bluuebunny/crossref_metadata_2025_split huggingface dataset:
Hsd11b2 Targeting in the rat
Authors: Mullins Linda Jane , Peter Audrey , Kenyon Christopher , Sourgia‐Koutraki Yolanda , Bailey Matthew , Mullins John 
Published: June 2021

We have used zinc finger nucleases (ZFNs) to target the rat Hsd11b2 gene by microinjection of ZFN mRNAs into single cell rat embryos. We generated multiple targeted alleles predicted to truncate the gene transcript. Having segregated the alleles by breeding, detailed metabolic and telemetric analyses of one line was undertaken.The Hsd2ZFNΔ120 line carries a 120bp deletion, removing half of exon 2. Animals homozygous for the deletion show symptoms of the syndrome of apparent mineralocorticoid excess (SAME). They are polydypsic and polyuric from an early age, but have increased haematocrit suggesting volume depletion. The increase in Na+/K+ ratio on sodium loading (0.3% Na+ to 3% Na+) is blunted, compared to controls.Homozygotes are hypertensive on 0.3% Na+ diet with a mean arterial blood pressure of 180mmHg (control MABP −115mmHg), and demonstrate a distinct lack of circadian rhythm (“non‐dippers”), though circadian rhythm for heart rate is normal. Sodium restriction (0.03% Na+) reduces BP to 140mmHg, but enhances its circadian rhythm. Meanwhile the concomitant tachycardia is sustained. We are currently investigating heart rate variability under different salt diet regimes. This rat model will extend experimental possibilities for exploring the mechanism underlying SAME.
Tumor Necrosis Factor induces cerebral edema and increased cerebrovascular permeability in normal pregnant rats
Authors: Warrington Junie Paula , Ryan Michael J , Drummond Heather A , Spradley Frank T , Granger Joey P 
Published: June 2021

Cerebrovascular events contribute to ~40% of preeclampsia/eclampsia deaths and preeclamptic patients commonly present with neurological symptoms. Our laboratory recently showed that placental ischemia, induced by reducing uterine perfusion pressure, closely mimics preeclamptic patients resulting in cerebral edema and increased inflammatory cytokines such as tumor necrosis factor (TNFα). In this study, we examined whether TNFα, infused via intraperitoneal implanted mini‐osmotic pumps (100ng/day from gestational day 14 to 19), contributes to cerebral edema and blood‐brain barrier (BBB) disruption in normal pregnant rats. Mean arterial pressure, measured via carotid catheters, was significantly increased in TNFα infused rats (112 vs. 107 mmHg; p=0.01). Brain water content, calculated as (wet weight ‐ dry weight)/ wet weight × 100, was also significantly increased in TNFα infused rats (78.2 vs. 77.9%; p=0.01). BBB permeability, assayed using the Evans blue extravasation method, was increased in the hippocampus of TNFα‐infused rats (p<0.05) but was unchanged in the striatum, cerebellum, and anterior brain. Our results indicate that TNFα released from ischemic placentas, could mediate cerebral edema through increased BBB permeability. TNFα may be an important clinical target for preventing cerebrovascular complications associated with preeclampsia.Funding: NIH Grant HL108618
Clinical and Genetic Determinants of Blood Pressure Under Ambulatory Conditions on Days With and Without Acute Exercise
Authors: Ash Garrett Igo , Eicher John D. , Tsongalis Gregory J. , Thompson Paul D. , Pescatello Linda S. 
Published: June 2021

OBJECTIVETo identify clinical and genetic factors accounting for the variability in the BP response to acute bouts of dynamic exercise of varying intensity.METHODS48 men (X±SEM 43.9±1.4yr, 29.5±0.7kg•m−2) with hypertension [systolic (SBP)/diastolic (DBP) 145.7±1.5/85.7±1.1mmHg] completed 3 randomly assigned experiments: a cycle exercise bout at LIGHT and VIGOROUS intensity and a CONTROL session of seated rest. Subjects left the laboratory wearing an ambulatory BP monitor. We obtained blood samples for the fasting cardiometabolic profile and genotyping. Multiple variable regression tested correlates of the daytime ambulatory BP response following exercise and CONTROL.RESULTSClinical (30/18%) and genetic (0/6%) factors accounted for 30/24% of the variability in the SBP/DBP response following LIGHT (p=0.01); 27/52% [clinical (13/31%) & genetic (14/21%)] in the SBP/DBP response following VIGOROUS (p=0.01); and 18/29% [clinical (18/29%) & genetic (0/0%)] in the SBP/DBP response following CONTROL (p=0.02).CONCLUSIONOur findings indicate that for every 10% increase in the intensity of a bout of acute dynamic exercise, the genetic contribution accounting for the variability in the SBP/DBP response increased by 1.2/2.0% (R2=0.779, p=0.08/R2=0.967, p=0.01), respectively. No such trends were found with clinical factors.Supported by American Heart Association Grant‐in‐Aid # 0150507N.
Angiotensin Receptor Blocker (ARB) prevents albuminuria and cardiac hypertrophy but does not protect the cuffed kidney in murine 2K1C hypertension
Authors: Hartono Stella , Knudsen Bruce E , Grande Joseph P 
Published: June 2021

Despite proven efficacy, ARB therapy reduces glomerular filtration in patients with unilateral renal artery stenosis (RAS). The objective of this study was to determine whether ARB exacerbates renal injury in 2K1C hypertension. RAS was established in mice by placement of a cuff on the right renal artery, which reduces blood flow by 70%. Both Hydralazine (H) and Valsartan (ARB) reduced blood pressure to a similar extent. RAS‐mediated cardiac hypertrophy (heart weight 134% sham (S) p<0.001) was prevented by ARB (102% S, p=ns) but not H (116% S, p=0.03); cardiac fibrosis was prevented in both ARB and H. Although ARB did not exacerbate renal atrophy (91.4 ± 2.1% atrophy vehicle (V) vs 75.3 ± 9.5% ARB, p=ns), H reduced renal atrophy (91.4 ± 2.1% V vs 28.4 ± 13.5% H, p<0.01). The contralateral kidney underwent compensatory hyperplasia (123% S; p<0.001), which was blunted with ARB or H treatment. No significant histopathologic alterations were observed in the contralateral kidneys of any group. RAS induced proteinuria in V mice. Urine protein excretion in ARB RAS mice was reduced to levels observed in S mice; H did not significantly alter urine protein excretion. Severe renal atrophy was observed in the cuffed kidneys of V mice, which was not exacerbated by ARB. However, ARB reduced proteinuria to baseline levels, indicating that ARB may protect overall renal function in unilateral RAS.These studies were supported by NIH grant P01 HL85307.
Using Drosophila to identify inhibitors of oxalate kidney stone formation
Authors: Tanamoto Nana , Hirata Taku , Cabrero Pable , Lieske John C , Ritman Erik L , Dow Julian AT , Romero Michael F 
Published: June 2021

BackgroundKidney stones are an expensive and painful health problem with calcium oxalate (CaOx) being 70% of cases. We developed a Drosophila model of CaOx stones [Hirata et al., AJP‐Renal Physiol, 2012]. This work showed that dPrestin (Slc26a5/a6) exchanges Cl− for oxalate (Ox). Thiosulfate (S2O3) and tannic acid (TA) are implicated to inhibit CaOx crystals in supersaturated urine. Here, we report transporter, renal tubule and whole body effects of S2O3 and TA on crystal formation.MethodsS2O3 and TA transport effects were examined by voltage clamping of dPrestin‐expressing Xenopus oocytes. Cis‐inhibition of oxalate transport was also examined in (a) dissected Malpighian tubules (MT) or flies fed ox‐enriched food plus S2O3 or TA. MT CaOx crystals were viewed by DIC, or whole flies by microCT.ResultsdPrestin transports S2O3 but not TA, yet both inhibited Ox transport (Ki(S2O3)~10mM; Ki(TA)~100μM). S2O3 competitively inhibits while TA blocks dPrestin. MT treated with S2O3 or TA ↓ crystal formation compared to MT soaked in Ox only. Flies fed with S2O3 or TA also showed ↓ CaOx in MTs.ConclusionsS2O3 and TA are both inhibitors of CaOx crystallization. These experiments show that these inhibitors have distinct mechanisms of action. Our Results indicate that (a) this Drosophila model can be used as a chemical screening assay for CaOx stone prevention and (b) TA, due to low Ki, might be a reasonable therapy for certain CaOx stones patients.[DK83007, DK92408]
Feedback inhibition of the epithelial Na+ channel (ENaC) during acute sodium loading in vivo
Authors: Patel Ankit Bharat , Frindt Gustavo , Palmer Lawrence G. 
Published: June 2021

The in vivo role of feedback inhibition of ENaC by intracellular [Na+] ([Na+]i) was studied by investigating the effect of amiloride during an acute sodium load to Na+‐deprived rats in the presence of an aldosterone mini‐pump. Amiloride blocks sodium entry into principal cells of the cortical collecting ducts (CCD) and presumably decreases [Na+]i. ENaC activity was evaluated by measuring amiloride‐sensitive currents (INa) in split‐open CCDs using whole‐cell patch clamp. ENaC surface expression was assessed by an in vivo biotinylation protocol. A four‐hour acute sodium load decreased INa by 70%. The presence of amiloride did not significantly change INa after an acute sodium load. Whole‐kidney expression of cleaved α, β, and cleaved γ ENaC decreased with an acute sodium load by 2.6, 3.4, and 6.2 fold, respectively. In addition, cell‐surface expression of β and cleaved γ ENaC decreased with an acute sodium load by 2.5 and 4.7 fold, respectively. Amiloride increased whole‐kidney expression of cleaved α and cleaved γ ENaC after an acute sodium load by 2.1 and 3.0 fold, respectively, and cell‐surface expression of β and cleaved γ ENaC by 2.2 and 5.2 fold, respectively. These results suggest that [Na+]i can regulate ENaC expression in vivo but changes in factors other than [Na+]i and aldosterone are responsible for down regulating ENaC activity in response to an acute sodium load.NIH RO1‐DK59659 (LGP) and T32‐GM07739 (ABP).
ASK1 and p38 MAP kinase regulate proteolytic processing of ENaC
Authors: Cook David Ian , Lee Il‐Ha , Song Sung‐Hee , Dinudom Anuwat 
Published: June 2021

These Ussing chamber studies were undertaken to investigate the mechanism by which hydrogen peroxide (0.5 mM) inhibits mouse epithelial Na+ channels (ENaC) transfected into FRT cell monolayers.We found the inhibition was blocked by expression of a dominant negative construct of the kinase ASK1, and was replicated by expression of a constitutively active mutant of ASK1. The effects of both hydrogen peroxide and of the constitutively active mutant of ASK1 were blocked by the p38 MAP kinase inhibitor, SB203580, and by expression of a dominant negative mutant of p38 MAP kinase.Western blotting with an antibody for phosphorylated p38 MAP kinase confirmed that ASK1 modulates the amount of phosphorylated p38 MAP kinase. Epithelial Na+ channels are regulated by the Nedd4–2 system that controls their surface expression. We found, using surface biotinylation methods, however, that neither ASK1 nor p38 MAP kinase affected surface expression of the channels.Epithelial Na+ channels are normally activated by proteolysis of the α‐ and γ‐subunits during their intracellular processing. When we used ENaC subunit constructs in which the N‐ and C‐terminals are labelled with V5 or FLAG tags to examine the effect of the kinases on proteolytic processing, we found that they inhibit proteolytic cleavage of both α‐ and γ‐ENaC.In summary, we have found that hydrogen peroxide inhibits epithelial Na+ channels expressed in FRT cell monolayers via the kinases ASK1 and p38 MAP kinase, which reduce the activity of the channels by inhibiting the normal activation of the channels by proteolysis.
Activation of rat and human organic anion transporter 1 and 3 by transcription factor B‐cell CLL/lymphoma 6 (BCL6)
Authors: Henjakovic Maja , Wegner Waja , Burckhardt Birgitta C. , Burckhardt Gerhard 
Published: June 2021

In rat kidneys, organic anion transporter 1 (Oat1) and Oat3 are male‐dominant and testosterone‐dependently expressed. Sex‐differences in the secretion of several drugs known to be substrates of human OAT1 and OAT3 suggest a male‐dominant expression of these transporters also in humans. The aim of this study was to identify the regulators of sex‐dependent rat Oat1 and Oat3 transcription and the effect of these regulators on human OAT1 and OAT3 promoters.RNA was isolated from renal cortical slices of four adult rats per sex. Expression profiling was done using SurePrint G3 rat GE 8×60K microarray. Selected candidate genes were verified using real‐time PCR. Luciferase assays were carried out by transient transfection of opossum kidney (OK) cells with promoter fragments and expression vectors.Our results suggest that the male‐dominant expression of Oat1 and Oat3 is not directly regulated by the testosterone/androgen receptor‐complex. The human OAT1 and OAT3 promoters were also not affected by testosterone. The transcription factor B‐cell CLL/lymphoma 6 (BCL6) was male‐dominantly expressed and activated the promoters of Oat1/Oat3 and OAT1/OAT3, independent of predicted BCL6‐binding sites, possibly mediated by the interaction with other transcription factors.BCL6 constitutes a promising candidate gene for the sex‐dependent regulation of rat Oat1/Oat3 and human OAT1/OAT3 transcription.
Rosetta modeling of the inner KCa3.1 pore, a hotspot for small molecule modulation
Authors: Jenkins David Paul , Wulff Heike , Yarov‐Yarovoy Vladimir 
Published: June 2021

The intermediate‐conductance KCa3.1 and the related small‐conductance KCa2 channels constitute potential pharmacological targets for immunosuppression, fibroproliferative disorders, hypertension and various neurological diseases. However, there currently is no available crystal structure or molecular model of these medically relevant channels that could be used for structure‐assisted drug design. Using the Rosetta modeling method we constructed a model of the KCa3.1 pore and energy minimized it in the presence of the KCa3.1 blocker TRAM‐34. The lowest energy confirmation of the KCa3.1 complex with TRAM‐34 identified Thr250 and Val275 as interaction sites, two residues which had been previously demonstrated to completely abolish triarylmethane sensitivity when mutated to the corresponding residues in KCa2.3. The model further correctly identified the interaction of the benzothiazinone‐type KCa3.1 blocker NS6180 with Thr250 and Val275 in KCa3.1 and modeled binding of the negative KCa2 channel gating modulator NS8593 to a KCa3.1‐Thr250Ser‐ Val275Ala mutant channel, which is blocked more potently by NS8593 than the WT‐KCa2.3 channel. The Rosetta KCa3.1 model can be used to understand the molecular mechanism of action of KCa2/3 channel modulators and assist with drug design of blockers and gating modulators.Supported by RO1 GM076063 (to H.W.) and UC Davis startup (to V.Y.).
Palmitoylation of large conductance calcium‐ and voltage activated potassium (BK) channel β‐subunits
Authors: Bi Danlei , Chen Lie , McClafferty Heather , Tian Lijun , Shipston Mike 
Published: June 2021

BK channels control diverse physiological functions including vascular tone and neuronal excitability. In these tissues, BK channel pore‐forming α‐subunits (α) typically assemble with regulatory β‐subunits (β). Though the properties of α are regulated by a range of post‐translational modifications (PTMs) like S‐acylation, whether β are also modified by this PTM is unknown. We predicted that both β1 and β4 were palmitoylated and confirmed it by 3H‐Palmitate incorporation and acyl‐RAC assays. We investigated the effect of palmitoylation of the β1 and β4 on the trafficking and activity of theα ZERO variant in HEK293 cells by quantitative immunofluorescence imaging and patch clamping. The cell surface expression of ZERO was significantly enhanced upon co‐expression of either β1 or β4 (>;100% by β1 and >;50% by β4). Depalmitoylation of β4, but not β1, by mutation of a single cysteine residue to alanine, significantly reduced co‐expressed ZERO variant surface expression. In macropatch recordings, co‐expression of wild‐type β1 and β4 significantly left‐shifted the V0.5max of the channels as expected at 10uM intracellular free calcium. However, depalmitoylation of either βdid not significantly affect the normal left‐shift of V0.5max. These data suggest that palmitoyaltion is crucial for trafficking of BKα upon β4, but not β1, but not their effect on V0.5max. The functional role of β1 pamitoylation is to be determined.The work is supported by Wellcome Trust and The China Scholarship Council.
Patch clamp screening of a7 nAChRs and the temperature dependence of allosteric modulation on nAChR and GABAA
Authors: Haedo Rodolfo Jorge 
Published: June 2021

The nicotinic acetylcholine receptor (nAChR) is a member of the ligand‐gated ion channel superfamily which includes GABAA, 5HT3, NMDA and glycine receptors. Neuronal nAChR are found in many areas of the mammalian peripheral and central nervous systems including the autonomic ganglia, hippocampus and thalamus. Despite their relative scarcity of expression in the CNS, when activated, nAChR have been shown to have profound effects on cognitive performance, locomotor activity, cardiovascular function and pain perception, amongst others.Additionally, the effect of PNU‐120596 was investigated at physiological temperatures (37 °C), where the current amplitude was significantly smaller, compared with using the same concentrations applied at room temperature. This supports the idea of a strong temperature dependence of the allosteric modulation by PNU‐102596 on nAChRs, also reported by others (Sitzia, Front. Pharmacol. 2011 2: 81). More importantly, we demonstrate for the first time an equal temperature dependence of GABAARs positive allosteric modulators, such as Diazepam, suggesting a general temperature dependence on positive allosteric modulation of ligand‐gated ion channels.
9‐Phenanthrol Inhibits Calcium Oscillations in HL‐1 Mouse Cardiomyocytes
Authors: Burt Rees , Graves Bridget M. , Li Chaunfu , Williams David L. , Fregoso Santiago P. , Hoover Donald B. , Wondergem Robert 
Published: June 2021

Transient Receptor Potential Melastatin 4 (TRPM4) is functionally expressed throughout the heart and has been implicated as a calcium‐activated nonselective cation channel that mediates membrane depolarization. The functional significance of TRPM4 in regards to Ca2+ signaling and its effects on cellular excitability and pacemaker function remains inconclusive. We show by Fura2 Ca‐imaging that pharmacological inhibition of TRPM4 in HL‐1 mouse cardiac myocytes by 9‐phenathrol (10 μM) decreases Ca2+ oscillations followed by an overall increase in [Ca2+]i. The latter occurs also in HL‐1 cells in Ca‐free solution and after depletion of sarcoplasmic reticulum Ca2+ with thapsigargin (10 μM). Furthermore, by whole‐cell voltage clamp we show that 9‐ phenanthrol reversibly inhibits TRP‐like membrane current; by fluorescence immunohistochemistry we demonstrate that HL‐1 cells display punctate surface labeling with TRPM4 antibody. We conclude that 9‐phenanthrol inhibits TRPM4 ion channels in HL‐1 cells, which in turn decreases Ca2+ oscillations followed by a compensatory increase in [Ca2+]i from an intracellular store other than the sarcoplasmic reticulum. We speculate that the most likely source is the mitochondrion.RB supported by APS summer undergrad fellowship.
Modulation of αENaC mRNA expression under different stresses: Implication of its 3′UTR.
Authors: Migneault Francis , Pascariu Mihai , Dagenais André , Berthiaume Yves 
Published: June 2021

Active sodium transport by the epithelial Na+ channel (ENaC) is important for fluid movement across the alveolar epithelium in the lungs and for the resolution of pulmonary oedema in acute respiratory distress syndrome (ARDS). It was shown that cellular stress induced by lipopolysaccharides (LPS) or cycloheximide (Chx) downregulate αENaC mRNA via different molecular mechanisms. The purpose of this project was to investigate the importance of the αENaC 3′UTR in the mRNA modulation of ENaC. Alveolar epithelial cells were co‐transfected with 3′UTR mutants (V5/ENaC±3′UTR and Luc±3′UTR) inserted in pTRE‐tight vector along with the Tet‐Off vector. The cells were treated with Chx (1.0μM), LPS (15μg/mL) or Actinomycin D (5μg/mL) along with Dox (1μg/mL) to inhibit transcription. We found that 3′UTR deletion caused a ~60% decrease of luciferase activity and expression compared to the complete recombinant. The T1/2 of V5‐αENaC mRNA was 60 min and the deletion of 3′UTR reduced T1/2 to 37min. Chx reduced αENaC mRNA T1/2 to 33min whereas LPS had no effect. Actinomycin D, a transcription inhibitor, increased T1/2 >; 120min. These results suggest that the αENaC mRNA half‐life is shorter then previously estimated and that 3′UTR seems to be important for the stability of αENaC mRNA.Supported by FRSQ, CFC and CIHR.
Effect of Relaxation Breathing on Glycemic Response in Type II Diabetic Subjects
Authors: Settell Megan Leigh , Gregor Angela L , Ackermann Kristin C , Segner Sara A , Anderson Melanie M , Anderson Ashley D , Wilson Ted 
Published: June 2021

BackgroundPharmacological treatments are often used to improve hyperglycemia in Type II diabetics. Stress is associated with development of insulin resistance and hyperglycemia. Non‐pharmacological interventions that reduce stress could improve glycemic response. This study determined if a standard relaxation breathing (RB) exercise could improve glycemic response in non‐insulin dependent Type II diabetics.MethodsSubjects (n=9; 62.0 ± 9.3 yrs; BMI 31.0 ± 6.5) were randomized in a double‐cross over design. Following a 9 hour fast from food/medications and a 30 min acclimation period of control breathing (CB) or RB, baseline blood glucose was measured (0 min) prior to an oral glucose tolerance test drink (OGTT; 50g/240mL). Blood glucose was measured 30, 60, 120 min post OGTT. RB consisted of sequentially increasing exhalation durations (1, 2, …10 sec) repeated every 10 min for 20 min pre‐OGTT and every 10 min for 120 min post OGTT.ResultsBlood glucose at 0, 30, 60 and 120 min was 158 ± 41, 251 ± 66, 307 ± 58, and 231 ± 57 mg/dl for control, and 156 ± 43, 260 ± 67, 309 ± 54, and 214 ± 60 mg/dl for RB. Blood glucose AUC for control and RB was 30683 ± 2201 and 30394 ± 2191. No significant differences between groups were observed for blood glucose, AUC, heart rate, or %SpO2.ConclusionAcute implementation of RB does not appear to improve glycemic response in non‐insulin dependent Type II diabetics.
Phenylephrine‐induced calcium oscillations in human mesenteric arterial smooth muscle
Authors: Tejerina Teresa , Navarro‐Dorado Jorge , Fameli Nicola , Garcia‐Alonso Mauricio , Ramajo Marta , Urraca Concha , Redondo Santiago , Medina Ursula , van Breemen Casey 
Published: June 2021

Phenylephrine (PE)‐induced oscillatory fluctuations in intracellular Ca2+ concentration ([Ca2+]i) of vascular smooth muscle (SM) have been observed in every blood vessel isolated from a wide variety of mammals. Paradoxically, until recently similar observations in humans have proven elusive. In this study, we report for the first time observations of adrenergically‐stimulated [Ca2+]i oscillations in human mesenteric artery SM. We partially characterized these oscillations by using the inhibitory drugs nifedipine, 2‐aminoethoxydiphenyl borate (2‐APB), cyclopiazonic acid (CPA), and HA‐1077. PE and one of these drugs were added to different arterial segments, which had previously been loaded with Fluo‐4 AM in PSS and mounted on a myograph on the stage of a confocal microscope. We observed PE‐induced oscillations in [Ca2+]i, which initiated and maintained vasoconstriction. After PE contraction, all drugs provoked relaxation of the vessel segments, sometimes only partial, and reduced or inhibited oscillations, except CPA, which rarely caused relaxation. These preliminary results point to a potential involvement of the sarcoplasmic reticulum Ca2+ and IP3R in the maintenance of the observed oscillations.FIS PI080920, RECAVA RD06/0014/1007