bluuebunny/crossref_metadata_2025_split huggingface dataset:
An essential role of cortactin in regulation of ENaC
Authors: Ilatovskaya Daria , Vinnakota Kalyan C , Pavlov Tengis S , Staruschenko Alexander 
Published: June 2021

Epithelial Na+ Channel (ENaC) is critical for maintenance of sodium balance. Cortactin is a key signaling protein for many cellular processes, including direct interaction with F‐actin. In this study we have addressed the role of cortactin in mediating regulation of ENaC. As assessed by Western blotting, cortactin is highly expressed in mpkCCDc14, M‐1 and MDCK cells. Immunohistochemistry analysis showed localization of cortactin in the cortical collecting duct in Sprague‐Dawley rat kidneys. Co‐immunoprecipitation analysis revealed that cortactin directly interacts with ENaC subunits when co‐expressed in CHO cells. We have supported these data with fluorescent microscopy co‐localization approach and fluorescence correlation spectroscopy analysis. Coexpression of cortactin with ENaC decreased channel's activity as measured by patch‐clamp. Surprisingly, cortactin's dynamin‐binding Src homology 3 domain is not required for ENaC regulation. Similarly cortactin mutants with deleted phosphorylation domain, or lacking ability to bind F‐actin decrease ENaC current. However cortactin mutant unable to bind Arp2/3 complex does not influence ENaC activity. Thus, we conclude that cortactin decreases ENaC activity via Arp2/3 complex.Supported by AHA and ASN.
Vesicle associated membrane protein (VAMP) 3 but not VAMP2 is involved in NKCC2 apical targeting and processing in thick ascending limbs (TALs)
Authors: Caceres Paulo S , Mendez Mariela , Ortiz Pablo A 
Published: June 2021

NKCC2 undergoes apical targeting to the cell surface in TALs in the absence of hormonal stimulation. VAMP2 and VAMP3 colocalize with NKCC2 in the apical membrane and subapical space of TALs and may differentially regulate protein processing, sorting and apical targeting of NKCC2. We hypothesized that both VAMP2 and VAMP3 regulate constitutive NKCC2 surface expression. We measured the effect of a) siRNAs against VAMP2 and VAMP3 and b) tetanus toxin; on surface and total NKCC2 expression by biotinylation of rat TAL suspensions and Western blot. VAMP2 siRNA decreased VAMP2 (85% decrease) without affecting VAMP3 and VAMP3 siRNA decreased VAMP3 (95% decrease) without affecting VAMP2 in control experiments. VAMP2 siRNA did not affect surface NKCC2 (81 ± 7 % of control) or total NKCC2 expression (93 ± 9 % of control). In contrast, VAMP3 siRNA decreased surface NKCC2 by 53 ± 13% (p<0.05) and total NKCC2 expression by 59 ± 8 % (p<0.01), suggesting that VAMP3 mediates both processing and apical targeting. We next used a low concentration of tetanus toxin, which cleaves and inactivates VAMP2 and VAMP3, to block apical targeting, but not processing or sorting. Fifty min treatment with 30 nM tetanus toxin decreased surface NKCC2 by 33 ± 5 % (p<0.05) without affecting total NKCC2 expression. Our data suggest that VAMP3 is involved in both NKCC2 processing and apical targeting in TALs whereas VAMP2 is not likely involved in these processes.NIH
Sorting of α2 and α3 Na+ Pumps in Glia and Neurons: Linkage with Na/Ca Exchanger‐1
Authors: Song Hong , Weber David J , Thompson Scott M , Blaustein Mordecai P. 
Published: June 2021

The α2 Na+ pump amino acid (AA) sorting sequence in astrocytes and arterial myocytes is: L27DELKKEVA35 (Song et al., J Biol Chem 281:12929, 2006). Plasmids encoding this sequence, with an N‐terminal Green Fluorescent Protein and C‐terminal Flag (f)‐tag (GFP‐LDELKKEVA‐f), when transfected (Lipofectamine) into mouse primary cultured astrocytes, were dominant negative (DN) for normal α2 expression (by immunocytochemistry). Mutations of AAs 28–34 to Ala did not prevent the DN effect, but L27A, A35S (as in α1 pumps) or K31P were not DN. Thus, the α2 sorting sequence corresponds to two turns of an α‐helix with attachment sites at Leu‐27 and Ala‐35. Loss of the DN effect by the K31P mutant is consistent because Pro disrupts α‐helices. Adenoviral transfection of α2(1‐130)/α1‐f chimera in astrocytes knocks down α2 Na+ pumps and, simultaneously, Na/Ca exchanger 1 (NCX1) expression, with a ~12 hr half‐time. NCX1 silencer RNA and α2 Na+ pump knock‐out mouse data also reveal linkage of α2 and NCX1 expression in astrocytes.The homologous neuronal α3 Na+ pump N‐terminal sequence is: LDDLKKEVA. We generated plasmids coding for the N‐terminal 120 AAs of α1 and α3 Na+ pumps with mCherry and flag tags [mCherry‐α1(1‐120)‐f and mCherry‐α3(1‐120)‐f]. Transfection and expression of the latter, but not the former, was DN for α3 Na+ pumps in rat primary cultured hippocampal neurons. This suggests that LDDLKKEVA is the α3 sorting sequence.
Functional expression of the epithelial sodium channel delta subunit in human respiratory epithelial cells
Authors: Schwagerus Elena , Buckley Stephen T , Ehrhardt Carsten 
Published: June 2021

The expression and function of the epithelial sodium channel δ subunit (δ‐ENaC) as well as its contribution to ion and fluid transport in human respiratory epithelial cells.Expression of δ‐ENaC was investigated in human alveolar (A549) and bronchial (Calu‐3, 16HBE14o‐) epithelial cell lines on genomic and protein levels. Reverse transcriptase and real‐time PCR were carried out as well as analysis by Western blot and immuno‐confocal laser scanning microscopy (CLSM). Ussing chamber studies were performed with Calu‐3 monolayers using Evans blue at 10 – 50 μM.Delta‐ENaC expression was confirmed in all cell lines on mRNA level with higher abundance in A549 and 16HBE14o‐ than in Calu‐3 cell monolayers. Generally, mRNA levels of δ‐ENaC were weaker than that of α, β and γ ENaC subunits. By Western blot and CLSM δ‐ENaC protein was detected in all cell lines, with the highest expression found in Calu‐3 and 16HBE14o‐ cells. CLSM also revealed the δ‐ENaC subunit located in the apical aspect of cellular membranes. Evans blue exhibited a concentration‐dependant increase of ISC up to 650% at 50 μM.These results indicate that ä‐ENaC is functionally expressed in human respiratory epithelial cell monolayers. Further experiments will investigate its role in ion and fluid transport processes across the respiratory epithelial barrier.ES is funded by an IRSCET postgraduate scholarship.
Regulation of ENaC by proprotein convertase subtilisin/kexin type 9(PCSK9)
Authors: Sharotri Vikas , Snyder Peter M 
Published: June 2021

The epithelial Na+channel ENaC forms a pathway for Na+ absorption across epithelia. Various proteases regulate ENaC by altering its activity. Here we tested the effect of the serine protease proprotein convertase subtilisin/kexin type 9 (PCSK9) on ENaC. We found that PCSK9 and ENaC interact using a coimmunoprecipitation assay. PCSK9 decreased amiloride sensitive Na+ current in Xenopus oocytes and in FRT epithelia expressing αβγENaC. This resulted from decreased ENaC cell surface expression. Using a cycloheximide chase assay we found that PCSK9 accelerated degradation of ENaC. The proteasomal inhibitor ALLN reversed this decrease in ENaC protein. In contrast, PCSK9 had no effect on endocytosis or degradation of the cell surface pool of ENaC. Together, the data indicate that PCSK9 regulates ENaC by increasing its degradation in the biosynthetic pathway.Supported by funding from American Heart Association 0920147G (VS) and NIH HL058812 (PMS)
Effects of cytochrome P450 metabolites of arachidonic acid on the epithelial sodium channel (ENaC)
Authors: Pavlov Tengis S , Ilatovskaya Daria , Roman Richard J , Staruschenko Alexander 
Published: June 2021

Sodium reabsorption via ENaC in mammalian distal nephron plays a central role in body fluid volume regulation. It was previously shown that arachidonic acid (AA) significantly decreased ENaC activity in cortical collecting duct (CCD) cells and that effect of AA is mediated by 11,12‐EET. CYP450 metabolites of AA have been implicated in the control of sodium and water excretion. LC/MS/MS analysis revealed the presence of EET‐8,9, EET‐11,12, EET‐14,15 and 5‐HETE, 12/8‐HETE, 15‐HETE, but not 20‐HETE in cultured renal collecting duct principal mpkCCDc14 cells. Coexpression of Cyp2c8 but not Cyp4a10 with all three ENaC subunits in CHO cells significantly decreased amiloride‐sensitive current density in whole‐cell experiments. Single channel analysis revealed that EET‐8,9, EET‐11,12 and EET‐14,15 acutely decrease ENaC activity in the mpkCCDc14 cells. In contrast, neither 5‐HETE nor 12/8‐HETE or 15‐HETE have effect on ENaC activity. We conclude that EET‐8,9, EET‐11,12 and EET‐14,15 are important for maintaining transepithelial Na+ transport and ENaC activity.Supported by ASN, AHA and MCW Research Affairs Committee.
Interaction Of COMMD Family Proteins With The Epithelial Sodium Channel.
Authors: Liu Yongfeng , Swart Marianne , McDonald Fiona 
Published: June 2021

The COMMD (Copper Metabolism gene Murr1 Domain) family of proteins consists of 10 members, COMMD1‐10. The prototype of the COMMD family, COMMD1, was found to be involved in copper metabolism, NF‐κB signaling and sodium uptake. The classic epithelial sodium channel (ENaC) in the distal kidney is made up of three similar subunits, α, β and γENaC subunits. Another ENaC family member, δENaC, is expressed throughout the brain. Previously our laboratory identified COMMD1 was an ENaC‐interacting protein and inhibited amiloride‐sensitive Na+ transport of the δβγENaC subunits and αβγENaC channels [1].Therefore, we hypothesized other COMMD proteins might (1) bind to ENaC, (2) regulate ENaC and (3) if other COMMD proteins do regulate ENaC activities, COMMD might be involved in the Nedd4‐2 ubiquitination pathway in the regulation of ENaC. Our GST pulldown and co‐immunoprecipitation results have shown that most COMMD proteins bound to α, β, γ and δENaC subunits. Using immunohistochemistry experiments we show that COMMD3 and COMMD9 are both expressed in kidney and overlay with αENaC expression. Functional data from Ussing chamber experiments has shown that COMMD3 and COMMD9 down‐regulate ENaC currents in FRT cells. Therefore COMMD3 and COMMD9 may be novel regulators of ENaC.
ATF3 is activated in ventilator induced lung injury
Authors: Dos Santos Claudia , Akram Ali , Masoom Hussain , Han Bing , Haitsma Jack J. , Peng Claudia , Shan Yuexin , Zhang Haibo , Liu Mingyao , Hai Tsonwin , Slutsky Arthur S. 
Published: June 2021

IntroductionMechanical ventilation (MV) has been shown to cause ventilator‐induced lung injury (VILI). We wanted to identify novel molecular targets involved in reducing the harmful effects of MV (VILI).MethodsTo identify potential novel genes and proteins involved in VILI we i) performed microarray analysis on previously published VILI animal model data, ii) exposed human bronchial epithelial cells (Beas‐2b) in vitro to mechanical cyclic stretch (30 cycles/min for 4 hours), iii) performed western blot analysis on these stretched and non‐stretched cells, and iv) validated the regulation of the newly discovered gene(s) in vivo using wild type (WT) and ATF3 (Activator transcription factor 3) knock‐out mice (ATF3 KO). These two groups of mice were exposed to either injurious or non‐injurious ventilation strategies in combination with LPS or saline.ResultsATF3 was identified by microarray analysis as one of the main genes involved in VILI. The presence of this factor was validated in vitro in the mechanically cyclic‐stretched Beas‐2b cells. Western blot assays confirmed expression of ATF3 in the stretched cells and their absence in the static cells. We further confirmed the regulation of ATF3 using WT and ATF3 KO mice in a model of VILI in vivo. ATF3 KO mice exposed to LPS in combination with injurious MV had more inflammation than WT mice of similar treatment or mice treated with saline.ConclusionExpression of ATF3 is stretch dependent and this gene seems to confer protection in combination with MV. Specific targeting of this factor in clinical settings could reduce VILI in patients receiving MV.
Functional expression of the hypo‐osmosensitive channels TRPV4 and TRPM3 in mouse airway epithelium
Authors: Lembrechts Robrecht , Brouns Inge , Schnorbusch Kathy , Pintelon Isabel , Timmermans Jean‐Pierre , Adriaensen Dirk 
Published: June 2021

TRPV4 and TRPM3, members of the transient receptor potential channel family, are membrane proteins that mediate Ca2+ entry in cells upon application of hypo‐osmotic solutions. This study aimed at investigating the expression and functionality of these osmosensitive channels in the epithelium of mouse intrapulmonary airways by combining confocal Ca2+ imaging and multilabel immunohistochemistry.Immunostaining revealed that TRPV4 and TRPM3 are colocalized on both ciliated cells and Clara cells, the most common cell types in the mouse airway epithelium, but also that the neuroendocrine cells, grouped as neuroepithelial bodies, are negative. Confocal live cell imaging of precision cut vibratome slices of mouse lungs loaded with Fluo‐4 showed that short term (30s) perfusion with a hypo‐osmotic solution (200 mOsm) results in a clear and reversible rise in intracellular Ca2+ in all Clara and ciliated cells.Both TRPV4 and TRPM3 channels may allow the cells to react to changes in extracellular osmolarity and therefore potentially play a central role in airway epithelial homeostasis by modulating epithelial water transport. The presented approach opens interesting new perspectives for unraveling functional aspects of diverse cell and tissue types in control lungs and disease models.Support: IWT fellowship SB/81162 (R.L); FWO grant G.0081.08 (D.A.); UA grants GOA BOF 2007 (D.A.) and KP BOF 2006 (I.B.)
LPS‐mediated Inflammation Alters Neonatal Central Respiratory Control
Authors: Balan Kannan V , Kc Prabha , Mayer Catherine A , Wilson Christopher G , Miller Martha J , Martin Richard J 
Published: June 2021

Perinatal inflammation is implicated in precipitating lung and brain injury in preterm infants, both of which are associated with impaired respiratory control (IRC). The mechanisms that link peripheral inflammation to central respiratory control have not been well studied. We hypothesized that an endotoxin‐induced inflammatory response in the immature lung triggers cytokine upregulation in the brainstem resulting in IRC. Lipopolysaccharide (LPS, 0.1μg/g) or saline was administered intratracheally into anesthetized vagal‐intact or denervated 10–13 day old F344 rat pups. In experiment 1, 2h post LPS, pups were sacrificed and the brainstem was assessed for mRNA expression of proinflammatory cytokines by RT‐PCR. In experiment 2, the effect of LPS exposure on ventilatory responses (VR) in carotid sinus nerve (CSN) intact and denervated rat pups was quantified using unrestrained whole body plethysmography (WBP). Rat pups exposed to LPS exhibited increased IL‐1β and IL‐6 mRNA and vagotomy decreased IL‐1β mRNA. WBP showed an attenuated VR to hypoxia but not hypercapnia in LPS exposed rat pups. LPS‐instilled CSN denervated pups also showed blunting of the hypoxic response. Our data suggest that pulmonary inflammation during early development and the resulting brainstem cytokine responses are associated with impaired hypoxic VR, which cannot be attributed to altered sensory trafficking from the carotid body.Supported by: HL50527, HL056470
Multiphoton calcium imaging of methylxanthine‐reversal of opioid depression of inspiratory‐related pre‐Bötzinger complex rhythm in newborn rat brainstem slices
Authors: Ruangkittisakul Araya , Panaitescu Bogdan , Ballanyi Klaus 
Published: June 2021

Breathing depression by opioids is reversed by methylxanthines (e.g. theophylline). Opioids block Ca2+ channels while methylxanthines cause Ca2+ release from cellular stores at high doses. We studied in newborn rat brainstem slices with 2photon imaging whether opioid block of inspiratory center pre‐Bötzinger Complex (preBötC) and its recovery by theophylline involved cytosolic Ca2+ change.DAMGO blocked bursting and related Ca2+ rises in most preBötC neurons. Ca2+ baseline fell by ~10% in these cells, but not in presumed astrocytes. Theophylline and phosphodiesterase‐4 (PDE4) blocker Rolipram restored rhythm with no effect on Ca2+ baseline. Ca2+ store blocker cyclopiazonic acid did not raise the baseline in preBötC neurons, but in astrocytes. Ca2+ rises became irregular upon block of L‐type Ca2+ channels with nifedipine or Ni2+(0.1mM) without effect on Ca2+ baseline or rhythm. At 2.5mM, Ni2+ blocked rhythm and Ca2+ baseline fell by 10%.SummaryOpioid depression is associated with a Ca2+ fall in preBötC cells that is not due to block of L‐type Ca2+ channels whereas actions of methylxanthines or PDE4 blockers do not involve a major change in Ca2+.Supported by CIHR, AHFMR, CFI‐ISRIP, MFN Health
Breathing Efficiency in Systolic Heart Failure: Impact of Hyperventilation and Dead Space
Authors: Woods Paul Robert , Olson Thomas P , Frantz Robert P , O'Malley Kathy A , Hulsebus Minelle L , Johnson Bruce David 
Published: June 2021

BackgroundPatients with heart failure (HF) often develop pulmonary system abnormalities. Specifically, an abnormal ventilation relative to metabolic demand (VE/VCO2, ventilatory efficiency) during exercise has been shown to be an important marker of disease severity and prognosis. The purpose of this investigation was to focus on the factors that underlie the abnormal breathing efficiency in this population.Methods14 controls (10 male) and 40 (36 male) mod‐severe HF patients, age 52±12 and 56±8 yrs, respectively, performed graded exercise on a cycle ergometer. Gas exchange and blood gas measurements were made during submaximal exercise (~65% peak VO2) and used to quantify the influence of hyperventilation (PaCO2) and dead space ventilation (VD) on VE/VCO2.ResultsThe VE/VCO2 ratio was lower in controls (30±4) than in HF (45±9, p<0.001) due to a lower PaCO2 and higher VD/VT that contributed an estimated 29% and 71%, respectively, to the increased VE/VCO2 (p<0.05). The elevated VD/VT in HF was in turn the result of a tachypneic breathing pattern (lower VT, 877±306 vs 2003±504ml, p<0.01) in the presence of a relatively normal VD (9.6±3.8 vs 11.9±5.7 L/min, p=0.094).ConclusionThe abnormal ventilation in relation to metabolic demand in HF during exercise is predominantly due to alterations in breathing pattern, rather than hyperventilation or changes in ventilation/perfusion relationships. HL71478
Regulation of arterial H+ during exercise in man
Authors: Wasserman Karlman , Sun Xing‐Guo , Stringer William W. , Beaver William L 
Published: June 2021

Despite a relatively high H+ production compared to total body H+, arterial H+ concentration ([H+]a) is well regulated during exercise. This is achieved by ΔVE of exercise. The study objective was to quantify [H+]a regulation below and above the lactic acidosis threshold (LAT) during exercise.MethodsTwo progressively increasing cycle exercise protocols were performed.ResultsIn the first, we related VE to [H+]a and PaCO2. All subjects developed an acidosis with exercise, respiratory below and metabolic above LAT. ΔVE correlated with Δ[H+]a up to the LAT, (ΔVE/Δ[H+]a ¡Ö15L/nM). The increase in [H+]a (2–3 nM) was attributable to PaCO2 increase. ΔVE correlated with ΔPaCO2 only up to the LAT. Above LAT, VE increased less with increasing [H+]a. The 2nd protocol compared a 4‐fold difference in work rate increase to determine the effect of time on the VE response. Below LAT, VE tracked VCO2 with equal sensitivity for the two rates. Above the LAT, VE was steeper for the more slowly increasing work rate.Conclusion[H+]a is regulated by sensitive ventilatory control mechanisms. ΔVE/Δ[H+]a below the LAT is of CO2 origin, rapid and steep, not‐detectibly affected by time. In contrast, ΔVE/Δ[H+]a above LAT is less and steeper with time. The difference between the ΔVE responses to the 2 H+ species is likely due to differences in chemoreceptor permeability of lipophilic and lipophobic forms of H+.
20 wk resistance training (RT) in 70 y olds improves glucose handling and leg blood flow (LBF) responsiveness to feeding and exercise‐plus‐feeding without reversing age‐related declines in protein kinase B (PKB) responses or increasing endothelial markers
Authors: Phillips Bethan , Hildebrandt Wulf , Williams John , Atherton Philip , Rankin Debbie , Smith Kenneth , Macdonald Ian , Greenhaff Paul , Rennie Michael 
Published: June 2021

Ageing blunts glucose tolerance and the LBF responses to exercise and feeding. We hypothesized that 20 wk RT would improve these and tested this in studies of healthy, normal weight (BMI 23–28 kg·m−2) young (Y) (n=12, 25±3 y, means±SE), middle‐aged (M) (n=12, 50±3 y,) and old (O) (n=8, 70±4 y) men and women (≈50:50). LBF was measured (Doppler) and muscle biopsies taken in post‐absorptive (PA) conditions and 150 min after beginning intermittent feeding (Fortisip: 16% protein, 49% carbohydrate, 35% fat; ≈6.5 kJ−1·kg−1·30 min−1), with one leg rested and the other recovering from exercise (6×8 reps, 75% 1RM). In O, plasma [glucose] before RT was greater (P≤0.05) than in Y in both PA (5.86±0.15 vs. 5.02±0.19 mM) and peak fed (8.82±0.70 vs. 7.94±0.42 mM) conditions and this was associated with blunted PKB kinase responses to feeding in O (+63% vs. +363%; P≤0.01). However, after RT, post‐feed glucose AUC fell in all (Y, −41±3; M, −34±3; O, −22±1 mM·min) without improvements in PKB responsiveness. After RT, LBF responses to exercise+feeding in M and O were enhanced (M: 147±32% vs. 101±16%; O: 115±47% vs. 59±15%, both P≤0.01) without changing whole‐muscle abundance of CD31, eNOS or α1/β2‐adrenoceptor proteins. Thus, RT rejuvenates glucose handling and LBF responsiveness in older people without resensitization of PKB or increases in endothelial markers.Support: UK BBSRC, EC EXEGENESIS
Are international guidelines for physical activity sufficient to improve health status?
Authors: Foulds Heather J A , Bredin Shannon S D , Charlesworth Sarah A , Ivey Adam C , Warburton Darren E R 
Published: June 2021

PURPOSES1) To determine whether international physical activity guidelines are adequate to change health status in inactive adults. 2) To examine directly the dose‐response relationship between exercise volume and indicators of health status.METHODParticipants (N=65; age=44±12 yr) were assigned randomly to one of five 13 week exercise training programs: 1) 10 min brisk walking 1x/week (n=11), 2) 10 min brisk walking 3x/week (n=10), 3) 30 min brisk walking 3x/week (n=16), 4) 60 min brisk walking 3x/week (n=11), and 5) 30 min running 3x/week (n=17). Health indicators were recorded before and after training including anthropometry, fasting glucose, total cholesterol (TC), high density lipoproteins (HDL) and blood pressure.RESULTSOne 10 min weekly bout of exercise was sufficient to cause significant changes in select measures of health status. There were similar changes in fasting glucose, TC/HDL ratio, and diastolic blood pressure to that seen with higher volumes of activity. However, higher volumes of exercise (30 min or more of brisk walking per week) were required to change body mass significantly.CONCLUSIONSExercise volumes as low as 10 min of brisk walking per week can lead to improved indicators of health status. However, higher volumes of exercise appear to lead to additional health benefits.Research Support Source: Michael Smith Foundation for Health Research, CIHR, NSERC Canada.