bluuebunny/crossref_metadata_2025 huggingface dataset:
Histamine receptor antagonists have the capacity to alter in vivo human sweating: implications for hypohidrosis
Authors: Wilson Thad E. , Meade Stephanie , Stump Corey , Skaggs Kendra , Banwait Sumeet , Galloway Samantha , Garrett Jason
Published: June 2021

An atopic dermatitis mouse model has identified histamine and the histamine type‐1 receptor (H1R) pathway to be a mechanism of disease‐induced hypohidrosis. To begin to translate these findings into in vivo human skin, we tested the hypothesis that intradermal perfused H1R antagonist pyrilamine maleate would accentuate local eccrine sweating induced sympathetically via whole‐body heat stress. Two intradermal microdialysis probes were placed into the dorsal forearm of 3 healthy female and 2 healthy male subjects (health determined by health history and physical exam; age = 25±1 yrs; and BMI = 25.3±3.5 kg/m2) to perfuse (5 μL/min) of pyrilamine maleate (3 mM) or the vehicle (lactated Ringer's) while measuring sweat rate (capacitance hygrometry) directly over the microdialysis membrane. Whole body heating (46°C water perfusion of a high‐density tube‐lined suit) increased intestinal temperature (pill telemetry) by 1.0°C from 36.9±0.2°C, heart rate (ECG) by 32±5 bpm, calf blood flow (venous occlusion plethysmography) by 7.8±1.4 ml/100 ml tissue, and whole‐body sweat loss of 1.5±0.2 kg but did not significantly alter mean arterial pressure. Mean skin temperature was clamped under the suit at 38–39°C, while uncovered forearm skin temperature increased from 29.6±0.3 to 35.1±0.6°C. Pyrilamine maleate increased local sweat rate compared to the vehicle control beginning at 35 min of whole‐body heating (intestinal temperature of 37.1±0.2°C) and this difference remained throughout the heat stress (group: P=0.008, time: P<0.001, and interaction: P<0.001). These data indicate that the H1R mechanism has the capacity to alter sweat rate during a physiological induced sudorific perturbation known to affect histamine concentration in human skin. Thus, it is possible that this mechanism of hypohydrosis could be a viable target in individuals with atopic dermatitis.

Support or Funding Information

Laboratory support: NIH AR069912

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Modulation of 5‐HT receptor and adenosine 2A receptor on intermittent hypoxia‐induced respiratory recovery following mid‐cervical contusion in the rat
Authors: Wu Ming‐Jane , Wen Ming‐Han , Lee Kun‐Ze
Published: June 2021

The present study was designed to evaluate the therapeutic effectiveness and mechanism of intermittent hypoxia on respiratory function at distinct injured stages following mid‐cervical spinal cord contusion. In the first study, adult male rats were received laminectomy or unilateral contusion at 3rd–4th cervical spinal cord at 9 weeks of age. The ventilatory behavior in response to mild acute intermittent hypoxia [10 episodes of 5 min of hypoxia (10 % O2, 4 % CO2, 86 % N2) with 5 min of normoxia intervals] was measured by the whole body plethysmography at the acute (~3 day), subchronic (~2 week) and chronic (~8 week) injured stage. The minute ventilation of contused animals could be significantly enhanced following intermittent hypoxia due to augmentation of the tidal volume at all time points post‐injury. However, this intermittent hypoxia‐induced ventilatory long‐term facilitation was only observed in uninjured animals at the acute stage. In the second study, the effect of intermittent hypoxia on respiration was examined in contused animals after blockade of serotonin receptors or adenosine 2A receptors. The result demonstrated that intermittent hypoxia‐induced enhancement of minute ventilation was attenuated by serotonin receptor antagonist (methysergide) but rather enhanced by adenosine 2A receptor antagonist (KW6002) at the subchronic and chronic injured stages. These results suggested that intermittent hypoxia can induce respiratory recovery from the acute to chronic injures stages, and the therapeutic effect were dampened by inhibition of serotonin receptors but could be amplified by blockade of adenosine 2A receptors.

Support or Funding Information

Ministry of Science and Technology (105‐2628‐B‐110‐002‐MY3), Higher Education Sprout Project (07C030111) and NSYSU‐KMU Joint Research Project (107‐I001)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Neurochemical Plasticity of Phrenic Motor Neuron Adenosine 2A Receptors: Effects of Cervical Spinal Injury and Intermittent Hypoxia
Authors: Smith Kristin , Allen Latoya L. , Seven Yasin B. , Ciesla Marissa , Asa Zachary , Simon Alec , Holland Ashley , Santiago Juliet , Stefan Kelsey , Ross Ashley , Gonzalez‐Rothi Elisa J. , Mitchell Gordon S.
Published: June 2021

Cervical spinal adenosine 2A receptor activation is necessary for certain forms of spinal respiratory motor plasticity, such as phrenic long‐term facilitation elicited by severe acute intermittent hypoxia (AIH; Nichols et al., 2012) or moderate AIH with acute cervical spinal injury (cSCI; Navarette‐Opazo et al., 2016). Whereas cervical spinal A2A receptor activation is sufficient to elicit phrenic motor facilitation (pMF; Golder et al., 2008), it undermines other, serotonin‐dependent forms of pMF via cross‐talk inhibition (Hoffman et al., 2010; Navarette‐Opazo et al., 2017). Thus, A2A receptors exert complex effects on phrenic motor plasticity. However, it is not known if conditions such as cSCI and/or repetitive exposure to intermittent hypoxia elicit neurochemical plasticity in A2A receptor expression (and associated phrenic motor plasticity). Thus, we characterized A2A receptor expression in phrenic motor neurons after chronic cSCI and prolonged exposure to different protocols of intermittent hypoxia. A2A receptor expression was determined in male Sprague Dawley rats with and without C2 spinal hemisection (C2Hx; 12 wks post‐injury) that were exposed to 28 days of: 1) normoxia; 2) daily AIH (10, 5‐min 10.5% O2 episodes with 5‐min intervals per day; 1.5 hrs/day); 3) moderate CIH (CIH 5/5: 5‐min 10.5% O2 episodes, 5‐min intervals; 8 hrs/day); and 4) high dose CIH (CIH 2/2: 2‐min 10.5% O2 episodes, 2 min intervals; 8 hrs/day). Rats were then perfused, and cervical spinal cords sectioned (40μm). C3 to C5 sections were processed for A2A receptor and Cholera toxin B subunit (CtB, injected 14 days before C2Hx). Using a custom MATLAB algorithm, optical density in CtB‐positive phrenic motor neurons was quantified. Statistical analysis was performed using MANOVA (SAS JMP). C2Hx decreased A2A receptor expression in phrenic motor neurons (p<0.05). Intermittent hypoxia exerted protocol‐specific effects on A2A receptor expression (CIH 2/2 > CIH 5/5 > daily AIH; p=0.014). Although intermittent hypoxia appeared to increase A2A receptor expression more after C2Hx, this effect was only marginally significant (injury/treatment interaction; p=0.061). The dual effects of injury and intermittent hypoxia on A2A receptor expression suggest that adenosine receptor plasticity may be important in the development of new therapeutic strategies to improve breathing after spinal cord injury.

Support or Funding Information

Supported by: NIH T32 HD043730 (LLA) OT2OD023854 (SPARC), NIH K12 HD055929 (EGR), McKnight Brain Institute.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
ErbB Family Members in Human Pulmonary Microvascular Endothelial Cells Respond Differently when Exposed to Hypoxia
Authors: Regan Christopher , Jin Yi Childrens , Pool Caitlyn Childrens , Liu Yusen , Chen Bernadette , Nelin Leif Childrens
Published: June 2021

Pulmonary hypertension (PH) is seen in diseases associated with hypoxia (WHO classification Group 3), and one of those diseases is bronchopulmonary dysplasia (BPD). BPD is the most common co‐morbidity following preterm birth, and the incidence is as high as 100% in babies born at 22 weeks' gestation. There are currently no available preventative or curative therapies for PH associated with BPD. Vascular remodeling is a hallmark of PH diseases including that seen with BPD. The proliferation of pulmonary endothelial cells, as well as release of pro‐proliferative substances from endothelial cells that can induce vascular smooth muscle cells to proliferate, are central to the pathogenesis of vascular remodeling in PH. We have previously shown that human pulmonary microvascular endothelial cells (hPMVEC) respond to hypoxia by proliferating. We have also shown that hypoxia‐induced proliferation depends on epidermal growth factor (EGF) release resulting in epidermal growth factor receptor (EGFR) activation. EGFR is a member of the ErbB family of receptor proteins, of which there are 4 described and EGFR is also known as ErbB1. We have recently shown that hPMVEC express ErbB2 and ErbB4, but that ErbB3 mRNA and protein are absent in hPMVEC. We hypothesized that hypoxia would also regulate ErbB2 and ErbB4 expression levels in hPMVEC. We used standard western blotting techniques to identify ErbB2 and ErbB4 protein levels in fetal hPMVEC incubated in normoxia (21% O2, 5% CO2) or hypoxia (1% O2, 5% CO2, balance N2) at 37° C for 24 hours. We found that ErbB4 protein levels were similar between normoxic and hypoxic hPMVEC's, while ErbB2 protein levels were lower in hypoxic hPMVEC than in normoxic hPMVEC. The absence of change in ErbB4 protein expression levels in hypoxia does not exclude the possibility for alteration in ErbB4 signaling in hypoxia. To begin to explore the role of ErbB4 signaling in hypoxia we determined if the ErbB4 ligand neuregulin‐4 would increase protein levels of ErbB4 in hPMVEC and we found that in normoxic hPMVEC addition of neuregulin‐4 increased protein levels of ErbB4. To determine if ErbB4 siRNA would knockdown ErbB4 specifically, i.e. without affecting ErbB1 or ErbB2 protein levels, we used an siRNA approach. Results show notable decreased protein expression level of ErbB4 exposed to siRNA as compared to cells exposed to scramble controls, but no notable changes in the expression levels of ErbB2 protein. We speculate that ErbB4 is involved in the hypoxic response of hPMVEC to hypoxia and may represent a potential novel therapeutic target for pulmonary hypertensive disorders associated with hypoxia.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Intermittent Hypoxia Induces Pulmonary Vascular Remodeling and Increases the Expression of STIM‐activated TRPC‐ORAI Channels in the Lung
Authors: Castillo‐Galan Sebastian , Arenas German A , Arias Paulina , Reyes Victor Roberto , Krause Bernardo , Iturriaga Rodrigo
Published: June 2021

Obstructive sleep apnea (OSA), a breathing disorder characterized by episodes of chronic intermittent hypoxia (CIH) and re‐oxygenation during sleep, is an independent risk for systemic hypertension. Furtheremore, 20–60% of OSA patients develop moderate pulmonary hypertension. Rats exposed to CIH developed pulmonary vascular remodeling, but the mechanisms underlaying the vascular remodeling are not well known. Stim‐activated TRP‐CORAI channel (STOC), cationic calcium‐permeable channels, are overexpressed in the lung and plays a key role in the development of pulmonary vascular remodeling in animals exposed to sustained hypoxia. However, whether these channels are involved in the pulmonary vascular remodeling induced by CIH have not been addressed. We studied the time‐course of the vascular pulmonary remodeling and the changes in STOC expression, in a rodent model of OSA. Male Sprague‐Dawley rats (~200g) were exposed for 14 to 28 days to CIH (5% O2, 12 times/h, for 8h). At 14, 21 and 28 days of CIH animals were euthanized and lungs dissected to determine the vascular remodeling index by histology analysis, and the pulmonary mRNA and protein levels of the STOC forming subunits TRPC1, TRPC4, TRPC6, ORAI 1, ORAI 2 and STIM1 by qPCR and western blot, respectively. Results were compared with age‐matched controls. CIH increased the pulmonary expression of TRPC 6, ORAI 1, ORAI 2 and STIM1 at 21 and 28 days compared with controls. In addition, CIH was associated to an increased in pulmonary arteries medial thickness at 21 and 28 days (66.6% ± 2.4, 66.3% ± 4.2 vs 45.4 ± 1.6, 21 and 28 days of CIH vs control respectively, p < 0.05). Present results shown that CIH increased the pulmonary STOC expression, and these changes paralleled the vascular remodeling.

Support or Funding Information

Fondecyt grants 1150040 and 1180341

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Intravascular Delivery of TAT‐conjugated Focal Adhesion Kinase Protects against Acute Lung Injury
Authors: Quadri Sadiqa , Das Shonit , Emine Memet , Mohammad Islam N , Bhattacharya Sunita , Bhattacharya Jahar
Published: June 2021

Acute lung injury (ALI), a major cause of high mortality in all age groups, occurs because of endothelial barrier failure. But it lacks a cure. We showed that endothelial focal adhesion kinase (FAK) is barrier protective (PMID: 23033075), suggesting FAK may be a cure for ALI. To test this hypothesis, we purified wild type FAK (FAKWT) and FAK with YàF mutation at tyrosine 925 (FAKMUT), which is expressed in FAK's focal adhesion targeting domain. Then we non‐covalently conjugated the proteins with the cell‐permeable peptide TAT (HIV‐1 transactivator of transcription). We intranasally instilled LPS in anesthetized mice. After 24h, we intravenously infused TAT‐FAKp or TAT‐FAKpY925F. After 4 h, we gave intravenous Evans Blue‐albumin (EBA). After a further 4 h, we assayed ALI in terms of EBA concentration and leukocyte counts in the bronchoalveolar lavage (BAL). Also, by live confocal microscopy of isolated, blood‐perfused lungs, we assayed endothelial F‐actin in terms of the lung endothelial fluorescence of Rhodamin Phalloidin. LPS increased EBA and leukocytes two‐times above baseline (p<0.05 each). TAT‐FAKWT, but not TAT‐FAKpMUT inhibited both responses (p<0.05). Endothelial F‐actin was markedly higher after TAT‐FAKWT than after TAT‐FAKpMUT infusion (p<0.05). LPS‐induced mortality in 5 days was 80% for untreated or TAT‐FAKpMUT infused mice. However, TAT‐FAKpWT infusion decreased the mortality to 25% (p<0.05). Our findings indicate that purified FAK can be effectively given by intravenous injection. TAT‐FAKWT, given well after the initiation of LPS‐induced ALI, markedly increased survival. The protective effect was due to FAK's Y925 residue that is likely responsible for enhancing cortical F‐actin, thereby strengthening the endothelial barrier (PMID:23033075). We propose that intravenous delivery of TAT‐FAKpWT might be effective therapy for ALI (HL36024).

Support or Funding Information

NIH: HL36024

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 effects of aging and exercise on lung mechanics, surfactant and alveolar macrophages
Authors: MCCAIG LYNDA , Pape Cynthia , Gill Sean , Veldhuizen Ruud
Published: June 2021

Introduction

It has been well established that advancing age leads to changes to the respiratory system and an associated susceptibility to lung diseases. Furthermore, exercise may counteract this pulmonary disease susceptibility. To start to investigate the underlying processes of these effects we explored the baseline characteristics due to aging and exercise on lung mechanics and on two important components of a healthy pulmonary environment, alveolar macrophage function and the pulmonary surfactant system. It was hypothesized that aging would impact lung mechanics, macrophage polarization and the status of the surfactant system, and that these changes would be mitigated by exercise.

Experimental approach: Male C57BL/6 mice were housed from 2–3 to 22 months, for the aged group, or until 4 months of age for young control mice. Mice in both the young and aged groups were randomized to either voluntarily running exercise or non‐exercise for a 2 month period. Mice were euthanized and lung mechanics were analyzed using a Flexivent ventilator. Subsequently, the lungs were lavaged to obtain pulmonary surfactant and alveolar macrophages. Pulmonary surfactant was analyzed for surfactant pool sizes and activity whereas alveolar macrophages were examined for a response to pro‐and anti‐inflammatory stimuli.

Results

Changes in lung mechanics, such as increased compliance and decreased airway resistance, were observed in the aged cohorts. These changes were not affected by exercise.

The quantity as well as the biophysical activity of the pulmonary surfactant system was unaffected by either aging or exercise. More alveolar macrophages were recovered from exercising aged mice as compared to both the young and non‐exercising groups.

Support or Funding Information

Western Strategic Support, Lawson Internal Research Funds

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
High Resolution Evaluation of Human Airway Cell Diversity Towards Understanding the Cellular Composition and Function of the Distal Human Airways
Authors: Basil Maria Ciocca , Zacharias William , Katzen Jeremy , Carl Justine , Li Shanru , Morley Michael , Englehardt John , Cantu Edward , Morrisey Edward
Published: June 2021

The mammalian lung is a complex organ comprised of epithelial cells, supporting stroma, and capillaries, which combine to allow the lung to perform its principle function of gas exchange. Our understanding of the cellular and molecular mechanisms that drive lung development and repair is based almost entirely on the study of murine models. While the human lung has many similarities to its murine counterpart, it also contains additional complexity. Distinctive structures, such as the respiratory bronchioles – the most distal portion of the human respiratory tree – are not present in small mammals. The cellular composition and function of these distal human airways are not well understood, but this region of the human lung is involved in many pathological processes, including subsets of interstitial lung disease, chronic lung allograft rejection, and COPD. There is a need to better define the nature of these human‐specific lung epithelial lineages. In this study, we utilized single cell RNA‐seq technology, combined with high resolution imaging, to characterize epithelial cells from distal respiratory airways and proximal conducting airway across postnatal development. Functional relationship between these cell types were assessed with induced pluriportent stem cell assays and in vitro organoid systems. The airway epithelium of the distal terminal and respiratory bronchioles represent a distinct cellular subset. The identification and analysis of these cells provides insight into the unique organization of the human lung, as well as the role these populations play in human disease.

Support or Funding Information

5T32HL007586‐32

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dexamethasone Improves Neuromuscular Junction Recovery From Ischemia‐Reperfusion Injury Induced by Tourniquet Application in Mouse Hindlimb
Authors: Tu Huiyin , Zhang Dongze , Wadman Michael C , Li Yu‐Long
Published: June 2021

Tourniquet‐induced ischemia‐reperfusion (IR) injuries impede long‐term recovery of skeletal muscle contraction. The neuromuscular junction (NMJ) is an important factor to influence skeletal muscle contractile function. Here, we observed the effect of dexamethasone (Dex) on morphological and functional recovery of the NMJ from tourniquet‐induced IR injuries in mouse hindlimb.

Unilateral hindlimb of C57/BL6 mice were subjected to 3 hours of ischemia by placing a rubber band and followed different periods of reperfusion (1 week, 2 weeks, 4 weeks, and 6 weeks). Dex treatment (1 mg/kg/day, i.p.) began on the day of IR induction and lasted for 7 days. After 3 hours of ischemia and different periods of reperfusion, NMJs in the gastrocnemius muscle showed significantly the morphological and functional damage. In the gastrocnemius muscle, TNFα and IL‐1β (two inflammatory cytokines) were increased after 1, 2 and 6 weeks of tourniquet‐induced IR. Treatment with Dex inhibited the elevation of TNFα and IL‐1β, mitigated fragmentation of nicotinic acetylcholine receptor (nAChR) clusters, promoted re‐innervation of nAChR clusters, and increased nerve‐stimulated end plate potentials (EPPs).

The data suggested that tourniquet‐related IR induced serious skeletal muscle inflammation for a long period. Dex treatment for 1 week inhibits IR‐caused inflammation in the early period of IR, and improves the morphological and functional recovery of the NMJ from tourniquet‐induced IR injuries.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Oxytocinergic Neurons in the Hypothalamic Paraventricular Nucleus Stimulate Brown Adipose Tissue Thermogenesis through Rostral Medullary Raphe
Authors: Fukushima Akihiro , Nakamura Kazuhiro
Published: June 2021

Hypothalamic neurons in the paraventricular nucleus (PVN) produce several neuropeptides, including oxytocin and vasopressin, and send their axons to various brain regions. These PVN neurons have been considered to serve neuroendocrine and autonomic functions, and many studies have demonstrated the involvement of oxytocin in social and emotional behaviors (e.g. maternal and sexual behaviors, and stress responses). Although these behaviors are accompanied with autonomic responses, it is unclear how oxytocin modulates the activities of the autonomic nervous system. To investigate the roles of PVN oxytocinergic neurons in the central regulation of autonomic function, in this study, we first performed neural tract tracing in rats from PVN oxytocinergic neurons to the rostral medullary raphe region (rMR), which contains sympathetic premotor neurons controlling brown adipose tissue (BAT) thermogenesis. In the rMR, oxytocin‐immunoreactive fibers, but not vasopressin‐immunoreactive fibers, were distributed, and oxytocin‐containing terminals were apposed to tryptophan hydroxylase‐expressing (i.e., serotonergic) neurons, potentially BAT sympathetic premotor neurons. Retrograde tracing from the rMR with cholera toxin b‐subunit (CTb) labeled a portion of oxytocinergic neurons in the caudal PVN, but not in the supraoptic nucleus, suggesting that the PVN is a major source of oxytocinergic fibers in the rMR. Then, we performed in vivo BAT sympathetic nerve recording combining optogenetic stimulation of the PVN‐derived oxytocinergic axon terminals in the rMR. Recombinant adeno‐associated viral vectors were injected into the PVN to express the engineered channel rhodopsin variant, ChIEF, under the oxytocin promoter. We confirmed that most of PVN neurons expressing ChIEF‐mCherry contained oxytocin and that their axon fibers were densely distributed in the rMR. Optogenetic stimulation of these fibers evoked BAT sympathetic nerve activities as well as increased expired CO2 and heart rate. Similar thermogenic and cardiac responses were elicited by nanoinjection of oxytocin into the rMR, and these responses were blocked by prior injection of the oxytocin receptor antagonist, L‐368,899 into the rMR. In addition, oxytocin injection into the rMR also potentiated NMDA‐evoked BAT sympathetic nerve activities. These findings indicate that PVN oxytocinergic neurons stimulate thermogenic sympathetic outflow to BAT through activation of rMR neurons. This oxytocinergic pathway may play an important role in emotion‐related autonomic responses.

Support or Funding Information

This work was supported by JSPS KAKENHI Grant Numbers JP16H05128, JP15H05932, JP26118508 (KN) and by AMED under Grant Number JP18gm5010002 (KN)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Kinin B1 Receptor Antagonism Attenuates DOCA‐Salt Hypertension by Modulating Angiotensin II Signaling in the Brain
Authors: Sriramula Srinivas
Published: June 2021

Deoxycorticosterone acetate (DOCA)‐salt hypertension is associated with increased expression of the pro‐inflammatory kinin B1 receptor (B1R) and elevated angiotensin II (Ang II) levels in the brain. We previously reported that DOCA‐salt hypertension is attenuated in global B1R knockout mice. However, the direct association between Ang II signaling and B1R has not yet been fully studied. In this study, we tested the hypothesis that central blockade of the B1R attenuates DOCA‐salt hypertension by altering angiotensin signaling within the brain. DOCA‐salt treatment (1mg/g body wt. DOCA, 1% saline in drinking water for 3 weeks) significantly increased blood pressure (BP; radiotelemetry) in male wild‐type (WT) mice (145 ±3 mmHg, n=8, p<0.01) compared to control mice (106 ±3 mmHg, n=6). Chronic intracerebroventricular (ICV) infusion of R715, a specific B1R antagonist, attenuated the DOCA‐salt‐induced hypertension (122 ±4 mmHg, n=8, p<0.01). Measurement of Ang‐II levels using ELISA revealed a 3‐fold increase in the hypothalamus (n=6, p<0.01 vs. Control) of hypertensive mice, which was blunted by ICV treatment with R715 for 3 weeks. In WT mice, DOCA‐salt treatment resulted in up‐regulation of angiotensin type‐1 receptor (AT1R; 3‐fold, n=6, p<0.01 vs. Control) and concomitant reduction of Mas receptor (30% decrease, n=6, p<0.01 vs. Control) gene expression in the hypothalamic paraventricular nucleus. Conversely, ICV treatment with R715 abrogated these receptor expression changes. To further confirm the pivotal role of neuronal B1R, hypothalamic primary neurons isolated from neonates were exposed to LDABK, a specific B1R agonist (300 nM, 24 h) after 14 days of culture. Treatment with LDABK significantly increased the gene expression of AT1R (2.5‐fold, n=6, p<0.01 vs. Vehicle) and this effect could be blocked by pretreatment with the B1R antagonist, R715 (10 μM, 30 min prior). Together, these data provide novel evidence that kinin B1R antagonism modulates angiotensin signaling in the brain and there by attenuates the development of DOCA‐salt hypertension.

Support or Funding Information

The American Heart Association (15SDG25720021)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Local ionotropic glutamate receptor activity is required for the ramp‐like increase of sympathetic activity induced by PVN TNFα
Authors: Mourao Aline Andrade , Pedrino Gustavo Rodrigues , Toney Glenn M.
Published: June 2021

Central nervous system actions of pro‐inflammatory cytokines (PIC) play a pivotal role in driving exaggerated sympathetic nerve activity (SNA), a major aggravating factor in cardiovascular and metabolic diseases. We previously reported that delivery of tumor necrosis factor alpha (TNFα), a prototypical early onset PIC, into the hypothalamic paraventricular nucleus (PVN) elicits a progressive, ramp‐like, increase of splanchnic SNA (SSNA) that resembles the response to sympathoexcitatory stimuli such as plasma insulin and leptin. Here we investigated the contribution of local ionotropic glutamate receptors both in triggering and sustaining this SNA response. In anesthetized (α‐chloralose/urethane) male Sprague‐Dawley rats, unilateral PVN injection of vehicle (aCSF) was without affect, but subsequent injection of TNFα (0.6 pmol/50 nl) elicited a significant (P<0.0001) ramp‐like increase of SSNA that averaged +55 ± 5.5% over a period of 60 min. Mean arterial pressure and heart rate were unaffected. Separate PVN pretreatment with the AMPA receptor antagonist NBQX (5.2 nmol/50 nl) and the NMDA antagonist AP5 (24 nmol/50 nl) (n = 5/group) each significantly attenuated (P<0.05) the TNFα elicited SSNA response (55 ± 5.5 % vs. NBQX: 24 ± 5% vs. AP5: 32 ± 6.5%) at the 60 min post‐TNFα time point. PVN pretreatment with a cocktail of NBQX and AP5 abolished the TNFα‐induced SSNA ramp (55 ± 5.5 % vs. 1.3 ± 2.1%; P<0.0001). When delivered separately into the PVN 60 min after TNFα, neither NBQX nor AP5 significantly altered the rate of SSNA ramping over an additional 60 min period. These results indicate that ionotropic glutamate receptor activity in the PVN is required to initiate the progressive increase of SSNA triggered by TNFα, and hint that sympathoexcitatory stimuli such as TNFα that elicit a PVN‐driven ramp‐like SNA response might do so by inducing a long‐term potentiation (LTP)‐like form of glutamatergic synaptic plasticity.

Support or Funding Information

Capes (AAM, GRP), NIH HL088052 & AHA 25710176 (GMT)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Neuropeptide Y Receptor Y1 (NPY Y1) Inhibition Attenuates Intestinal Inflammation in Murine Colitis and Human IBD Biopsy Cultures
Authors: Chandrasekharan Bindu , Saeedi Bejan , Dhere Tanvi , Neish Andrew S
Published: June 2021

Rationale

Inflammatory bowel disease, characterized by intestinal inflammation, is commonly treated with drugs targeted against the pro‐inflammatory cytokine tumor necrosis factor (TNF). However anti‐TNF drugs are expensive, and some patients stop responding to anti‐TNF's over time.

Aim

Based on our published findings that neuropeptide Y (NPY) can regulate TNF levels in the gut, we investigated the efficacy of neuropeptide Y (NPY) inhibition in attenuating intestinal inflammation by selectively blocking NPY receptor Y1 (NPY Y1) in mouse model of dextran sodium sulfate(DSS)‐induced colitis, and in colonic biopsies from patients with inflammatory bowel disease (IBD) ex vivo.

Methods

Colitis was induced in mice (129S strain) by administration of 3% DSS in drinking water for 7 days. NPY Y1 inhibition was achieved via intra rectal administration of NPYY1 antagonist BIBP‐3222 (@ 10 mg/kg of mouse body weight) on alternate days during the DSS treatment. Mice were monitored for weight loss, fecal blood and diarrhea (clinical score), colon length, histological score, TNF expression (by real time PCR and enzyme linked immunosorbent assay/ELISA) and on day 7 subjected to endoscopy. Colonic biopsies were obtained from healthy volunteers (n = 10) and IBD patients (n = 8), and biopsies were cultured ex vivo in presence or absence of BIBP‐3222 (0.1mM) for 24 h (at 37°C, 5% CO2).

Results

In mouse models of (DSS)‐induced colitis, NPY Y1 inhibition resulted in significant protective effects as determined from the clinical score and histological scores, colon length and endoscopic colitis score (p < 0.05). TNF mRNA was also significantly downregulated in the colon of DSS + BIBP‐treated mice as compared to DSS ‐ treated mice (p < 0.05). Colonic biopsies from IBD patient's ex vivo depicted significant increase in TNF levels as compared to biopsies from healthy subjects (p < 0.05). Incubation of IBD patient biopsies with BIBP‐3222 was effective in significantly inhibiting TNF release by at least 50 % as determined by ELISA (p < 0.05).

Conclusions

Taken together, our data suggests that NPYY1 inhibition is effective in attenuating intestinal inflammation in murine colitis and human IBD biopsies ex vivo.

Support or Funding Information

We acknowledge the support from Litwin‐IBD Pioneers Initiative grant from Crohn's & Colitis Foundation (B.C) and the U.S. National Institutes of Health grants AI64462 & DK089763 (A.S.N.)

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 Role of Sensory Nerves in Modulating Antigen Specific Immune Responses
Authors: Tynan Aisling , Gunasekaran Manojkumar , Tsaava Téa , Imperato Gavin H , Chavan Sangeeta S , Tracey Kevin J
Published: June 2021

The nervous and immune systems are the two major organ systems of memory and host defense. Both systems sense changes in the environment, mobilize host responses, and establish memories of threatening insults. Immunological memory is developed by T and B cells, which retain antigen‐specific information for decades. Upon subsequent challenge with the same antigen, antigen‐specific B cells proliferate resulting in a rapid and robust secondary antibody response that neutralize and eradicate the antigen. However, little is known about the role of sensory neurons in modulating antigen‐specific B cells responses. We have previously demonstrated that local sensory neural network plays a vital role in regulating antigen trafficking through the lymphatic system in vivo. Here, we show that TRPV1‐expressing sensory neurons are involved in the regulation of immune responses. Specific genetic ablation of TRPV1‐lineage neurons results in a failure to generate a robust antigen‐specific antibody response following immunization (Anti‐KLH IgG levels Day 28, Wild type 8243.64 U/ml versus TRPV1 ablated 1068.60 U/ml). However, in vitro stimulated B‐cells from TRPV1‐ablated mice produce similar levels of IgG as wild type littermate controls suggesting that there is not a B‐cell intrinsic defect in TRPV1‐ablated mice. Co‐culture of splenocytes with sensory neurons results in an increased level of antibody production. Direct activation of TRPV1+ signals by selective optogenetic stimulation (5 minutes, 473nm laser, 10Hz, 50% duty cycle) results in a significat decrease of antigen trafficking to the popliteal lymph node as compared to sham controls (Sham 11012691 Average Fluorescent Volume versus Photostimulated 5330646 Average Fluorescent Volume, p<0.05). These findings, together with our previous studies, implicate a neuro‐immune crosstalk in both physiological and pathological conditions, and raises the exciting possibility that the nervous system collaborates with the immune system to regulate antigen‐mediated responses.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Muscle Sympathetic Activity Kinetics during One‐leg Cycling in Men and Women with and without Heart Failure: Evidence for Preserved Cardiopulmonary Baroreflex Sympathoinhibition
Authors: Millar Philip J , Notarius Catherine F , Picton Peter , Haruki Nobuhiko , Floras John S
Published: June 2021

Microneurographic recordings of muscle sympathetic nerve activity (MSNA) are generally analyzed over discrete epochs, usually in minutes. This is sufficient for determining resting sympathetic outflow but may obscure physiologically relevant information regarding temporal changes during an acute stress, such as exercise. To address this limitation, we applied a moving average to examine continuously the effects of one‐leg cycling on MSNA in 22 participants with (n=11) and without (n=11) heart failure with reduced ejection fraction (HFrEF). MSNA burst frequency and incidence were acquired over an 8 minute period: 2 minutes of baseline, zeroload cycling, moderate intensity cycling (50% of VO2 peak), and recovery. MSNA was analyzed in 30 second epochs, advancing in five‐second increments to create a 91 point overlapping moving average plot. Area under the curve analysis demonstrated that both MSNA burst frequency and incidence were higher in HFrEF than controls during zeroload and moderate intensity cycling (both, p<0.05). To estimate the reflex sympathoinhibitory consequences of skeletal muscle‐pump induced increases in cardiopulmonary blood volume at the start of exercise, we calculated the nadir reduction in MSNA burst frequency and incidence during the first 30 seconds of zeroload (HFrEF vs. controls: −6±7 vs. −7±4 bursts/min; −12±11 vs. −15±5 bursts/100 heartbeats) and moderate intensity (HFrEF vs. controls: −7±5 vs. −6±7 bursts/min; −11±7 vs. −8±9 bursts/100 heartbeats) cycling; no differences were observed between HFrEF and controls (all, p>0.05). Such initial reductions in MSNA burst frequency were observed in 9/11 HFrEF and 11/11 controls with zeroload cycling; reductions occurred in 11/11 HFrEF and 7/11 controls with moderate intensity cycling (both, p>0.05). These similar group‐mean magnitude reductions in MSNA at the onset of exercise are consistent with preserved cardiopulmonary‐reflex mediated sympathoinhibition in HFrEF; the subsequent increase in MSNA is consistent with an exaggerated muscle metaboreflex contribution. As peripheral neurogenic vasoconstriction contributes to exercise intolerance in HFrEF, exercise regimens with shorter durations (e.g. high intensity interval training) may be better tolerated and more efficacious elements of conditioning protocols for such patients.

Support or Funding Information

This study was supported by Grants‐in‐Aid from the Heart and Stroke Foundation of Ontario (T4938, NA6298). P.J.M. was supported by a Canadian Institutes of Health Research Postdoctoral Fellowship. J.S.F. was supported by the Canada Research Chair 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.
Vasopressin V1a Receptor of Renal Collecting Duct Intercalated Cells Promotes Urinary Proton Secretion
Authors: Giesecke Torsten , Koshimizu Taka‐Aki , Kawahara Katsumasa , Gimber Niclas , Schmoranzer Jan , Himmerkus Nina , Isermann Julian , Bleich Markus , Assay Niklas , Leipziger Jens , Fähling Michael , Smorodchenko Alina , Bachmann Sebastian , Mutig Kerim
Published: June 2021

Antagonists of the V1a vasopressin receptor (V1aR) are emerging as a therapeutic strategy for retardation of chronic kidney disorders. Physiologically, V1aR signaling has been linked with acid‐base homeostasis, although the underlying mechanisms remain debatable. In this study, we tested the hypothesis that V1aR activation in type A intercalated cells (A‐ICs) induces urinary H+ secretion.

We generated an anti‐V1aR antibody and verified its specificity using V1aR‐knockout tissues. Localization studies in mice, rat and human tissue, were preformed using immunofluorescence and confocal microscopy or 3D‐structured illumination microscopy (3D‐SIM). For functional studies in vivo, V1aR‐specific agonist, AO‐4‐67, was administered in AVP‐deficient Brattleboro rats and C57/BI6J mice (0.2, 2, or 10μg/kg b.w.; 1 to 4h). Urine was collected in metabolic cages or via ureteral catheter and plasma samples were taken. Isolated, microperfused mouse collecting ducts were analyzed for V1aR‐dependent luminal pH changes and cultured inner medullary collecting duct cells for proton‐secreting vH+‐ATPase regulation.

Localization of V1aR in mouse, rat and human kidneys produced basolateral signal in A‐ICs and perinuclear to subapical signal in type B intercalated cells of connecting tubules and collecting ducts throughout these species. In addition, basolateral V1aR signal was detected in macula densa cells of mouse but not rat or human kidneys. Administration of the V1aR agonist, A0‐4‐67, significantly decreased urinary pH in vasopressin‐deficient Brattleboro rats (10 μg/kg body weight for 4 hours i.p.; pH 7.38 to 6.71 after 1 hour, P<0.01) and C57/BI6J mice (2 μg/kg body weight for 1 hour i.p.; pH 7.18 to 6.78 after 20 minutes, P<0.05). Basolateral treatment of isolated perfused medullary collecting ducts with the V1aR agonist or vasopressin increased intracellular Ca2+ levels in intercalated cells and decreased luminal pH (pH −5% after 3 minutes, P<0.05) suggesting V1aR‐dependent Ca2+ release and stimulation of proton secreting proteins. Basolateral treatment of inner medullary collecting duct cells with the V1aR agonist induced luminal translocation of vacuolar H+‐ATPase in A‐ICs (apical signal intensity +93%, P<0.001).

In summary, our results show that activation of V1aR contributes to urinary acidification via H+ secretion by A‐ICs, which may have clinical implications for pharmacological targeting of V1aR.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
miR‐137 mediates TNF‐dependent inhibition of Aquaporin 2 expression
Authors: Hao Shoujin , DelliPizzi AnnMarie , Hao Mary , Ferreri Nicholas R.
Published: June 2021

We previously showed that TNF produced by renal epithelial cells inhibits NKCC2 activity as part of a mechanism that attenuates increases in blood pressure in response to increases in sodium intake. We also showed that ambient urine osmolality was higher in TNF deficient mice compared with age‐ and sex‐matched wild type (WT) mice. As the role of TNF in the kidney is still being defined, the effect of TNF produced by renal epithelial cells on aquaporin‐2 (AQP2) expression was determined in this study. TNF levels were higher in urine, but not plasma, of mice that ingested 1% NaCl (HS) in the drinking water for three days compared with tap water (40±4 vs 18±5 pg/24 h). The increase in urinary TNF levels was associated with an approximately 5‐fold increase in TNF mRNA abundance in the inner medulla excised from mice given HS. Interestingly, we observed that miR‐137 levels also increased approximately 2‐fold in the inner medulla isolated from mice given HS. Six days after intrarenal injection of a lentivirus construct designed to silence TNF, EGFP‐shTNF‐ex4, renal but not splenic TNF mRNA levels were significantly reduced. Injection of control lentivirus (U6) did not alter TNF mRNA in either kidney or spleen. RT‐PCR analysis showed that AQP2 mRNA levels were significantly decreased in the inner medullary tissue from mice given HS. However, renal silencing of TNF in mice given HS exhibited a 3‐fold increase in AQP2 mRNA and protein expression in the inner medulla compared with WT. In contrast, EGFP‐shTNF‐ex4 but not U6 decreased miR‐137 levels in the inner medulla in mice given HS. Moreover, target seed regions of miR‐137 were conserved in the 3′‐untranslated region of mouse AQP2 mRNA. Accordingly, intrarenal injection of miR‐137 inhibited the increase of AQP2 mRNA abundance in the inner medulla induced by silencing TNF in the kidneys of mice given HS. As TNF production in the kidney was induced by administration of HS for 3 days, the effects of TNF silencing on urine volume were determined. Mice were acclimated in metabolic cages for 3 days after receiving intrarenal injections of either EGFP‐shTNF‐ex4 or U6. Food, body weight, water intake, and urine output was then measured in mice given HS or tap water for 3 days. Both water intake and urine output were reduced in mice injected with EGFP‐shTNF‐ex4 compared with mice given U6 lentivirus. The decrease in urine volume was associated with an increase in AQP2 mRNA accumulation. Collectively, these findings suggest that the intrinsic effects of TNF produced within the kidney in response to HS intake include an inhibitory effect on AQP2 expression that is mediated via a mechanism involving miR‐137.

Support or Funding Information

NIH(HL133077)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Aldosterone‐Induced MicroRNAs Act as Negative Feedback Regulators for the Mineralocorticoid Receptor in Kidney Distal Nephron Epithelia
Authors: Ozbaki‐Yagan Nejla , Liu Xiaoning , Bodnar Andrew J , Ho Jaqueline , Butterworth Michael Bruce
Published: June 2021

Regulated sodium (Na+) homeostasis in the kidney is achieved by the action of the renin‐angiotensin‐aldosterone signaling cascade. The terminal signaling hormone, aldosterone binds to the mineralocorticoid receptor (MR) to increase Na+ uptake across tubule epithelial cells by altering the expression and function of channels, transporters and regulatory proteins acting in concert. Along with a change in gene and protein expression, aldosterone regulates the expression of non‐coding RNAs including microRNAs (miRs). The primary function of miRs is to repress target mRNA expression and decrease protein abundance. Extended aldosterone stimulation (1–7 days) is known to reduce MR expression (protein and mRNA) in cell and animal models. To investigate if miRs were involved in this negative feedback regulation of the MR, we profiled the expression of miRs predicted to bind to the MR‐3’untranslated region (UTR) in mouse cortical collecting duct (mCCD) epithelial cells stimulated with aldosterone (50nM). Of the significantly altered miRs, the miR‐466a‐e family members were most highly upregulated (>5 fold). This upregulation was verified in vivo in CCD cells isolated from mouse kidneys of animals placed on low Na+ diets for 3 days. Luciferase assays confirmed miR‐466 binding to the MR‐UTR. Preventing the upregulation of miR‐466 after aldosterone stimulation, using miR inhibitors increased MR protein expression. Aldosterone‐stimulated sodium transport via the epithelial sodium channel in miR‐466 inhibited CCD cells was significantly higher than control transfected cells. The Na+‐stimulation was sustained over extended periods (24–72hrs) and miR‐466 inhibited cells had increased aldosterone sensitivity. In conclusion, we have identified a role for the miR‐466's in the negative feedback regulation of MR after extended aldosterone stimulation. Inhibition of miR‐466 released this feedback regulation to allow for a larger, more sensitive and more sustained response to aldosterone stimulation.

Support or Funding Information

NIDDK R01DK103776 (JH) NIDDK DK102843 (MB)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
G‐protein Pathway Suppressor 2 (GPS2) Enhanced the Big Potassium (BK) Channels Activity through modulating interaction between GPS2 and WNK4
Authors: Wang Yunman , Luo Tongwang , Li Ruidian , Cai Hui
Published: June 2021

The Large Conductance Ca2+ Activated Potassium (BK) Channels plays an important role in renal potassium secretion. G‐protein pathway suppressor 2 (GPS2) is a multifunctional protein and transcriptional regulation factor that is involved in the G‐protein mitogen‐activated protein kinase (MAPK) signaling pathway. We have previously reported that WNK4 inhibits BK channel activity by stimulating ERK1/2 and p38 signaling pathways. We have recently shown that GPS2 enhances BK channel activity and its protein expression by reducing ERK1/2 signaling‐mediated degradation of the channel. We therefore hypothesized that GPS2 stimulates BK channel activity through its interaction with WNK4. We first investigated whether GPS2 interacts with WNK4. In HEK293 cells cotransfected with HA‐WNK4 and either myc‐vector or myc‐GPS2, co‐immunoprecipitation (co‐IP) experiments showed that myc‐GPS2 is able to co‐immunoprecipitate WNK4, whereas myc‐vector does not. To confirm their interaction, we performed the reciprocal co‐IP experiments in HEK293 cells co‐transfected with HA‐GPS2 and either myc‐vector or myc‐WNK4. We found that myc‐WNK4 co‐precipitates HA‐GPS2 while myc‐vector does not. To further assess the effects of GPS2 on WNK4‐mediated inhibitory effects on BK, we performed the western blot analysis in HEK293 cells cotransfected with BK with or without WNK4 in the presence or absence of GPS2. We found that WNK4 significantly inhibits BK protein expression as expected. However, in the presence of GPS2, BK protein expression is restored to the control level. To explore whether GPS2 affects WNK4 protein degradation we did WNK4 ubiquitination experiment. In Cos‐7 cells co‐transfected with WNK4 with or without GPS2, we showed that GSP2 significantly enhances WNK4 ubiquitination, suggesting that GPS2 promotes WNK4 degradation. We further showed that GPS2 reversed the WNK4‐mediated inhibition of BK protein expression by partially reducing the BK degradation through both proteosomal and lysosomal pathways. These data suggested that GPS2 stimulates BK activity through its interaction with WNK4, leading to enhancing WNK4 ubiquitination and degradation via both proteosomal and lysosomal pathways.

Support or Funding Information

VA Merit 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.