bluuebunny/crossref_metadata_2025 huggingface dataset:
Renal arterial blood flow in conscious, freely‐moving animals via wireless telemetry
Authors: Adreani Christine Marie , Pettinger Steve , Mills William , Trinh Tu , Cunningham Paul , Sun Shu-Yu , Forrest Michael
Published: June 2021

Our objective was to develop and validate a fully implantable telemetry device to measure renal arterial blood flow in laboratory animals under physiologically appropriate conditions. Novel flow sensors were developed and tested acutely in vivo in a stepwise fashion with external electronics to validate the sensor design and to collect data to facilitate the design of the fully implantable blood flow electronics. Infusion of dopamine (3 μg/kg/min) increased renal blood flow by 14.3 +/−2.8% in dogs implanted with the prototype novel flow sensors. Two additional female beagles were instrumented with the fully implantable device and monitored weekly for 12 weeks. A flow sensor was placed around the left renal artery and a solid state blood pressure transducer was placed in the abdominal aorta. Electronic components consisting of the signal processing circuits, telemetry transmitter and battery were implanted in the peritoneal cavity. Stable recordings of renal arterial blood flow values of ~10 ml/min/kg were obtained for both dogs. This device enables evaluation of renal arterial blood flow, blood pressure, ECG, and core temperature following pharmacologic intervention while eliminating physiologic stress associated with animal restraint.
Determining how 4‐phenylbutyrate inhibits tunicamycininduced acute kidney injury
Authors: Carlisle Rachel E , Brimble Elise , Ingram Alistair J , Austin Richard C , Dickhout Jeffrey G
Published: June 2021

It has been demonstrated that many nephrotoxic drugs, such as tunicamycin, induce endoplasmic reticulum (ER) stress as part of the mechanism by which they cause acute kidney injury (AKI). There are three main mechanisms by which the low molecular weight chemical chaperone 4‐phenylbutyrate (4‐PBA) can modify AKI: (1) as a histone deacetylase inhibitor, (2) as an ER stress inhibitor, and (3) as a mediator of waste nitrogen excretion. We hypothesized that 4‐PBA would inhibit tunicamycin‐induced acute kidney injury using one of these three mechanisms. C57BL/6J mice were either untreated or pre‐treated with 4‐PBA for one week, at a dose of 1 g/kg, delivered in the drinking water. Proximal tubular cell injury was then induced by intraperitoneal injection of tunicamycin (0.5 mg/kg) for 3 days. In the whole animal, tunicamycin was found to induce proximal tubule injury in the juxtamedullary region, demonstrated by the presence of tubular epithelial cell vacuolization, in PAS‐stained sections. 4‐PBA pretreatment reduced the injury score associated with tunicamycin‐induced AKI. The mechanism by which 4‐PBA prevents tunicamycin‐induced kidney injury is currently under investigation. Supported by CIHR OSO‐115895.
HSP27 regulation in acute unilateral obstructed kidney, along with RMIC and collecting duct cells subjected to mechanical, oxidative, and inflammatory stress
Authors: Carlsen Inge Gram , Nilsson Line , Frøkiær Jørgen , Nørregaard Rikke
Published: June 2021

In vivo, renal medullary interstitial cells (RMIC) and collecting duct (CD) cells are subjected to mechanical, oxidative and inflammatory stress as a result of ureteral obstruction. Heat shock protein 27 (HSP27) is expressed in RMIC and CD cells in vivo and in vitro, and plays a role in stabilization of actin filaments. Both HSP27 and the phosphorylated‐form (pHSP27) have anti‐apoptotic functions.

In vivo studies using rats subjected to acute unilateral ureteral obstruction (UUO) for 6h and 12h showed unchanged HSP27 protein level in kidney inner medulla (IM). pHSP27 protein level was increased in obstructed kidney IM compared to both sham and contralateral kidney. Further examination of HSP27 protein abundance in response to mechanical, oxidative, and inflammatory stress in vitro, we subjected RMIC and CD cell to pressure, H2O2 and interleukin 1â (IL‐1â) stimulation for 2–24h. Applying oxidative stress to RMIC and CD cells increased HSP27. Inflammatory stress with IL‐1â increased HSP27 and pHSP27. Pressure of 60 mmHg was explored directly in RMIC which increased HSP27.

These data indicates that in response to acute UUO different forms of cellular stress increase expression of HSP27 and pHSP27, which may play a role in the cytoprotection of RMIC and CD cells.
Protective effects of Plantago asiatica L. extract against ferric‐nitrilotriacetate (Fe‐NTA) induced liver oxidative stress in Wistar rats
Authors: Hong Chung-Oui , Hong Seung-Teak , Lee Yan-Houy , Lee Kwang-Won
Published: June 2021

The Plantago asiatica L.(PA), a native plant in East Asia (Korea, Japan and China etc.), traditionally used as a popular folk for the treatment of liver diseases. The purpose of this study was to evaluate the liver protective effects of PA extract(PAE) on the Ferric‐nitrilotriacetate(Fe‐NTA)‐induced oxidative hepatotoxicity. Fe‐NTA is a potent hepatotoxic agent and has been reported to induce hepatocyte necrosis. The study of liver protection effect that pretreatment with PAE(1, 2, 4 g/kg b.w.) by gavage to Wistar rats for 7 days before a single dose of Fe‐NTA(13.5 mg Fe/kg b.w., i.p.). Only Fe‐NTA treated group showed significantly increased the serum levels of the hepatic enzyme, aspartate aminotransferase(AST) and alanine aminotransferase(ALT) which is representative liver damage marker, and liver tissue malondialdehyde(MDA), product of lipid peroxidation. Moreover, the level of biomarkers indicate the antioxidants status, glutathione(GSH), glutathione‐S‐transferase(GST), glutathione reductase(GR) and superoxide dismutase(SOD) in liver tissue were decreased. However, PAE pre‐treated group showed decreases in the levels of serum AST and ALT and liver tissue MDA in concentration dependent manner and increases in the level of GSH, GST, GR and SOD. These results are significantly different(p<0.05) to compare with only Fe‐NTA treated group. Thus, our study suggested that PAE might contribute to the chemopreventive material against Fe‐NTA‐mediated liver oxidative damage.
NADPH oxidase 4 induces cardiac arrhythmia in zebrafish through ROS
Authors: Zhang Yixuan , Shimizu Hirohito , Siu Kin L , Chen Jau-Nian , Cai Hua Linda
Published: June 2021

Indirect evidences implicate that oxidative stress might play an important role in cardiac arrhythmogenesis. Recently, we identified an intriguing link between NADPH oxidase 4 (NOX4) and atrial fibrillation in humans. The present study utilizes cardiac specific GFP transgenic zebrafish to examine effects of NOX4 injected RNA on cardiac rhythm. Full‐length human NOX4 was cloned, transcribed, and RNA injected to one‐cell stage fish embryos. Remarkably, 51.0% of the fish developed arrhythmia (irregular heart beat) after 24–30 hrs. To quantify the intervals among heart rates of each embryo, we recorded consecutive digital images (30 frames/second) of each embryo and analyzed by custom‐made software. The variation of intervals in NOX4 embryos was significantly bigger than that of control embryos (0.038±0.0020 vs. 0.027±0.0026 second/beat, n=11–17, all data as Mean±SEM, p=0.0028). The NOX4 embryos also had increased superoxide production determined by electron spin resonance (5.3±0.7 vs. 10.9±1.5 μM/min/mg, n=9–12, p<0.01). Co‐injection of an ATG morpholino led to reduced arrhythmogenesis (to 20.2%) and blunted superoxide production. We also generated catalytically inactive NOX4 RNA (proline 437 substituted with histidine in the NADPH binding domain), which failed to induce arrhythmia and overproduction of superoxide (n=5, p=0.67). In conclusion, our data have identified a novel phenotype of cardiac arrhythmia in NOX4 overexpressed zebrafish, providing new insights into mechanistic studies of arrhythmogenesis.

This study has been supported by National Heart, Lung and Blood Institute (NHLBI), and American Heart Association.
Searching for the Na1 site of hSGLT1
Authors: Loo Donald D.F. , Hirayama Bruce A. , Jiang Xuan , Wright Ernest M.
Published: June 2021

The crystal structure of vSGLT has provided the location of a Na+ binding site that is conserved in the LeuT structural family, the Na2 site. In human SGLT1 there is strong functional evidence for two Na+ binding sites, one of which (Na2) is conserved in homology models. The location of the Na1 site is unknown in hSGLT1 but in the LeuT family it is colocalized with the substrate binding site. To search for the Na1 site in hSGLT1, we measured the intrinsic Na+ affinity of sugar binding site mutants and the gating and ligand coordinating residues (H83C, Y290C, W291C and Q457C) from the [Na+] dependence of the SGLT1 capacitive currents (charge movement) in the absence of glucose. For wild‐type and Q457C hSGLT1 the Na+ sensitivity of the charge movement was ≈−100 mV per 10‐fold decrease in external [Na+]. This was dramatically reduced for H83C, Y290C and W291C hSGLT1, ≈−50 mV per 10‐fold decrease in external [Na+]. In addition, the Na+/glucose coupling coefficient for hSGLT1 decreased from 2 to 1 for mutants Y290F and W291F. The loss of Na+ affinity for mutants H83C, Y290C and W291C, and the reduction in coupling indicates that these resides may be involved in Na+ binding. We hypothesize that the Na1 site in hSGLT1 is close to the substrate binding site as in LeuT and BetP.

Supported by NIH grant DK19567.
Reconstitution of transepithelial Amino Acid Transport in MDCK Epithelia
Authors: Guetg Adriano Antonio Doriano , Torrente Marta , Mariotta Luca , Camargo Simone M. R. , Verrey François
Published: June 2021

Amino acids (AAs) are (re)absorbed across intestine and kidney proximal tubule epithelia by a machinery composed of luminal and basolateral AA transporters (AATs). These AATs have been characterized as yet mostly separately in non‐polarized expression systems. We aim now at reconstituting transepithelial neutral AA transport in Madin‐Darby Canine Kidney (MDCK) cells to test the cooperation of these AATs. For the luminal uptake, we expressed the Na+/neutral AA symporter B0AT1 (SLC6A19) together with its kidney associated protein TMEM27 using a vector encoding both proteins separated by an internal ribosome entry site (IRES). These cells displayed increased intracellular amounts of aromatic and β‐branched AAs as measured by HPLC. For the basolateral efflux, we expressed the uniporters that transport aromatic (TAT1, SLC16A10) and β‐branched (LAT4, SLC43A2) AAs as well as the heterodimeric neutral AA exchanger LAT2 (SLC7A8) with accessory glycoprotein 4F2hc (SLC3A2) as a fusion protein. Expression and function of these transporters was verified by Western blotting and/or immunofluorescence and by uptake of radiolabeled AAs. Using this newly established expression model, we are currently testing the transport of AAs across epithelia expressing all and/or part of the AA transport machinery to understand the cooperation of the involved AATs. Supported by the Swiss NSF grant 31‐130471 to FV.
Transport of organic cations across human bronchial and bronchiolar epithelial barriers in vitro
Authors: Salomon Johanna Jessica , Ehrhardt Carsten
Published: June 2021

In this study, we aimed to characterise the function of organic cation/carnitine transporters (OCT/Ns) in human bronchial (Calu‐ 3) and bronchiolar (H441) epithelial cells by using radiolabelled model substrates. The effect of time on uptake of [14C]‐TEA (TEA) and [3H]‐acetylcarnitine (Ac‐Car) was studied. In addition, bi‐directional transport studies were carried out. TEA uptake was found to be time‐dependent in both cell lines, however, H441 cells exhibited a higher level of uptake. Transport experments revealed net‐absorption of both compounds investigated. The apparent permeability coefficient (Papp) for apical‐to‐basolateral transport was 1.93±0.03×10−6 cm/s (TEA) and 1.36±0.01×10−5 cm/s (Ac‐ Car) across Calu‐3 cells. The corresponding basolateral‐to‐apical values were 1.05±0.02×10−6 cm/s (TEA) and 2.32±0.05×10−6 cm/s (Ac‐Car), respectively. In the case of H441 cells, Papp values for TEA were 2.09±0.03×10−5 cm/s (a‐to‐b) and 8.99±0.06×10−6 cm/s (b‐to‐a), and for Ac‐Car 2.71±0.02×10−5 cm/s (a‐to‐b) and for Ac‐ Car 1.14±0.02×10−5 (b‐to‐a), respectively. These data further support the hypothesis that organic cations are actively transported at the human air‐blood barrier, and that the involved transporter sites might play a role in processing of positively charged pharmaceuticals. This work is supported by SFI grant 07/SRC/B1154.
Discovery of an inward rectifying potassium channel inhibitor with preference for Kir2.3, Kir3.X and Kir7.1
Authors: Raphemot Rene , Lonergan Daniel , Nguyen Thuy T. , Utley Thomas J. , Gogliotti Rocco , Hopkins Corey R. , Lewis Michelle , Lindsley Craig W. , Weaver C. David , Denton Jerod S.
Published: June 2021

Inward rectifying potassium (Kir) channels play key physiological roles in diverse cell types and may represent novel drug targets for disease. In cardiac myocytes, Kir channels contribute to the resting membrane potential and the late repolarization phase of the action potential. Homomeric Kir2.3 and heteromeric Kir3.1/3.4 channels are expressed almost exclusively in the atria and are postulated drug targets for atrial fibrillation, a common arrhythmia that predisposes patients to stroke. Efforts to assess the utility of Kir2.3 and Kir3.1/3.4 channels as therapeutic targets have been hampered by the lack of drug‐like compounds targeting these channels. We therefore performed a fluorescence‐based high‐throughput screen for small‐molecule inhibitors of Kir3.X channel function. Several novel inhibitors were discovered. One compound, termed VU573, inhibits Kir3.X channels with a half‐maximal inhibition concentration of 1.9 μM. Subsequent electrophysiology‐based counterscreens revealed that VU573 inhibits Kir2.3 and Kir7.1 with similar potency, but is less active toward Kir1.1, Kir2.1, and Kir4.1. VU573 is the first selective inhibitor for Kir2.3 and most potent Kir7.1 inhibitor known to date. We are currently using medicinal chemistry to improve the potency and selectivity of VU573 for Kir2.3 and Kir3.X and understand the structural requirements for inhibition of these channels.

NIH R01 award to Jerod S. Denton DK082884
Ano6 functions as a positive modulator of volume‐regulated anion channels
Authors: Owsianik Grzegorz , Prenen Jean , Hermans Diane , Eggermont Jan , Voets Thomas , Nilius Bernd
Published: June 2021

Anoctamin 6 (Ano6) is a homologue of Ano1 that functions as a calcium‐activated chloride channel. Recently, Ano6 has been identified as an essential component of outwardly rectifying chloride channels. It has been also shown that Ano6 and Ano1 are activated by hypotonic cell swelling (HTS) and might be a part of an epithelial volume‐regulated anion channel (VRAC). Using quantitative PCR, we showed that Ano6 expression levels correlated with detection of large VRAC currents in smooth muscle‐like BC3H1 and colon carcinoma Caco‐2 cells. Overexpression of Ano6 in HEK293 cells resulted in significant increase of HTS‐evoked anion currents when compared to untransfected cells. In contrast to Ano1, Ano6 was not activated by elevation of intracellular Ca2+. Moreover, co‐expression with caveolin‐1, a main component of caveolae plasma membrane and a positive regulator of VRAC, significantly increases HTS‐evoked currents in Ano6‐overexpressing HEK293 cells. Similarly, transient Ano6 expression in Caco‐2 cells generated large increase of HTS‐evoked currents when compared to the untransfected cells. Recorded currents displayed known characteristics of VRAC such as a rapid deactivation at high positive potentials and block by tamoxifen and mibefradil. Our results suggest that Ano6 is a positive modulator of VRAC function. Supported by Interuniversity Poles of Attraction Program and Program Financing, KU Leuven.
Studies on the functional expression of epithelial sodium channel delta subunit (δ‐ENaC) in human respiratory epithelial cells in vitro
Authors: Schwagerus Elena , Illek Beate , Fischer Horst , Ehrhardt Carsten
Published: June 2021

While epithelial sodium channel α‐, β‐ and γ‐subunits are crucial for the ion and fluid balance in the lung, the role of the δ‐subunit remains unclear. Here, we studied δ‐ENaC expression and its contribution to ion transport function in human respiratory epithelial cells. Expression of the δ‐ENaC was investigated in human respiratory epithelial cell lines (A549, H441 and Calu‐3) as well as in alveolar epithelial cells in primary culture (hAEpC) on both gene and protein levels. In Ussing chamber studies, the effects of reported δ‐ENaC modulators (i.e., Evans blue, capsazepine and icilin) were studied in H441 and Calu‐3 cell monolayers. All investigated cell types showed presence of both δ‐ENaC mRNA and protein, as revealed by PCR, Western blot and immunofluorescence microscopy. All pharmacological modulators resulted in blockade of Na+ current in H441 cell monolayers. Interestingly, in Calu‐3 cell monolayers only capsazepine showed an inhibitory effect, whereas Evans blue and icilin increased ISC, which were mostly Cl− dependent currents. These data suggest a physiological role for δ‐ENaC in human respiratory epithelium. However, further studies need to determine δ‐ENaC regulation and the specificity of the observed pharmacological effects. ES is funded by an IRCSET postgraduate scholarship.
The Selective TRPV4 Antagonist GSK2263095A Attenuates High Venous Pressure‐Induced Lung Injury In Murine And Canine Lung
Authors: Townsley Mary I. , Jian Ming-Yuan , Cheung Mui , Willette Robert N. , Thorneloe Kevin S.
Published: June 2021

We have shown that the lung permeability response to high venous pressure (HiPv) is abrogated in mice lacking the vanilloid transient receptor potential channel TRPV4. The current study evaluated the efficacy of a novel selective TRPV4 antagonist GSK2263095A in limiting HiPv‐induced lung injury. Lungs isolated from anesthetized C57BL/6 mice or dogs were perfused with buffer/4% albumin or autologous blood, respectively, at constant flow. The filtration coefficient (Kf, an index of endothelial permeability) was measured at baseline (BL) and during 15‐min challenge with 25 or 40 cmH2O HiPv. In mouse lung, 25 cmH2O HiPv increased Kf 4.9‐fold, vs 2.9‐fold in lungs pretreated with 300 nM GSK2263095A (p<0.05). In canine lung, 25 cmH2O HiPv had no significant impact on Kf: 2.0‐ (vehicle) and 1.6‐fold BL (100 nM GSK2263095A). However, 40 cmH2O HiPv increased Kf significantly to 6.1‐fold baseline, an effect attenuated (final Kf 3.0‐ fold. baseline) by pretreatment with 100 nM GSK2263095A (both p<0.05). Further, the TRPV4 antagonist markedly attenuated the HiPv‐induced increase in extravascular lung water (p<0.05) and tended to blunt blood extravasation in canine lung. We conclude that TRPV4 is required for the permeability response to HiPv in both mouse and canine lung, and that the TRPV4 antagonist GSK2263095A is protective against lung injury induced by high pulmonary venous pressure. Supported by GlaxoSmithKline.
Regulation of ion transport and ASL height by the anti‐inflammatory mediator, lipoxin A4 in normal and cystic fibrosis bronchial epithelium
Authors: Urbach Valerie , Higgins Gerard , Al-Alawai Mazen , Costello Richard W , McNally Paul , Verriere Valia , Harvey Brian J
Published: June 2021

Mutations of CFTR results in defective Cl− secretion and Na+ hyperabsorption which contributes to a reduction of the airway surface liquid layer height (ASLh) thus promoting bacterial colonization and inflammation. Lipoxin A4 (LXA4) which plays a central role in resolving inflammation is decreased in cystic fibrosis (CF). We have investigated the role of LXA4 in modulating ion transport and ASLh in CF and non‐CF bronchial epithelial cell lines (CuFi‐1 and Nuli‐1) and primary cultures grown under an air‐liquid interface. ASLh were measured using confocal microscopy and ion transport using electrophysiological techniques.

LXA4 (1nM) significantly increased ASLh in CF and non‐CF epithelia. This effect was inhibited by bumetanide, amiloride, Boc‐2 (FPR2 antagonist), extracellular hexokinase, reactive blue 2 and NF340 (P2Y and P2Y11 receptor antagonist). LXA4 stimulated a carbenoxolone (Panexin‐1 inhibitor)‐sensitive apical ATP release, intracellular Ca2+ mobilization and Cl− secretion and inhibited Na+ absorption. These effects of LXA4 involving apical ATP release, inhibition of Na+ absorption and stimulation of Cl− secretion to enhance ASL dynamics open up a new therapeutic avenue to promote mucociliary clearance in CF airways.
Claudin‐4 interacts with the small GTPase Rap1 and accelerates epithelial repair
Authors: Sutherland Katherine Marie , Bentley Trevor , LaFemina Michael , Ho Gwen , Hirsch Jan , Frank James
Published: June 2021

Epithelial repair is a requirement for recovery in patients with acute lung injury. In human lungs rejected for transplantation, higher cldn4 levels are associated with preserved epithelial barrier function, but the role of claudins in epithelial repair is uncertain. To determine if claudin‐4 is important to epithelial repair, we examined claudin‐4 expression and function in two in vitro model of epithelial repair: scratch wounding and tight junction resealing after calcium depletion. In both models, epithelial disruption was followed by an induction of claudin‐4. The increase in claudin‐4 was dependent of EGFR as inhibition with AG1478 prevented the increase in claudin‐4. Knock down of claudin‐4 with siRNA resulted in delayed epithelial repair in both models. To determine the contribution of claudin‐4 to repair we used an immuoprecipitation and mass spectrometry approach to identify potential claudin‐4 interaction partners in primary rat type 2 cells and the human airway cell line 16HBE. In wounded epithelial monolayers the small GTPase Rap1 was identified and this association was confirmed with co‐immuoprecipitation. Because Rap1 is known to mediate cell junction maturation, adhesion, and cell spreading, we hypothesize that claudin‐4 interacts with Rap1 to accelerate epithelial repair potentially by recruiting Rap1 to tight junctions. Funded by NIH/NHLBI HL88440 and T32 HL007185
Hypoxia modulates pregnancy‐mediated transcriptional regulation of BKca β1 subunit expression in ovine uterine arteries
Authors: CHEN MAN , DASGUPTA CHIRANJIB , ZHU RONGHUI , HUANG XIAOHUI , ZHANG LUBO
Published: June 2021

BKca channels play a key role in the increased uterine artery blood flow in pregnancy, which is attenuated by chronic hypoxia during gestation. We examined transcriptional mechanisms of BKca β1 subunit promoter in ovine uterine arteries. We cloned a 2‐kb promoter of β1 gene in sheep, which contains several transcription factor binding sites with CpG dinucleotides in their core binding sequences, including ERα/SP1, SP1, and AP1. ERα/SP1, but not SP1 or AP1, sites were hypomethylated in uterine arteries of pregnant as compared with nonpregnant animals, which was reversed by chronic hypoxia. The binding of ERα and SP1 to the site was confirmed by EMSA with super shift and ERα and SP1 bind simultaneously to the site. CpG methylation inhibited the binding. Deletion of the ERα/SP1 binding site significantly attenuated reporter gene activity of β1 promoter. ChIP assay showed an increase in ERα and SP1 binding to β1 promoter in pregnant uterine arteries, which was abrogated by chronic hypoxia. In accordance, β1 protein expression was increased in pregnant uterine arteries, which was blocked by hypoxia. The results demonstrate a novel mechanism of promoter methylation in transcriptional regulation of BKca β1 subunit gene expression patterns in uterine arteries in response to pregnancy and hypoxia. (Supported in part by NIH grants HL89012 and HL110125).
Hypothalamic Global Gene Expression in Response to Hypoxia in Late‐Gestation Fetal Sheep
Authors: Wood Charles E , Chang Eileen , Rabaglino Maria Belen
Published: June 2021

This study was designed to identify gene networks in the fetal hypothalamus that are up‐ or down‐regulated in response to a 30 minute period of hypoxia (decreased fetal PaO2 to ~10 mm Hg). Chronically catheterized fetal sheep (130–135 days gestation, term=147d) were euthanized 1 hr after onset of a 30 min hypoxia (n=3) or normoxia (n=3). mRNA was extracted and analyzed using the ovine Agilent 15.5k array, which was annotated in this lab. 241 genes were significantly (p<0.005) upregulated and 880 genes were significantly downregulated. GO terms associated with upregulated genes included ubiquitination, Cu homeostasis, oxidative metabolism. GO terms associated with downregulated genes included cell cycle, immune development, and apoptosis. KEGG analysis indicated a shift from aerobic to anaerobic metabolism and decreased gene expression in hematopoietic cell lineages. None of the regulated genes were controlled by HIF, and were therefore most likely influenced by changes in neurotransmission. We conclude that fetal hypothalamic genomic response to hypoxia reflects appropriate responses in cellular energetics and that it is not likely the result of direct cellular hypoxia.
The ABC of Communication: Easy as 123?
Authors: Evans Darrell John
Published: June 2021

The ability to get a message across to others and to convey specific thoughts and ideas effectively is becoming increasingly important as our communities become more interconnected. Despite the growing emphasis placed upon good communication skills, many scientists, educators and students continue to grapple with how best to engage their audience. There are multiple drivers for developing effective communication skills, including capturing the attention and interest of the audience, avoiding miscommunication and achieving the desired goal. Communicating successfully, whether in verbal or written format, therefore relies on three main steps: 1 ‐ identifying the audience, 2 ‐ defining what they need to know and why, and 3 ‐ delivering the message appropriately and effectively. Developing the skills and expertise necessary to follow such a strategy should enable specialists to engage and interact with each other as well as the non‐specialists, capitalise on any opportunities that present themselves and ensure that any message is not misinterpreted.
Epigenetic Reprogramming of Mitochondrial Dysfunction in hyperhomocysteinemia
Authors: Tyagi Neetu , Narayanan Nithya , Mishra Paras K , Qipshidze Natia , Givvimani Srikanth , Tyagi Suresh C
Published: June 2021

An elevated level of homocysteine (Hyperhomocysteinemia) is associated with increased risk factor of neurological disorder. Homocysteine (Hcy) modulates cellular methylation reactions. Previously we have demonstrated that HHcy caused significant metabolic changes and altered mitochondrial function in mouse brain endothelial cells (bEND3). However, the mechanisms behind such alterations remain unclear. The present study examined if the effects of Hcy on mitochondria dysfunction are mediated through an epigenetic mechanism. Following exposure to Hcy for 72 h, global DNA methylation and histone H3 lysine 9 (H3K9) acetylation were examined using flow cytometric analysis. Total DNA methyltransferase activity (DNMT) and protein levels of DNMT 3 were measured. Mitochondrial respiratory capacity and mitochondrial permeability transition (MPT) were measured by fluorescence dye. The result suggested that HHcy caused global DNA hypomethylation and histone hyperacetylation. Total DNMT activity significantly decreased which was accompanied by a significant reduction in protein levels of DNMT 3 and loss of MPT. Treatment of bEND3 with the DNMT inhibitor, 5‐aza‐2′‐deoxycytidine, mimicked the functional changes induced by Hcy. This study suggests that the Hcy related metabolic changes including altered mitochondrial function may be mediated through an epigenetic mechanism.
Central TRPV1 signaling regulates systemic blood glucose levels and hepatic PEPCK protein expression
Authors: O'Hare James D , Miyata Kayoko , Fourrier Torrey L , Krantz Amanda M , Derbenev Andrei V , Zsombok Andrea
Published: June 2021

The autonomic nervous system participates in the regulation of glucose homeostasis. Because transient receptor potential vanilloid type 1 (TRPV1) plays an important role in regulating excitatory neurotransmission in neurons of the dorsal motor nucleus of the vagus and in liver‐related neurons in the paraventricular nucleus (PVN) of the hypothalamus it has been suggested that TRPV1 activity mediates autonomic control of liver glucose homeostasis. We tested this hypothesis by measuring the effect of TRPV1 activation on systemic blood glucose levels and expression of hepatic gluconeogenic enzymes in mice. Activation of TRPV1 by injecting capsaicin (0.5 nmol) bilaterally into the PVN significantly lowered systemic blood glucose levels within 10 min by ~30 mg/dl. This decrease remained for up to 30 min following capsaicin injection and its effect was diminished in the presence of the glutamate receptor antagonist, kynurenic acid. Protein expression of hepatic gluconeogenic enzymes (PEPCK, G6Pase) was determined by Western blot. TRPV1 activation in the PVN decreased hepatic PEPCK levels, while G6Pase levels were unaltered. Our data suggest that TRPV1 signaling in the PVN controls systemic blood glucose levels, possibly by altering parasympathetic regulation of hepatic gluconeogenesis. Supported by AHA 10GRNT4540000, Tulane BIRCWH 2K12HD043451.
Hydrogen peroxide (H2O2) modulates membrane properties in second‐order nucleus tractus solitarii (nTS) neurons
Authors: Ostrowski Tim Daniel , Hasser Eileen M , Heesch Cheryl M , Kline David D
Published: June 2021

H2O2, a stable reactive oxygen species, is a potent neuromodulator of cellular and synaptic activity. H2O2 is normally present in low concentrations, but rapidly increases during bouts of hypoxia‐reoxygenation. Chemoafferent fibers terminate within the nTS, a center vital for cardiorespiratory responses to hypoxia. Thus, we determined the role of H2O2 on synaptic and membrane properties in the nTS using the patch clamp technique. Synaptic transmission was not altered by 10–500 μM H2O2, but membrane properties were modulated by 500 μM H2O2. Input resistance (IR) decreased with simultaneous hyperpolarization of membrane potential (MP) and action potential threshold (THR). Action potential discharge (APD) to depolarizing current was reduced with H2O2. Preventing the H2O2‐induced hyperpolarization of MP (holding MP at −60 mV) resulted in augmented APD to current injection. During washout, MP returned to pre‐H2O2 levels whereas THR remained hyperpolarized. This reduced the difference between MP and THR and increased APD excitability to depolarization compared to pre‐H2O2. Catalase within the recording pipette ablated the effects of extracellular H2O2, indicating an intracellular site of modulation. Taken together, H2O2 reduces excitability of nTS neurons and upon removal results in hyperexcitability that may play a profound role in cardiorespiratory reflexes. Support: RO1 HL098602.
Perivagal capsaicin alters the response of vagal motoneurons to Thyrotropin Releasing Hormone (TRH)
Authors: Holmes Gregory M. , Swartz Emily M , Travagli R. Alberto
Published: June 2021

A supposedly selective degeneration of vagal afferent C‐fibers by perivagal capsaicin (CAP) has often been used to identify the mechanism of action of GI‐ peptides. The actions of many of these peptides have been attributed to activation of vagal afferent fibers based on the dramatic reduction of their effects following perivagal CAP pretreatment. We have reported recently that, similar to surgical vagotomy, perivagal CAP induces dendritic degeneration, reduction of ChAT‐immunoreactivity and, possibly, death of a subpopulation of vagal preganglionic (DMV) neurons.

We tested the hypothesis that perivagal CAP treatment induces functional impairment of rat DMV neurons.

Monolateral perivagal CAP treatment (0.1 and 1%) was performed 7–10 days prior to monitoring gastric motility response induced by DMV microinjection of TRH (100pmoles), which increases motility via actions exclusively at DMV.

Microinjection of TRH in DMV reduced gastric motility by 42±16 and 76±5% in rats pretreated with 0.1 or 1% CAP vs control (N=5 for all). Conversely, gastric motility in response to microinjection of TRH in the contralateral DMV was not altered (N=3–4).

Our data show that perivagal CAP treatment induces functional impairment of DMV neurons and indicates that perivagal CAP treatment should not be used as an exclusive means to investigate the site of action of gastrointestinal peptides.

Funds: NIDDK 55530
The Role of Melanin Concentrating Hormone in Central Chemoreception: a Knock‐down Study by siRNAs in the Lateral Hypothalamus
Authors: Li Ningjing , Li Aihua , Nattie Eugene
Published: June 2021

Melanin concentrating hormone (MCH), a neuropeptide in the lateral hypothalamus (LHA), has been implicated in regulating food intake, energy balance, and cardiovascular control. Hypothalamic MCH neurons are known to have multisynaptic connections with diaphragm motoneurons, and to send projections to many central chemoreceptor sites although there are no physiological observations on whether MCH is involved in respiratory control. To test the hypothesis that MCH play a role in central chemoreception, we knocked down the expression of MCH by injecting a pool of siRNAs that target the MCH precursor gene (pMCH) into the LHA in rats and then measured changes in breathing, body weight, blood glucose, blood pressure, and heart rate. RT‐PCR data demonstrated a 60% reduction of pMCH mRNA expression in rats injected with pMCH siRNAs compared to rats received control scrambled siRNAs (n=5, P<0.001). Compared to pretreatment baseline, knock‐down of pMCH resulted in 1) a 20–30% increase in the VE/VO2 in response to 5 & 7% CO2 72hr after pMCH siRNA injection; 2) a 10% decrease in body weight and 30% reduction in blood glucose level; 3) an increase in both heart rate and blood pressure (n=4). There are no significant findings observed in the rats that received scrambled siRNAs. This study uses a siRNA approach to demonstrate the involvement of a hypothalamic peptide, MCH, in central chemoreception (HL 28066).
Serotonin 2A receptors augment synaptic transmission in the nucleus tractus solitarii (nTS)
Authors: Austgen James R. , Kline David D.
Published: June 2021

The nTS is the primary termination and integration point for cardiorespiratory afferents in the brainstem. Serotonin (5‐Hydroxytryptamine, 5‐HT) in the nTS plays a profound role in modulating synaptic and cardiovascular function. This study examined the role of the 5‐HT 2A receptor subtype in mediating neurotransmission in the nTS. Excitatory postsynaptic currents in monosynaptic nTS neurons were recorded in the horizontal brainstem slice in the presence of a 5‐HT2A receptor agonist (TCB‐2) or antagonist (MDL11939). TCB‐2 augmented solitary tract (TS) evoked EPSC amplitude elicited at 0.5 and 20 Hz. Conversely, MDL11939 depressed TS‐EPSCs amplitude evoked at 0.5 and 20 Hz. TCB‐2 and MDL11939 did not alter the TS‐EPSC paired‐pulse ratio, magnitude of depression from the first event or decay time. TCB‐2 did not alter miniature (m)EPSC amplitude or frequency, whereas MDL11939 increased mEPSC frequency but not amplitude. These data suggest the 5‐HT2A receptor is an excitatory neuromodulator in the nTS. Support: HL 085108.
Exercise training suppresses central command‐evoked sympathoexcitation in rats with heart failure
Authors: Koba Satoshi , Inoue Yumiko , Hisatome Ichiro , Watanabe Tatsuo
Published: June 2021

Sympathoexcitation evoked by central command, a parallel activation of the locomotor and autonomic neural circuits in the brain, has been shown to become exaggerated in heart failure (HF). In the present study, we tested whether exercise training (ET) in HF normalizes central command‐evoked sympathoexcitation. HF was induced in rats > 6 wks after coronary artery ligation. A subset of the HF rats was treadmill trained (20 m/min, 5% grade, 60 min/day, 5 days/wk, 8–14 wks) (HF+ET). Central command was activated by fictive locomotion in decerebrate rats. Fictive locomotion induced by 30‐s electrical stimulation at 35 μA of the mesencephalic locomotor region evoked a larger (P<0.05) increase in renal sympathetic nerve activity in HF rats [269 ± 45 arbitrary unit (AU), fractional shortening (FS) = 20 ± 3%, N12] than that in HF+ET (121 ± 33 AU, FS = 20 ± 2%, N10) and sham (111 ± 37 AU, FS=46 ± 3%, N6) rats. The sympathoexcitation in the HF rats was significantly reduced by bilateral injection into the rostral ventrolateral medulla of Tempol (10 mM, 100 nl), a superoxide dismutase mimetic. In the HF+ET and sham rats, Tempol had no effect on the response. These results suggest that ET has a therapeutic effect to suppress sympathoexcitation evoked by central command in HF. Further, the role played by central superoxide in amplifying central command‐evoked sympathoexcitation in HF may be altered by ET. Supported by JSPS Kaken 22790226 (SK).
Effect of acetylcholinesterase inhibition with pyridostigmine on cardiovascular parameters in mice with myocardial infarction
Authors: Durand Marina Toledo , do Carmo Jussara M. , Corrêa Wesley G. , Oliveira Mauro , Fazan Rubens , Salgado Helio Cesar
Published: June 2021

The benefit of pyridostigmine (PYR), an acetylcholinesterase inhibitor, on mean arterial pressure (MAP), heart rate (HR) and intrinsic heart rate (IHR) was evaluated in mice (C57Bl/6J) submitted to myocardial infarction (MI). Mice were anesthetized (isoflurane) and telemetry transmitters (DSI) were implanted into the left carotid artery. After 5–7 days of recovery, basal MAP (mmHg) and HR (bpm) were recorded for 48 hours (10s every 10min). Further, basal MAP and HR were recorded continuously followed by methylatropine and propranolol administration. After basal recordings, mice were submitted to sham surgery or left anterior descending (LAD) coronary occlusion and received drinking water with or without PYR (3 mg/kg p.o. for 7 days). Five days after LAD occlusion, or sham surgery, recordings were repeated. Sham surgery did not affect MAP, HR and IHR (n=6). LAD occlusion (n= 3) promoted a MI of 11±1 %, decreased MAP (91±1 vs 106±1 before), increased HR (555±11 vs. 481±18 before) and did not affect the IHR (481±11 vs. 466±7 before). LAD occlusion combined with PYR (n=6) did not show any alteration in MAP (101±6 vs. 100±2 before), HR (489±20 vs. 469±15 before) and IHR (448±41 vs. 453±27 before). The results demonstrate that MI caused a decrease in MAP and an increase in HR combined with no alteration in IHR. In addition, PYR prevented the alterations in MAP and HR caused by MI, and did not change the IHR.
Rho Kinase Inhibition Lowers Sympathetic Nerve Activity in Conscious Rabbits with Chronic Heart Failure
Authors: Haack Karla Kristine Vincent , Gao Lie , Curry Pamela , Zucker Irving H
Published: June 2021

Rho‐Associated protein kinase (RhoK) is a serine/threonine kinase that is involved in calcium sensitization and vascular smooth muscle cell contraction. Recent work has implicated RhoK overactivation in cardiovascular diseases. Furthermore, the RhoK pathway in the brain stem has been shown to contribute to blood pressure regulation via the sympathetic nervous system (SNS). It has been extensively shown that a hallmark of chronic heart failure (CHF) is increased SNS activation. We investigated the effect of central RhoK blockade on renal sympathetic nerve activity (RSNA) in a pacing rabbit model of CHF. Briefly, we induced CHF in a rabbit model by placement of left ventricular pacing leads, and pacing at 360–380 BPM for 1–2 weeks. CHF was confirmed by echocardiography, and characterized by a reduction in ejection fraction to ~45%. Nerve recording electrodes were then implanted on the renal nervealong with an intracerebroventricular cannula and osmotic minipump containing sterile saline or 1.5 mg/kg/mL fasudil (Fas, a RhoK inhibitor) in saline at a rate of 1 μL/h. After 3 days, infusion with Fas lowered resting heart rate by 25 ± 2.1 BPM (p<0.03) and significantly reduced RSNA frequency (Fas: 13.1±4.7 Hz vs Veh: 39.1 ± 5.2 Hz; p<0.02). Change in RSNA in response to oropharyngeal smoke was increased in CHF Fas animals (93 ± 18%) vs CHF Veh treated animals (45 ± 9%) (p<0.03). These data suggest that central RhoK activation may contribute to sympatho‐excitation in the setting of CHF. Supported by PO‐1 HL62222.
Inactivity‐induced phrenic motor facilitation following intermittent neural inactivity is dependent on spinal atypical PKC
Authors: Baertsch Nathan Andrew , Baker-Herman Tracy L
Published: June 2021

Prolonged reductions in respiratory neural activity in Harlan Sprague Dawley (HSD) rats elicit an atypical PKC (aPKC) dependent form of spinal plasticity called inactivity‐induced phrenic motor facilitation (iPMF), a rebound increase in phrenic burst amplitude that is apparent when respiratory neural activity is restored. In Charles River Sprague Dawley (CRSD) rats, iPMF is pattern sensitive and is only induced by intermittent, but not sustained neural apneas. We hypothesized that iPMF in CRSD requires spinal aPKC activity. Anesthetized and ventilated CRSD were hyperventilated to create five brief (~1 min) neural apneas, separated by 5 min. 10 min following intermittent neural apneas, rats received an aPKC inhibitor (ZIP) or scrambled ZIP in the intrathecal space over C4. As expected, significant iPMF was observed prior to intrathecal ZIP or scrambled ZIP (5 min post‐apnea: 56 + 8 and 57 + 12 %baseline; p<0.01). Subsequent intrathecal ZIP significantly attenuated iPMF (p<0.001), such that phrenic amplitude was no longer different from baseline at 60 min post‐intermittent apnea (6 + 7 %baseline; p>0.05). By contrast, rats receiving scrambled ZIP continued to express significant iPMF (69+8 %baseline; p<0.01). These data suggest that similar to iPMF following a prolonged apnea in HSD, increased phrenic burst amplitude following intermittent neural apnea in CRSD is dependent on spinal aPKC activity. NIH 105511
The opioid‐induced suppression of augmented (‘sigh’) breaths is dose‐dependent and prevented by co‐administration of naloxone
Authors: Bell Harold James , Pankuch Glenn
Published: June 2021

We tested whether the morphine (MOR) induced suppression of augmented breaths (ABs) can be prevented by the non‐specific, competitive opioid receptor antagonist naloxone (NX). Further we determined whether the suppression of ABs follows a dose‐response pattern in the low‐end recommended dose range. We studied breathing in 8 rats on 4 separate days, giving four dosages of MOR s.c. in randomized order: 0 mg/kg (saline control), 1, 3, and 5 mg/kg. In a second set of 8 animals, we co‐administered either {MOR (5mg/kg) + NX (5 mg/kg)}, or {MOR (5mg/kg) + saline control (1 ml/kg)} on two separate days and monitored animals for 2 hours. MOR suppressed ABs in a typical doseresponse fashion, from 6.5 ± 1.6 ABs/15mins (control) to 1.5 ± 1.5, and 0.6 ± 1.8 ABs/15mins in the 3 and 5 mg/kg dose conditions respectively (both p<0.001). 5 mg/kg MOR caused ABs to be completely eliminated in all but one of the 8 animals we studied. In the second experiment, MOR caused a rapid and potent suppression of ABs across a two‐hour monitoring interval that was prevented by NX co‐administration. There were significantly more ABs when NX was co‐administered with MOR, regardless of the time interval across the 2 hours at which they were assessed post‐injection (p<0.001). In summary, MOR suppresses ABs in a dose‐response manner in the low‐end of the recommended dosage range, and this effect occurs via typical opioid‐receptor mediated pathways.

This study received financial support from the Department of Medicine, Penn State College of Medicine, in the form of laboratory start‐up funds.
The effects of two common injectable laboratory anesthetics on the regulation of augmented (‘sigh’) breaths
Authors: Bell Harold James , Moore Jacob , Haouzi Philippe , Van De Louw Andry
Published: June 2021

In this study, we investigated whether the link that we have recently established between hypocapnia and AB production is only observable in unanesthetized animals, possibly explaining why other laboratories using anesthetized preparations have not also reported this obvious effect. Ten male rats were studied on three separate days, receiving one of three injections each day in randomized order: ethyl carbamate (‘urethane’; 1.2 mg·kg−1); a ketamine/xylazine combination (ket/xyl; 80/10 mg·kg−1), or normal saline (control). Following each of the three interventions, breathing was monitored during 15 minute exposures to normoxia (room air), hypoxia (10% O2) and hypoxia+CO2 (10% O2, 5% CO2). Urethane anesthesia completely eliminated ABs from the breathing rhythm in room air conditions (0.0 ± 0.0 vs 4.0 ± 1.8 ABs·15min−1 in saline control, P<0.001), and decreased the hypocapnia‐dependent component of the hypoxia‐induced facilitation of ABs (p<0.001). Ket/xyl left the normal incidence of ABs in room air breathing intact (4.5 ± 2.2 ABs·15min−1, p=0.689 vs. saline control) but significantly suppressed the hypoxia‐induced facilitation of ABs (Δ = 9.6 ± 6.1 vs 32.7 ± 14.4 ABs·15min−1 in saline control, p=0.0015). Our results provide the first clear evidence that laboratory anesthesia can profoundly alter the regulation of ABs including the hypocapnia‐dependent component of their facilitation. This research was supported by laboratory research start‐up funds provided by the Department of Medicine, Penn State College of Medicine. JM was supported in part through the SURIP program of the Department of Cellular and Molecular Physiology of Penn State University.
Localization of nuclear and membrane progesterone receptors in the brainstem of adult mice
Authors: Joseph Vincent , Habbal Karim , Goulemetova Roumiana
Published: June 2021

We used immunohistochemistry to determine the localization of progesterone receptors (PR) in respiratory‐related areas in the brainstem of adult male mice. Serial slices were incubated with antibodies against nuclear PR (nPR), or membrane α and β PR (mPRα and mPRβ). A prominent staining for nPR, mPRα, and mPRβ appeared in caudal and rostral parts of the nucleus tractus solitarius (NTS), X and XII nuclei, but while nPR and mPRα stained cell bodies, mPRβ stained fibers. With double fluorescence labeling we showed that mPRα is co‐localized in catecholaminergic neurons (TH+) in NTS, and in motoneurons in X and XII nuclei (VGLUT2+). mPRβ is expressed in TH+ and VGLUT2+ fibers in these regions. A dense staining for nPR and mPRβ appeared in the ventrolateral medulla, in a region corresponding to the pre‐botzinger complex, and 3β‐HSD (an enzyme involved in progesterone synthesis) was also densily expressed in these regions. This suggests that local progesterone synthesis modulate respiratory function.
Ventilatory long‐term facilitation is altered in tryptophan hydroxylase 2 knock out mice
Authors: Hickner Stephen James , Angoa-Perez Mariana , Kuhn Donald M , Mateika Jason H
Published: June 2021

Purpose

This study was designed to examine the impact of serotonin (5HT) in the central nervous system (CNS) on ventilatory long‐term facilitation (vLTF) in spontaneously breathing mice.

Methods

We exposed six mice with a null mutation for tryptophan hydroxylase 2 (KO) and six wild type mice (WT) to 12 episodes of hypoxia (10 % oxygen) and measured ventilation via whole body plethysmography.

Results

KO
WT

Baseline VT
0.21 ± 0.03*
0.17 ± 0.01

Baseline Bf
150.5 ± 7.1*
169.5 ± 6.0

Recovery VT
0.22 ± 0.02*
0.16 ± 0.01

Recovery Bf
152.9± 11.2*
186.2 ± 4.8§

VT – tidal volume (ml); Bf – breathing frequency (breaths/min);

– significantly different from WT mice (p < 0.05);

– significantly different from baseline (p < 0.05).

KO mice displayed an elevated VT and a decreased Bf under baseline conditions. LTF was evident in the WT (see Bf measures) but not the KO mice following exposure to hypoxia.

Conclusions

Lack of 5HT in the CNS is accompanied by the absence of LTF following acute exposure to intermittent hypoxia.