renal sympathetic nerve activity (RSNA), mean arterial pressure (MAP) and heart rate (HR) responses to AVP microinjected in the PVN is significantly attenuated when the rats were pretreated with V1b receptor antagonist nelivaptan. In this study, we set out to identify differences in baseline RSNA, MAP and HR between rats exposed to the chronic mild unpredictable stress protocol (CMS) and their normal counterparts, as well as their responses to AVP microinjected into the PVN in the presence of the V1a receptor antagonist, Manning compound, and the V1b receptor inhibitor, nelivaptan. We observed that baseline MAP is higher in CMS rats (110 ± 2 mmHg vs. 115 ± 1 mmHg; P < 0.05). Furthermore, in control rats, combined inhibition of AVP with nelivaptan and Manning compound resulted in near complete abolition of the rise in MAP and baroreflex‐mediated decreases in HR and RSNA. CMS rats exhibited exaggerated baseline RSNA, 43.6 ± 9.97 μV.ms compared with control rats, 8.04 ± 3.24 μV.ms (P < 0.025). PVN AVP injection increased RSNA by 52.1 ± 3.3 %baseline in controls and was blocked by the dual blockade. RSNA did not increase significantly after PVN AVP in CMS rats and V1a receptor and V1b receptor blockade did not change this response. CMS rats displayed higher baseline RSNA 43.6 ± 9.9 μV.ms vs control rats 8.0 ± 3.9 μV.ms P < 0.025). Moreover, dual blockade significantly decreased RSNA from baseline values only in CMS rats: −12.6 ± 0.4 μV.ms compared with control rats −1.1 ± 0.6 μV.ms (P < 0.05). These findings are consistent with the concept that arterial pressure and RSNA are increased during chronic stress via dendritic release of AVP within the PVN thereby contributing to sympathoexcitation and hypertension.

Support or Funding Information

Department of Veterans Affairs Merit Award to NFR

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sex Differences in Autonomic Response to Hand Grip Exercise Among Individuals with Intellectual Disability
Authors: Sherman Sara R , Hilgenkamp Thessa I.M. , Schroeder Elizabeth C , Fernhall Bo , Baynard Tracy
Published: June 2021

Individuals with an intellectual disability (ID) are at increased risk for cardiovascular disease (CVD), which may be attributed to autonomic dysfunction. Sex differences in autonomic function are apparent in the general population but have not been investigated in persons with ID, and may help to further explain the high prevalence of CVD. Heart rate variability (HRV) is a non‐invasive measure for autonomic modulation and using a sympatho‐excitatory stimulus such as isometric handgrip (HG) exercise, may reveal altered autonomic function not observed at rest.

PURPOSE

To assess sex differences in cardiac autonomic function in individuals with and without ID using an isometric handgrip (HG) exercise test.

METHODS

HRV was assessed in 18 individuals with ID (Male: 9; 28±6yrs, 33±11.1kg/m2) and 23 without ID (Male: 13; 30±9.0yrs, 26±3.8kg/m2) in response to isometric HG exercise at 30% of maximal voluntary contraction. Heart rate was continuously recorded via ECG and the last 2 minutes of quiet rest and HG exercise were analyzed. HRV was analyzed in both the time (root mean square of successive differences (RMSSD) and percentage of RR intervals differing by <50ms (PNN50)) and frequency domains (low and high frequency (LF, HF), and LF/HF ratio). A change score was calculated from HG exercise ‐ baseline.

RESULTS

There were no differences between controls and ID. However, males in both groups demonstrated a blunted response to HG exercise in RMSSD, pNN50, and LF (p<0.05) compared to females.

CONCLUSION

Males in both groups appear to have a suppressed vagal withdrawal to HG when compared to the females. Future research should investigate autonomic regulation in sex‐specific groups with and without ID in a larger sample size.

Control
ID
ANOVA

Male
Female
Male
Female
Group
Sex
Group*Sex

Time‐Domain

RMSSD (ms2)
−8±9
−21±24
−0±14
−12±12
0.05
0.00
0.35

pNN50 (%)
−6±8
−13± 17
−4± 13
−9± 12
0.26
0.02
0.25

Frequency‐Domain

LF (ms2)
−337± 1098
−1401± 1638
125± 1038
−245±361
0.56
0.00
0.29

HF (ms2)¥
−211±254
−350±428
−118±510
−221±277
0.27
0.10
0.27

LF/HF Ratio§
6± 1
6± 1
6±0
6± 1
0.64
0.65
0.39

Data presented as mean change ± standard deviation.

Control= M (n=13), F (n=9); ID= M (n=9), F (n=9)

Log‐transformed for the case of non‐normal data

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Altered Cardiopulmonary Reflexes Evoked by Irritants in Spontaneously Hypertensive Rats – Effect of Route of Administration and Anesthetics
Authors: Hooper Justin Shane , Taylor‐Clark Thomas
Published: June 2021

Inhalation of noxious substances such as allyl isothiocyanate (AITC) evoke reflex bradycardia in conscious, healthy animals following the activation of airway nociceptive afferents. These afferents are also activated by air pollutants including particulate matter and ozone. Nevertheless, in patients with cardiovascular disease such pollutants evoke sympathoexcitation (tachycardia, hypertension). We hypothesize that preexisting cardiovascular disease remodels the pulmonary‐cardiac reflexes stimulated by noxious airway stimuli. Here, we studied the cardiopulmonary responses to two separate administration methods of AITC (inhalation and intravenous (i.v.) injection) in conscious and anesthetized normotensive Wistar‐Kyoto rats (WKY) and Spontaneously Hypertensive rats (SH). Both methods of AITC administration evoked bradycardia with increased RR‐interval (RRi) and atrial‐ventricular (AV) block in conscious WKY rats. AITC i.v. evoked similar bradycardic responses in conscious SH rats, however, AITC inhalation evoked a complex brady‐tachycardia which alternated between increased and decreased RRi and both AV block and premature ventricular contractions (PVCs). AITC inhalation caused bradypnea with sporadic episodes of tachypnea in both strains, while only bradypnea was seen after AITC i.v. administration. These responses were shown to be vagally mediated as they were abolished by bilateral vagotomy in a decerebrate model. Anesthesia was also found to prevent the effect of AITC inhalation on HR in both strains, while the bradycardic response to AITC i.v. persisted in the WKY but not in the SH. Furthermore, anesthesia prevented breathing rate responses to inhaled AITC in the SH, but not in the WKY. Differences were also seen in the effects of anesthesia on breathing rate responses to AITC iv between the two strains as bradypnea persisted in the WKY, while the SH was found to have bouts of tachypnea in addition to the bradypnea seen in the conscious studies. The differential responses evoked by AITC in the SH along with the differences in effects of anesthesia on these responses lead us to conclude that multiple unique circuits work independently to control cardiopulmonary reflexes and that preexisting hypertension remodels nociceptive cardiopulmonary reflexes towards sympathoexcitation.

Support or Funding Information

NIH SPARC Award

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Impact of Caffeine on Dynamic Cerebral Autoregulation in Habitual Consumers
Authors: Pizzey Faith K , Tourula Erica N , Theorin Tyler J , Subudhi Andrew W , Pearson James
Published: June 2021

Caffeine is an adenosine receptor antagonist that is consumed daily by 90% of the United States population and can lead to adenosine receptor upregulation. In caffeine‐naïve individuals, drinking caffeine increases dynamic cerebral autoregulation as assessed by the rate of regulation (RoR) but the influence of caffeine in habitual users is unclear. We aimed to investigate whether dynamic cerebral autoregulation in middle and posterior cerebral arteries was altered by caffeine consumption in habituated consumers. Nine healthy subjects (age: 27 ± 6 yrs, Ht: 179 ± 14 cm, Wt: 76.1 ± 13.6 kg) completed three trials where they drank (6.25ml/kg body mass) either caffeinated coffee (CAF; 4mg caffeine/kg body mass), decaffeinated coffee (DECAF), or water (WATER). Before subjects drank anything (pre drink) we measured mean arterial pressure (MAP; mmHg), middle (MCAv; cm/s) and posterior (PCAv; cm/s) cerebral artery blood velocities at baseline and then during two successive thigh occlusion/release protocols to examine dynamic cerebral autoregulation (Rate of Regulation: RoR, 1/s). Subjects were then given one of the three drinks in a double‐blind, counterbalanced design. Thirty minutes later baseline measurements and the assessment of RoR were repeated (post drink). MAP decreased 14 ± 4 mmHg across all trials following thigh cuff deflation in the assessment of RoR pre drink. Pre drink RoR in the MCA was CAF: 0.19 ± 0.13, DECAF: 0.21 ± 0.14 and WATER: 0.21 ± 0.09 1/s. Pre drink RoR in the PCA was CAF: 0.24 ± 0.18, DECAF: 0.16 ± 0.15 and WATER: 0.23 ± 0.12 1/s. MAP increased from pre to post drink (CAF: 84 ± 7 to 98 ± 6 mmHg; DECAF: 88 ± 10 to 95 ± 6 mmHg and WATER: 89 ± 9 to 97 ± 5 mmHg; p = 0.002), but was not different between trials (p > 0.05). MAP decreased 14 ± 3 mmHg across all trials following thigh cuff deflation in the assessment of RoR post drink. Post drink, MCA RoR demonstrated an interaction effect between trial and time (p = 0.047) and a main effect of time (p = 0.042). Post drink MCA RoR increased in the CAF trial relative to pre drink (0.40 ± 0.17 1/s; p = 0.008) but was unchanged in DECAF (0.23 ± 0.12 1/s) and WATER trials (0.23 ± 0.11 1/s) (both p > 0.05 vs. pre drink). Post drink, PCA RoR was not different relative to pre drink (CAF: 0.35 ± 0.13, DECAF: 0.20 ± 0.11 and WATER: 0.21 ± 0.10 1/s) and did not demonstrate a main effect of time (p = 0.335), trial (p = 0.081) nor an interaction effect (p = 0.414). These findings suggest that caffeine consumption increases dynamic cerebral autoregulation in habituated consumers and supports the increased RoR found in the MCA following caffeine administration in caffeine naïve individuals.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Intraluminal Nutrients Modulate Intracellular Calcium Activity in the Enteric Nervous System of Adult Mice
Authors: Cavin Jean‐Baptiste , Glover Joel , MacNaughton Wallace K. , Sharkey Keith A.
Published: June 2021

Background

It has long been hypothesized that enteric neurons are able to detect nutrients present in the intestinal lumen, but direct evidence in intact preparations of the intestine is lacking. Understanding how enteric neurons react to changes in dietary constituents will open new avenues for discoveries on the relation between nutrition, modulation of gut function, and health.

Aim

To measure neuronal activity in the enteric nervous system in response to luminal nutrients.

Methods

We have designed chambers allowing us to visualize, record and quantify intracellular calcium activity in 3D live‐cell confocal recordings of neurons from intact, whole thickness segments of mouse intestine. Twenty mice expressing the genetically encoded calcium reporter Gcamp6 under the control of the pan‐neuronal Wnt1 promoter were used to record intracellular calcium activity in neurons from the jejunum and colon, while perfusing different nutrients through the lumen. Krebs solution was perfused as control and Ensure®, 10% Intralipid® or 20mM Glucose were used as stimuli. 3D videos of calcium fluorescence in the neuronal network were acquired with a Nikon A1R confocal microscope and calcium dynamics were analyzed using the Imaris software (Bitplane). Results are expressed as relative changes in fluorescence intensity (ΔF/F) ± SEM among the neuronal populations studied.

Results

Perfusion of the intestine with Ensure® (liquid meal replacement) increased the fluorescence intensity in reactive neurons by 670 ± 270% in the jejunum and 403 ± 62% in the colon. Peak amplitude was measured on average 5–6 minutes after the initiation of the nutrient perfusion. Perfusion with 10% Intralipid® (lipid emulsion) increased the fluorescence intensity in reactive neurons by 300 ± 180% in the jejunum and 150 ± 15% in the colon. On the other hand, perfusion of the intestine with Glucose (20mM in Krebs solution) lowered baseline fluorescence intensity in a population of neurons by −91 ± 3% in the jejunum and −80 ± 3% in the colon. The percentage of neurons reacting to intraluminal stimuli was dependent on the stimulus and was different between the jejunum and the colon, indicating that subpopulations of neurons might differentially react to different nutrient stimuli.

Conclusions

We report the existence of slow intracellular calcium changes in the neurons in the intestine that vary depending on the nutrient content of the intestinal lumen. The origin and functional consequences of these changes in intracellular calcium activity as well as the identity of responding neurons are currently under investigation.

Support or Funding Information

Funding sources: Canadian institutes of Health Research, Alberta Innovates, Human Frontier Science Program.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Regulation of NKCC2 Ubiquitination by the Skp1‐Cullin1‐FBox Complex in Thick Ascending Limbs
Authors: Ares Gustavo
Published: June 2021

NaCl reabsorption by the thick ascending limb (TAL) is mediated by the Na/K/2Cl cotransporter NKCC2. Nitric oxide and atrial natriuretic peptide decrease NaCl absorption in the thick ascending limb (TAL) by increasing the second messenger cyclic guanosine monophosphate (cGMP). We recently showed that NKCC2 is constitutively ubiquitinated, and that cGMP enhances the rate of ubiquitination. Ubiquitination is a post‐translational modification mediated by the ubiquitin‐activating enzyme (E1), the ubiquitin‐conjugating enzyme (E2), and the ubiquitin ligase (E3). The SCF (Skp1, Cullin‐1, F‐box protein) complex, is the largest family of E3 ubiquitin ligases in mammals. Therefore, we hypothesized that the SCF complex mediates the cGMP‐stimulated NKCC2 ubiquitination in rats TAL. To test our hypothesis we first tested whether we could inhibit neddylation of cullin‐1 with the inhibitor MLN4924 (1 μM). TALs samples were incubated for 60 minutes at 37°C in the presence or absence of the inhibitor, samples were lysed and we measured neddylated cullin‐1 by Western blot. As expected, neddylation of cullin‐1 was blunted in MLN4924 treated samples (baseline: 100%; MLN4924: 11.0 ± 3.5%, p<0.05). We then study whether inhibition of cullin‐1 inhibits the cGMP‐stimulated NKCC2 ubiquitination. TALs suspension from Sprague‐Dawley rats were aliquoted in 4 samples in the presence of the proteasomal inhibitor (MG132 20μM) to stop protein degradation. Then aliquots were incubated at 37°C in the presence or absence of MLN4924 for 10 minutes, and treated with vehicle or db‐cGMP 500 μM at 37°C for an extra 50 minutes. We found that blocking neddylation of cullin‐1, inhibits the cGMP‐stimulated NKCC2 ubiquitination (baseline: 100%; db‐cGMP 500μM: 162.5 ± 10.3%; MLN4924: 75.1 ± 20.2%; MLN4924 + db‐cGMP 500μM: 96.8 ± 19.6%, p<0.05). To further test our hypothesis, we generated a peptide containing the conserved sequence of F‐Box proteins, called the F‐Box domain, to compete for the binding site between native F‐Box proteins to the SCF complex. We used a chariot to facilitate the delivery of the peptide into the TAL cells. We found that the F‐Box peptide binds to the SCF complex by the pull down of cullin‐1, and inhibits the cGMP‐stimulated NKCC2 ubiquitination (F‐Box peptide: 100%; F‐Box peptide + db‐cGMP 500μM: 89.8 ± 3.9%, n.s.). Our data indicates that blocking neddylation of cullin‐1 or impairment the binding of the native F‐Box proteins to the SCF complex inhibits the cGMP‐dependent increase in NKCC2 ubiquitination. We conclude that the cGMP‐dependent increase in NKCC2 ubiquitination is mediated by the SCF complex. To our knowledge, this is the first evidence pointing to the E3 ubiquitin ligase complex involved in the ubiquitination of NKCC2 in native TALs.

Support or Funding Information

Institutional Mentored Grant Program‐Henry Ford Health System

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.