bluuebunny/crossref_metadata_2025 huggingface dataset:
Multiple red cell volumes (RCV) can be independently determined in sheep in vivo using red cells (RBCs) labeled at 4 biotin densities
Authors: Mock Donald M , Matthews Nell I , Strauss Ronald G , Burmeister Leon F , Schmidt Robert , Widness John A
Published: June 2021

Here we report our further development and validation of a method for independent, simultaneous measurement of RCV in sheep using flow cytometric enumeration of multiple populations of RBCs labeled at distinct biotin densities.

Method

The density of biotin on the surface of the RBCs was incrementally increased to produce 4 discrete populations of biotinylated RBCs (BioRBCs). BioRBCs and RBCs labeled with 14C‐cyanate were infused into the sheep, and blood samples were drawn at 10 min.

Results

At biotinylation reagent concentrations < 96 µg/mL RBC, the biotin method reproducibly determined RCV; mean error = 4.2% versus the reference 14C‐cyanate method (n=2 sheep). RCV values agreed among the densities; mean CV= 2% for 4 sheep. For reagent concentrations > 96 µg/mL RBC, BioRBCs were rapidly removed from circulation; this rapid removal caused an overestimate in RCV.

Conclusions

BioRBCs at densities low enough to survive in the circulation can be used for simultaneous in vivo measurements of RCV in sheep.

Speculation

In humans, BioRBCs can be used to repeatedly measure RCV in the same individual. This method will be useful in the investigation of the physiology and pathophysiology of the pregnant woman, fetus, and infant because only small sample volumes are required and because the individual is not exposed to radiation. NIH P01 HL046925 and Thrasher Research Foundation 02825‐3.
Seed based systematic discovery of specific transcription factor target genes
Authors: Mrowka Ralf , Blüthgen Nils , Kielbasa Szymon , Persson Pontus Börje , Fähling Michael
Published: June 2021

Reliable prediction of specific transcription factor target genes is a major challenge in functional genomics. Current sequence‐based methods yield many false predictions due to the short and degenerated DNA‐binding motifs.

Here, we describe a new systematic genome‐wide approach, the seed‐distribution‐distance method that searches large scale genome wide expression data for genes that are similarly expressed as known targets. This method is used to identify genes that are likely targets and thereby sequence‐based methods can focus on a subset of genes, giving rise to fewer false positive predictions. We show by cross‐validation that this method is robust in recovering specific target genes. Further, this method identifies genes with typical functions and binding motifs of the seed. The method is illustrated by predicting novel targets of the transcription factor NFkB. Among the new targets is optineurin, which plays a key role in the pathogenesis of acquired blindness caused by adult‐onset primary open‐angle glaucoma. We show experimentally that optineurin as well as other predicted genes are targets of NFkB.

We present a robust and reliable method to enhance the genome‐wide prediction of specific transcription factor target genes that exploits the vast expression information available in public databases today. A public web interface for customized searches will be available.
Role of Rac 1 and cAMP in endothelial barrier stabilization and thrombin‐induced barrier breakdown
Authors: Spindler Volker , Baumer Yvonne , Werthmann Ruth , Buenemann Moritz , Waschke Jens
Published: June 2021

Barrier stabilizing effects of cAMP as well as of the small GTPase Rac 1 are well established. Moreover, it is generally believed that permeability‐increasing mediators such as thrombin disrupt endothelial barrier functions primarily via activation of Rho A. In this study, we provide evidence that decrease of both cAMP levels and of Rac 1 activity contribute to thrombin‐mediated barrier break‐down. Treatment of human dermal microvascular endothelial cells (HDMEC) with Rac 1‐inhibitor NSC‐23766 decreased transendothelial electrical resistance (TER) and caused intercellular gap formation. These effects were reversed by addition of forskolin/rolipram (F/R) to increase intracellular cAMP but not by the cAMP analogue O‐Me‐cAMP which primarily stimulates protein kinase A‐independent signaling via Epac/Rap 1. However, both F/R and O‐Me‐cAMP did not increase TER above control levels in the presence of NSC‐23766 in contrast to experiments without Rac 1 inhibition. Next, we tested the role of Rac 1 and cAMP in thrombin‐induced barrier break‐down. Thrombin‐induced drop of TER and intercellular gap formation were paralleled by a rapid decrease of cAMP as revealed by fluorescence resonance energy transfer (FRET). The efficacy of F/R or O‐Me‐cAMP to block barrier‐destabilizing effects of thrombin was comparable to Y27632‐induced inhibition of Rho kinase but was blunted when Rac 1 was inactivated by NSC‐23766.
Extracellular ATP‐induced Akt activation stimulates NO production by NOS3
Authors: Silva Guillermo B , Garvin Jeffrey L
Published: June 2021

ATP simulates NO production in thick ascending limbs by activating purinergic receptors (P2R). However the signaling cascade involved is unknown. We hypothesized that ATP enhances NOS3 activity by stimulating PI3 Kinase and Akt. We measured NO production by thick ascending limb suspensions using the NO‐sensitive dye DAF‐2 DA and Akt activity by fluorescence resonance energy transfer (FRET). ATP (100 μM) increased NO production by 32±8 arbitrary units (AU). In the presence of selective NOS1 and NOS2 inhibitors, 7‐NI (10 μM) and 1400W (100nM), ATP stimulated NO production by 22±9 AU and 31±9 AU, respectively. In contrast, ATP only stimulated NO production by 6±5 AU in NOS3 ‐/‐ mice (p<0.04 vs. wild‐type). In the presence of the PI3 kinase inhibitor LY294003 (10 μM), ATP did not increase NO production (5±2 vs. 28±4 AU; p<0.02). With the PDK1 inhibitor (5 μM), ATP did not induce NO production (6±3 vs. 47±8 AU; p<0.02). ATP alone increased Akt activity by 120±12 FRET units (FU) and only by 11±17 FU in the presence suramin, a P2R antagonist (300 μM; p<0.01). In the presence of the Akt‐selective inhibitor (5 μM), ATP‐induced NO production was blocked by 90±4% (p<0.04). In vivo transduction of thick ascending limbs with a dominant‐negative Akt decreased ATP‐induced NO production by 60±6% (p<0.05). We concluded that ATP stimulates NO production in the thick ascending limb by activating the PI3 kinase/ PDK1/Akt signaling pathway.
Immunofluorescent localization of cystic fibrosis transmembrane conductance regulator (CFTR) and epithelial sodium channel (ENaC) in human sweat ducts
Authors: Brown Mary Beth , Vincent Karla K , Millard‐Stafford Mindy , McCarty Nael A
Published: June 2021

A functional link between the chloride channel cystic fibrosis transmembrane conductance regulator (CFTR) and the epithelial Na+ channel (ENaC), has been suggested based on evidence from electrophysiological and biochemical methods in heterologous expression systems. Their interaction in native tissue is not clearly defined but is implicated in the pathophysiology of cystic fibrosis. The present study utilized immunofluorescence and laser confocal microscopy on cryosections of healthy human skin specimens to characterize CFTR and ENaC localization in sweat ducts. Ductal segment morphology was identified with 4',6‐diamidino‐2‐phenylindole nuclear staining. Throughout the duct there was one to two fold less staining of CFTR compared to ENaC. The proportion of lumen‐specific staining was significantly (one to ten fold) greater for CFTR than for the more ubiquitous ENaC, but varied widely between tissue samples. There is additional corroborating evidence of some co‐localization of CFTR and ENaC confined primarily to the ductal lumen, a principal site of transepithelial NaCl reabsorption. This study is the first example of immunofluorescent staining for CFTR and ENaC localization in native human tissue. Future studies will help identify the spatial relationship between these two channel proteins, and how their potential interaction correlates to fluid and electrolyte transport in health and disease.
Palm domains of the epithelial sodium channel and sodium self‐inhibition
Authors: Chen Jingxin , Sheng Nan , Okumura Sora , Winarski Katie L. , Kleyman Thomas R. , Sheng Shaohu
Published: June 2021

Epithelial Na+ channels (ENaC) play an essential role in body fluid volume homeostasis by mediating apical Na+ entry in epithelial cells. The structure of chicken acid sensing ion channel 1 (cASIC1) homologous to ENaCs reveals that the palm domain offers the sole link between the extracellular domain and the channel pore within a subunit. We investigated the functional roles of the two charged residues within a palm domain β‐sheet (β12) in the mechanism of Na+ self‐inhibition by site‐directed mutagenesis. The Na+ self‐inhibition responses for wild type and mutant channels expressed in Xenopus oocytes were examined by two electrode voltage clamp. Charge reversal mutations at a homologous site (αK561E and γR520E) of β12 significantly reduced the magnitude and speed of Na+ self‐inhibition response. The Na+ inhibition was absent in oocytes expressing the double mutant channel (αK561E βγR520E). Mutation of the other charged residue in γ subunit (γE524R) also significantly suppressed Na+ self‐inhibition whereas the homologous mutation in α(α E565R) did not alter the inhibition. Our results suggest that certain charged residues in αand γpalm domains are critical determinants for Na+ self‐inhibition. Based on our observations and the insights from the cASIC1 structure, we propose that the palm domains play a pivotal role in Na+ self‐inhibition of ENaC. (Supported by NIH ES14701, DK54354 and DK079307)
Differential trafficking of NaPi2a and NaPi2c in response to PTH at the apical surface of renal proximal tubular cells
Authors: Blaine Judith Tamara , Breusegem Sophia , Giral‐Arnal Hector , Wilson Paul , Levi Moshe , Barry Nicholas
Published: June 2021

Disordered phosphorous homeostasis is a significant cause of cardiovascular morbidity and mortality. Parathyroid hormone (PTH) is a critical regulator of phosphorous homeostasis and the kidney plays a crucial role in maintaining phosphorous balance. Renal excretion of phosphorous is controlled by the sodium phosphate cotransporters NaPi2a and NaPi2c. The goal of this study is to examine the mechanisms governing the differential regulation of renal NaPi2a and NaPi2c in response to PTH. Using a novel application of total internal reflection fluorescence microscopy (TIR‐FM) to the apical membrane of renal proximal tubular cells we have found that, in response to PTH, NaPi2a is removed from the apical surface 3 times faster than NaPi2c. These findings have been confirmed using more traditional methods such as confocal microscopy and Western blot analysis. The advantage of TIR‐FM is that it allows selective examination of the apical surface of cells. We are currently using this technique to examine the role of apical brush border components including actin and myosin VI to the differential regulation of NaPi2a and NaPi2c by PTH. Funded by American Heart Association SDG 0830394N.
Tricellulin, a tight junction protein which reduces permeability for macromolecules or ions depending on its localization in tricellular or bicellular tight junctions
Authors: Krug Susanne , Amasheh Salah , Richter Jan , Milatz Susanne , Günzel Dorothee , Westphal Julie , Huber Otmar , Schulzke Jörg , Fromm Michael
Published: June 2021

The tight junction protein tricellulin‐a (TRIC‐a) is preferentially localized in tricellular (tTJ), but also in bicellular tight junctions (bTJ). TRIC‐a was cloned and gradually overexpressed into MDCK II cells. Methods included two‐path impedance spectroscopy, permeability measurements for ions and paracellular markers, confocal microscopy, and freeze fracture EM. After slight overexpression, TRIC‐a was localized mainly in tTJ. There, it did not alter paracellular resistance and permeability for mid‐sized solutes, but substantially reduced permeability for macromolecules between 4 and 10 kDa. As demonstrated by life cell imaging microscopy, macromolecular passage occurred across tTJ. In contrast, strong overexpression led to TRIC‐a localization in both, tTJ and bTJ, as a result of which also the paracellular resistance was increased and the permeability for mono‐ and divalent ions was decreased without charge preference. Ultrastructurally, TRIC‐a overexpression in bTJ led to linear bTJ strands and reduced the number of strand breaks. In conclusion, TRIC‐a proves to be a barrier‐forming TJ protein which, if localized in bTJ, consolidates the bTJ strand meshwork and reduces the permeability for ions and mid‐size solutes. However, at its predominant location, the tTJ, the main effect of TRIC‐a is to impede the passage of macromolecules. (Supported by DFG FOR 721)
SGK1 and insulin reduce the activity of mammalian electrogenic Na+/Monocarboxylate Transporters (SMCTe/Slc5a8)
Authors: López Adriana , Vázquez Noma , Torres Nimbe , Zandi‐Nejad Kambiz , Mount David B , Gamba Gerardo , Tovar Armando R , Romero Michael F , Plata Consuelo
Published: June 2021

SMCTe (SLC5A8) transports several monocarboxylates, including short‐chain fatty acids, ketone‐bodies, pyruvate, lactate, and keto‐isocaproic acid (KIC). We found SMCTe mRNA in zebrafish pancreas (in situ hybridization); other groups have demonstrated that SLC5A8 gene silencing is associated with pancreatic cancer. Extracellular KIC stimulates insulin secretion in pancreas, suggesting a potential role for SMCTe. However, the physiological role of SMCTe in pancreas is unknown. Using RT‐PCR with rat SMCTe‐specific primers, we found that SMCTe mRNA is expressed in rat pancreas. Using Western Blot analysis with specific SMCTe antibodies, we also detected SMCTe protein in rat pancreas (~75 kDa). We next tested the effect of insulin on SMCTe function using 22Na+ uptake in Xenopus oocytes injected with human SMCTe (hSMCTe) or co‐injected with hSGK1 cRNA. Specifically, we evaluated transport 22Na+ uptake elicited by 40 U insulin in presence or absence of KIC. Insulin reduced KIC‐mediated 22Na+ uptake transport by ~25% in hSMCTe‐oocytes. Human SGK1 also reduced KIC‐mediated 22Na+ uptake transport by ~25% in hSMCTe‐oocytes. Co‐expression of hSMCTe and hSGK1 in oocytes did not further reduce KIC‐mediated 22Na+ uptake. These results indicate that the signal transduction pathway by which insulin and SKG1 reduce SMCTe transport activity overlaps.

CONACYT48709MJ(CP), EY017732(MFR), DK070756(DM), DK64635(GG)
Assembly, maturation, delivery, and functional characterization of C. elegans Na,K‐ATPase α‐subunit (i.e. Eat‐6)
Authors: Gatto Craig , Otsuka Anthony J , Yaragatupalli Siddhartha , Artigas Pablo , Templeton Christopher L
Published: June 2021

Na,K‐ATPase (NKA) is responsible for actively transporting 3Na+ (out) and 2K+ (in) of most eukaryotic cells. These ionic gradients play a vital role in cell excitability, contractility, and osmotic balance. C. elegans is valuable genetic tool for studying eukaryotic protein maturation and delivery. Thus, in order to investigate further the membrane delivery of NKA we have constructed fluorescently tagged alpha and beta subunits of the worm NKA (i.e. eat‐6) for expression in C. elegans. The fluorescently tagged constructs were engineered behind a heat shock promoter so protein expression could be controlled. In addition, to verify the role of the α‐subunit, we performed double‐stranded RNA interference against the α‐subunit in worms containing the α‐YFP fusion protein. We found that RNA interference of endogenous worm NKA‐ β decreased the appearance of α‐YFP at the PM, demonstrating the requirement for βin proper NKA delivery consistent with our previous observations in insect cells. Interestingly, C. elegans Eat‐6 has significant sequence divergence from mammalian NKAs (~50% a.a. similarity) and has received limited functional characterization. Therefore, in addition to endogenous assembly and trafficking determinations, we are also functionally characterizing Eat‐6 via heterologous expression in X. laevis oocytes. Supported by NIH grant #GM061583 to CG and GM060190 to CLT.
Immnoprecipitation of Gastric H+/K+ATPase Beta Subunit in Hyperglycemic Mice
Authors: Elmore Zac , Muncie Christopher , Higashi Sakura , Wei Guojun , Chen Xuelian , Nakamura Suguru
Published: June 2021

An upregulation of gastric H+/K+ATPase has been observed in kidney epithelial cells with higher glucose medium. It also has been reported that gastric H+/K+ATPase (HKA) associates with type 1 diabetic patients. We examined the gastric HKA beta subunit protein levels in the isolated kidney medulla (OMCD) in streptozocin‐induced diabetic mice. Hyperglycemia was induced by intraperitoneal injections of streptozocin (50mg/kg) and monitored for 5 days. The mouse kidney medulla was dissected and lysed with a lysis buffer (pH 8). Cell lysate was incubated with monoclonal antibody (HKA beta subunit, 34 kDa) overnight under agitation at 4 °C. After incubation, protein A agarose macrobeads (85 μL) were added to the cell lysate and the lysate/bead mixture was incubated at 4 °C. Beads were washed with lysis buffer to remove protein. The amount of HKA beta subunit was quantified by Bradford protein assay. Serum glucose was 160 ± 25.78 mg/dL in normal mice and was 475 ± 60.90 mg/dL in the hyperglycemic group. At a wavelength of 540 nm, the HKA beta subunit protein absorbance was 0.056 ± 0.019 (n=16) in normal mice. In hyperglycemic mice, the HKA beta subunit absorbance was 0.070 ± 0.024 (n=28, p=0.05). The increased absorbance of gastric H+/K+ATPase beta subunit protein in hyperglycemic mice suggests that hyperglycemia stimulates the production of gastric H+/K+ATPase in the mouse kidney.
Nhe1 is a luminal protein in the mouse choroid plexus epithelium that translocates to the basolateral surface in Ncbe‐null mice
Authors: Praetorius Jeppe , Hübner Christian A , Damkier Helle H
Published: June 2021

The choroid plexus epithelium, CPE, secretes a major fraction of the cerebrospinal fluid, CSF. The Na+;HCO3− transporter, Ncbe/Nbcn2, in the basolateral membrane of CPE cells is important for Na+ dependent pHi increases and probably for CSF secretion. The Na+/H+ exchanger‐1, Nhe1, have been proposed to mediate similar functions in CPE. Nhe1 protein was immunolocalized to the luminal membrane domain in CPE and was absent in CPE from Nhe1 knockout, ko, littermates. The Na+‐dependent pHi recovery in the absence of CO2/HCO3− was dramatically decreased in the Nhe1‐ko mice compared to Nhe1‐wild type, wt, (ko 1.55% of wt, n=6, p=0.02) by fluorescence microscopy. In Ncbe‐ko mice, immunoreactive Nhe1 was targeted to the basolateral membrane, but luminal Na+ dependent pHi recovery was in fact increased in Ncbe‐ko compared to wt littermates (ko 146.1% of wt, n=5, p=0.007). In Ncbe‐wt mice, the luminal Nhe activity was expectedly inhibited by the Nhe blocker, 5′‐(N‐ethyl‐N‐isopropyl)‐amiloride (EIPA, 10µM), while in Ncbe‐ko, Nhe activity was insensitive to the inhibitor (ko 939.0% of wt, n=5, p=0.036). This indicates that a luminal, EIPA‐insensitive Nhe was induced and that EIPA‐sensitive Nhe activity was basolateral in Ncbe‐ko CPE. We speculate that the Nhe1 translocation in Ncbe‐ko CPE reflects a compensatory response, which provides the cells with better means of regulating pHi in the absence of Ncbe.
At LSU Health Sciences Center, Fresh is Best
Authors: Whitworth Richard H.
Published: June 2021

At LSUHSC, fresh unembalmed cadaveric specimens are the best for enhancing the educational experiences of medical students, residents and practicing clinicians. Fresh cadaveric tissue closely mirrors that of the living patient. The appearance, texture and pliability of fresh tissues make them ideal for teaching anatomy and operative procedures at all levels of medical education. The proper presentation of these tissues requires following a set of specific procedures developed by the autopsy supervisor in the department. Fresh specimens have proven to be essential for successful implementation of vertical integration within our clinical anatomy teaching curriculum. Fresh is best for teaching first and second year medical students the anatomical basis of invasive clinical procedures, such as lumbar puncture, endotracheal intubation, thoracocentesis, Foley catheterization and basic suturing techniques. Clinical faculty and anatomists are able to combine efforts using fresh specimens to realistically demonstrate the anatomical basis of operative procedures in the different surgical subspecialties to fourth year medical students in a senior Surgical Anatomy Elective. In the LSUHSC "Center for Advanced Practice" fresh cadaveric specimens are best for hands‐on training of residents in different surgical techniques and for postgraduate continuing education courses for practicing clinicians held in conjunction with major physicians' organizations and medical technology companies. Responses by students and clinicians have been unanimous in proclaiming that such tissues provide an excellent and realistic learning experience.
Chloride transport induced by the substitution of Asp555 with a Glu in the Na/HCO3 cotransporter SLC4A4 (NBCe1)
Authors: Lee Soojung , Yang Han Soo , Nichols J. Wylie , Rajbhandari Ira , Choi Inyeong
Published: June 2021

By structure/function analysis, we previously demonstrated that the substitution of Asp555 in NBCe1 with a Glu (D555E) causes the mutant transporter to produce currents by other anions. In this study, we further characterized the chloride transport of D555E by using two‐electrode voltage clamp and fluorescence measurements. In Xenopus oocytes expressing D555E, the application of a series of Cl concentrations without CO2/HCO3 produced outward currents in voltage‐clamp recordings. A similar application did not evoke outward currents in oocytes expressing wild type human NBCe1. The chloride currents induced by D555E were markedly diminished in CO2/HCO3 solution, consistent with the previous finding that D555E produces Cl currents in the absence of HCO3. To address the issue of whether the chloride conductance is not derived from endogenous oocyte proteins but is intrinsic to the mutant transporter, we expressed NBCe1 and D555E in HEK293 cells and measured chloride transport by using the fluorescence dye 6‐methoxy‐N‐(‐sulphopropyl) quinolinium SPQ. The expression of the transporters in cells was confirmed by immunoblot and immunocytochemistry with the antibody specific to the C‐terminal end of the protein. SPQ‐loaded cells were washed with a solution containing nitrate, and intracellular SPQ was then quenched by perfusion with iodide. After achieving basal fluorescence, iodide was replaced with nitrate to determine the iodide efflux. The fluorescence in D555E‐transfected cells significantly increased compared to that in NBCe1‐transfected cells. These data indicate that the chloride conductance associated with D555E is most likely to be intrinsic to the mutant transporter.
Identification of 4 charged amino acids necessary for Slc4a10‐derived protein function
Authors: Damkier Helle Hasager , Praetorius Jeppe
Published: June 2021

The Slc4a10 gene product (Ncbe/Nbcn2) imports Na+ and HCO3− into cells in an electroneutral manner. The aim of this study was to investigate the functional importance of 5 charged amino acids in rat Ncbe. Negatively charged amino acids, possibly involved in Na+ translocation, should be conserved among Na+‐dependent Slc4a‐derived proteins. Two candidate amino acids were mutated in a region of Ncbe homologous to the loop between TM9 and TM10 of Ae1 (topology model by Casey and coworkers): E890A and E892A. E584A in the end of TM3 served as negative control. Similarly, positively charged amino acids are presumed important for HCO3− translocation. We mutated two amino acids in Ncbe corresponding to the loop between TM11 and TM12 of Ae1: H976L and H980G. NIH‐3T3 cells were stably transfected with the 5 mutated Ncbe forms and compared functionally to wild type Ncbe and negative controls (dsRed‐transfected). The net Na+‐dependent HCO3− influx after acidification, in E890A and E892A mutants, was reduced to 36 and 31% of wild type Ncbe, respectively (n=5, p<0.05). The Na+ dependent HCO3− flux was also reduced in H976L and H980G (42 and 30% of wild type Ncbe, n=5, p<0.05). The Na+‐dependent net HCO3− influx in E584A mutant cells was not changed (96% of wild type, n=5, p>0.05). In conclusion, four of the tested charged amino acids were necessary for normal Ncbe function. We speculate that these are sites attracting ions for translocation.
Gene expression profile in human mesangial cells stimulated with aldosterone
Authors: Romero Damian G
Published: June 2021

Primary hyperaldosteronism is the most common cause of secondary hypertension and is associated with renal injury. Mesangial cells (MC) regulate glomeruli structure and are involved in multiple renal diseases. It has been previously reported that MC express the mineralocorticoid receptor and aldosterone (ALDO) regulates cell proliferation and oxidative stress among many other physio‐ and pathophysiological actions in MC. However, the molecular mechanisms that trigger the onset and progression of MC‐mediated renal injury by excess ALDO are not clear. We studied whole genome gene expression in human MC (HMC) to analyze which are the intracellular pathways involved in ALDO‐mediated glomeruli injury. HMC were treated with ALDO (10 nM) for 3 or 24 h and their transcriptome analyzed with Illumina BeadChips. Significantly regulated genes were used to analyze the early and late intracellular pathways modulated by ALDO. ALDO treatment for 3 h upregulated glycosphingolipid biosynthesis, and the Notch and Hedgehog signaling pathways, while significantly downregulated genes involved in cell cycle and division. ALDO treatment for 24 h upregulated ubiquitin‐mediated proteolysis and MAPK signaling pathways, and several transcription factors (PML, ELK4, SP6, ZNF323, ZXDC, MIZF). This study highlights new early and late intracellular pathways triggered by ALDO in HMC that may be involved in ALDO‐mediated renal injury
Quantitative trait loci for exercise capacity and response to training in FVB/NJ and C57BL/6J mice
Authors: Massett Michael P , Berk Bradford C
Published: June 2021

The genetic factors determining the magnitude of the response to exercise training are poorly understood. The aim of this study was to identify quantitative trait loci (QTL) associated with adaptation to exercise training in mice. Mice completed an exercise performance test before and after a 4‐week treadmill running program and changes in exercise capacity, expressed as work (kg‐m), were calculated. The change in work with exercise training was significantly different between inbred C57BL/6J (B6) and FVB/NJ (FVB) mice (0.34±0.12 vs. 1.78±0.17 kg‐m, P<0.05). FVBxB6 F1 mice had an intermediate phenotype (1.21±0.35). In F2 mice (n=188), changes in work ranged from ‐1.67 to +4.55 kg‐m. All F2 mice were genotyped at 20 cM intervals using 103 SNPs and genome scans were performed. Suggestive QTL (P=0.63) for changes in work were found on chromosomes 3, 11, and 14. In addition, significant QTL (P<0.05) were identified for pre‐ and post‐training work. QTL for pre‐training work were located on chromosomes 8 (peak at 60.1 cM, 4.47 LOD), 14 (15.3 cM, 4.16 LOD), and 19 (40.6 cM, 3.68 LOD). QTL for post‐training work were located on chromosomes 3 (72.2 cM, 4.83 LOD) and 14 (21.3 cM, 4.88 LOD). These data indicate that one or more QTL determine exercise capacity and training responses in mice. Furthermore, these data suggest that the genes that determine pre‐training work and training responses may differ.

Supported by AHA SDG and HL‐085918 (MPM) and HL‐62826 (BCB).
Cyclooxygenase (COX)‐1 derived prostaglandin E2 (PGE2) acting on its type 1 receptor (EP1R) mediates slow‐pressor angiotensin‐II (AngII) hypertension
Authors: Peterson Jeffrey , Burmeister Melissa A , Marden Jennifer , Poor Daniel , Nwakeze Chiamaka , Sharma Ram V , Anrather Josef , Iadecola Costantino , Davisson Robin L
Published: June 2021

COX‐derived prostaglandins have long been implicated in the cardiovascular effects of AngII. Recently a role for PGE2 acting on EP1R, has emerged in AngII‐dependent vasopressor effects. However, the role of this pathway in slow‐pressor AngII hypertension has not been investigated. Here we hypothesized that slowly developing AngII hypertension is mediated by PGE2 acting on EP1R. We also explored the enzymatic source of PGE2 involved. COX‐1, COX‐2 or EP1R null mice and wildtype (WT) controls were implanted with telemeters for mean arterial pressure (MAP) recordings during slow‐pressor AngII infusion (600ng/kg/min, 2 wks). Baseline MAP was similar between groups (WT 95±2, n=7; COX‐1−/− 93±2, n=5; COX‐2−/− 96±7, n=4; EP1R−/− 100±2 mmHg, n=4, p>.05). In WT mice, AngII induced the typical slow rise in MAP that peaked during the second week of infusion (ΔMAP 32±6, p<.05). This response was completely prevented in EP1R−/− mice (ΔMAP 2±4, p<.05). The hypertension was also blocked in COX‐1−/− mice (ΔMAP 5±1, p<.05), whereas the rise in MAP was intact in COX‐2−/− mice (ΔMAP 34±4, p>.05). Furthermore, power spectral analysis revealed a significant increase in sympathetic activation in WT mice that was absent in EP1R−/− (LF/HF WT 2.2±.4, n=3; EP1R−/− 0.7±.3 fold baseline, n=4, p<.05). These results demonstrate that COX‐1‐derived PGE2 acting on EP1R is critical in AngII slow‐pressor hypertension, and suggest that this pathway is required for the accompanying AngII induced sympathetic activation in this model of hypertension. Supported by HL063887, HL084624, AHA 0540114N.
Role of Phosphodiesterase 1A in Cold‐induced Hypertension and Cardiac Hypertrophy
Authors: Sun Zhongjie , Zhang Zhonge
Published: June 2021

The calcium/calmodulin‐dependent phosphodiesterase 1A (PDE1A) hydrolyzes the intracellular second messengers cAMP and cGMP. Cold exposure to (6 hr/day) results in hypertension and cardiac hypertrophy. PDE1A expression was up‐regulated in resistance arteries, kidneys and hearts of cold‐exposed rats. We hypothesized that RNAi inhibition of PDE1A would attenuate cold‐induced hypertension (CIH) and cardiac hypertrophy (CICH).

We constructed AAV carrying PDE1AshRNA, ControlshRNA, and LacZ driven by a CMV promoter. Four groups received an intravenous injection of AAV.PDE1A‐shRNA, AAV.Control‐shRNA, AAV.LacZ, and PBS, respectively, before exposure to cold (5°C), while 1 group received PBS and was kept at room temperature as a control. BP was increased significantly in all cold‐exposed groups except the group treated with PDE1AshRNA, indicating that PDE1AshRNA prevented CIH. PDE1AshRNA enhanced hypothesive responses to acetylcholine in cold‐exposed rats, indicating improved endothelial dysfunction. The in vivo left ventricle wall thickness was also decreased significantly in the cold‐exposed rats treated with PDE1AshRNA. PDE1A‐shRNA attenuated the cold‐induced increases in heart weight, cardiomyocyte sizes, and vascular wall thickness. PDE1AshRNA increased intracellular cGMP and cAMP levels and decreased VSMC cell proliferation and migration. Thus, PDE1A plays a critical role in the pathogenesis of CIH and CICH.
Superoxide formation in human intrarenal arteries is mainly due to NADPH oxidase activity and contributes to endothelium‐dependent vasodilation
Authors: Schlüter Torsten , Protzel Chris , Zimmermann Uwe , Klebingat Klaus‐Jürgen , Rettig Rainer , Grisk Olaf
Published: June 2021

We investigated the contribution of the NADPH oxidase (NOX) to superoxide formation and the modulation of agonist‐induced constriction as well as of endothelium‐dependent dilation by superoxide anions in human intrarenal arteries. Interlobar, arcuate and interlobular artery segments were dissected from kidneys of patients who underwent nephrectomy because of a renal tumor. Sequential inhibition of oxygen radical forming systems revealed that 75% of the superoxide formation was NOX‐dependent. NOX activity was two‐fold higher in distal than in proximal artery segments. The mRNA expression of the isoenzymes NOX2 and NOX4 and of the small NOX subunits p22phox and p47phox was similar in interlobar, arcuate and interlobular arteries. Phenylephrine (PE) and endothelin‐1 (ET‐1) induced similar maximum tension relative to depolarisation‐induced tension in arcuate and interlobular arteries (pD2 6.25 ± 0.25 and 8.22 ± 0.07, respectively) that was not affected by the superoxide scavenger tiron. When precontracted with 3 µmol/l PE, acetylcholine reduced arcuate and interlobular artery tension concentration‐dependetly to 40% of precontraction values. Tiron significantly blunted maximum acetylcholine‐induced vasodilation in both artery segments. In human intrarenal arteries NOX activity is the major source of superoxide formation which contributes to endothelium‐dependent vasodilation.
Regulation of the ADMA/PRMT/DDAH pathway in vascular smooth muscle cells by the p22phox component of NADPH oxidase
Authors: Luo Zaiming , Welch William , Teerlink Tom , Griendling Kathy , Wilcox Christopher
Published: June 2021

We reported increased reactive oxygen species (ROS) and asymmetric dimethylarginine (ADMA) in chronic kidney disease or hypertension. Arginine is methylated by protein arginine methyltransferases (PRMTs). ADMA is metabolized by dimethylarginine dimethylaminohydrolases (DDAHs). We tested the hypothesis that ROS dictate cellular arginine methylation. DDAH‐1 and ‐2 protein expression were increased by 37 ± 6 and 50 ± 4% (p<0.05) and PRMT‐3 protein increased 41 ± 7%(p< 0.05) in aortas of mice transgenic for p22phox in VSMCs (Tgp22smc), despite unchanged plasma ADMA (0.59 ± 0.1 vs 0.56 ± 0.1 μmol‐ L−1; ns). Rat preglomerular vascular smooth muscle cells (PGVSMCs) stably transfected with p22phox (S‐p22phox) which generated two‐fold more ROS had a 62± 2% (p<0.01) reduced DDAH activity, and a 51 ± 5% and 58 ± 4% (both p<0.05) reduced protein, but increased mRNA expression, for DDAH‐1 and ‐2. Western blots demonstrated substantial, but selective, protein carbonylation and asymmetric dimethylation of arginine moieties in lysates from S‐p22phox cells that were prevented by PEG‐catalase (500 units · ml−1) or tempol (10−4M). In conclusion, ROS produced by NADPH oxidase in VSMCs enhances PRMT and cellular accumulation of arginine methylated proteins. Protein carbonylation may contribute to decreased DDAH protein expression, despite increased mRNA. The similar changes in blood vessel of Tgp22smc mice demonstrates that these effects could underlie endothelial dysfunction.
Folic acid supplementation inhibits homocysteine‐induced superoxide anion production and chemokine expression in the kidney
Authors: Hwang Sunyoung , Sarna Lindsei , Siow Yaw L. , O Karmin
Published: June 2021

Hyperhomocysteinemia (HHcy), a condition of elevated blood homocysteine (Hcy) levels, is a metabolic disease. Kidney dysfunction is a common risk factor causing HHcy. Recent studies indicate that HHcy also lead to renal injury by inducing oxidative stress and expression of inflammatory markers. Monocyte chemoattractant protein‐1 (MCP‐1) is a potent chemokine that stimulates monocyte/macrophage accumulation in the kidney. Upregulation of this chemokine is associated with inflammatory injury. Although folic acid supplementation can reduce blood Hcy levels, its effect on the kidney is not clear. The aim of this study was to investigate the effect of folic acid on Hcy‐induced superoxide generation and chemokine expression in the kidney. HHcy was induced in Sprague Dawley rats fed a high‐methionine diet. The NADPH oxidase‐mediated superoxide production and MCP‐1 mRNA expression were significantly increased in HHcy rat kidneys. Folic acid supplementation effectively abolished HHcy‐induced oxidative stress by inhibition of NADPH oxidase activation. This, in turn, prevented HHcy‐induced chemokine expression in the kidney. Such a beneficial effect of folic acid is not directly related to Hcy reduction. Our results suggest that folic acid supplementation may offer a renal protective effect by antagonizing oxidative stress‐mediated inflammatory response.

(This study was supported by NSERC and CIHR)
Methodology for Measuring Steady‐State Glomerular Filtration Rate (GFR) Using Continuous Intravenous Infusion of FITC‐Inulin in Conscious Mice
Authors: Bivona B , Park S , Harrison‐Bernard L M
Published: June 2021

Studies were performed to determine the extent of renal disease in type II diabetic mice. GFR was measured in conscious chronically instrumented male 18 wk old diabetic (db/db; 53±1g; n=7) and control (db/m; 18±0.5g; n=6) mice. Under isofluorane anesthesia and aseptic conditions, a canula (RenaPulse) was implanted into the right jugular vein, advanced 5mm, sealed, routed subcutaneously to the scapular region, exteriorized, passed through a tether (PE320), and sutured into place using 6‐0 silk. Upon waking, mice were placed into metabolic cages and the tether attached to a counter‐balanced arm and stainless steel swivel (Instech; 375/D/22QM). Mice were infused continuously with 0.9% saline (48hr) and FITC‐inulin (24hr; Sigma, F3272; 15mg/ml) at 4.5 μl/min. Blood (300 μl) was collected from the submandibular plexus using a lancet (GoldenRod) into microtainer tubes (BD; 20U heparin) and spun in 3 microHCT tubes. Plasma was diluted (1:4) in 10mM HEPES (pH 7.4) and assayed for FITC‐inulin fluorescence (excitation 490, emission 520 nm). GFR was calculated based on the renal clearance of inulin: inulin infusion rate/plasma [inulin]. Fed blood glucose averaged 496±46 and 100±9mg/dl (p<0.05) in diabetic and control mice, respectively. HCT was not different between the groups (0.40±0.02 vs 0.44±0.05). GFR averaged 0.65±0.04 and 1.00±0.13ml/min/gKW in diabetic and control mice, respectively (p<0.05). GFR expressed/g BW was significantly reduced in the diabetic compared to control mice (5.8±0.5 vs 12.0±1.9). Measurement of GFR in the conscious mouse removes the influence of anesthesia on this parameter. In addition, GFR measured in anesthetized diabetic mice requires higher rates of fluid replacement compared to control. In summary, the db/db mouse exhibits a 35% reduction in GFR and represents an ideal animal model of human type II diabetic renal disease.
Effect of gender on cardiovascular responses to orthostatic stress – a modeling approach
Authors: Iliescu Radu , Hester Robert , Summers Richard , Coleman Thomas
Published: June 2021

Women are more predisposed to orthostatic intolerance than men. The mechanisms for this difference to orthostatic challenges may involve reduced neural sympathetic activation and/or lower blood volume in women. We used the Female and Male models developed under the Quantitative Human Physiology model to investigate these mechanisms. The female model was scaled based on current data in terms of plasma volume, organ and body structure and hemodynamics. The Female and the Male were subjected to head‐up tilt (HUT) of 60deg for 6 min and cardiovascular variables monitored continuously by time‐dependent iterations. The data were compared with published studies (Am.J.Physiol.Regul.Integr.Comp 289:R109‐116) where an identical protocol was used in men and women. We found that the increase in blood pressure was lower (1 vs. 5 mmHg) and the decrease in SV more pronounced (‐4 vs. ‐1 mL) in the Female vs. the Male, paralleling the changes observed in the human study. Cerebral perfusion was decreased more severely in the Female despite similar sympathetic outflow responses to orthostatic challenge. The absolute volume of blood sequestered in the veins of the lower torso region of the model was similar in the Female and Male, but % of total blood volume (9% vs. 6%) was higher in the Female. Since estrogens may increase the sensitivity of the baroreflex (BRS), we modeled the response to HUT during a 2‐fold increase in BRS in both the Male and Female. While the cardiovascular dynamics were improved in the Male, the Female had a more severe decrease in SV (‐6 mL), despite increased sympathetic outflow. The results of the model and the experimental data suggest that cardiovascular compensatory responses in women during orthostatic challenges are impaired primarily due to lower overall blood volume than in men. Supported by HL 51971, and a NSF EPSCoR.
Hemodynamic Responses to Prenatal Stress and/or Restraint Stress in Rats
Authors: Chen Fangping , Mullenax Heather , Sharma Puneet , Finkel Mitchell S
Published: June 2021

We reported that a combination of prenatal stress (PS) and restraint stress (PS+R) resulted in echocardiographic evidence of myocardial dysfunction in rats. We now report hemodynamic measurements in PS, R, or PS+R. PS, R and PS+R blunted baseline systolic and diastolic function under anesthesia. However, no differences were observed in +dp/dt and ‐dp/dt in PS or R in awake state. In contrast, the differences in +dp/dt and ‐dp/dt persisted with the PS+R in the awake state. Attenuated responses to isoproterenol were still apparent in PS, R and PS+R. The p38 MAPK inhibitor, SB 203580, reversed the blunted +dp/dt and ‐dp/dt. These findings support a central role for p38 MAP kinase in stress‐induced reversible myocardial dysfunction.

Mean±SE; * p<0.05 ,** p<0.01, control+R vs PS+R; # p<0.01, control vs PS+R
Prevention of Volume Overload‐Induced Adverse Myocardial Remodeling in Neurokinin‐1 Receptor Knockout Mice
Authors: Levick Scott P , Melendez Giselle C , Li Jianping , Supowit Scott C , Brower Gregory L
Published: June 2021

The neurokinin‐1 (NK‐1) receptor is the high affinity receptor for the neuropeptide, substance P, which is predominantly released from sympathetic afferent nerve fibers and is important in many diseases involving inflammation. We sought to determine the importance of the NK‐1 receptor to adverse myocardial remodeling in response to volume overload induced in 3 month old NK‐1 knockout (KO) (n=6) mice by creation of an aortacaval fistula. Age‐matched C57B/6 mice (fistula, n=5; shams, n=3) served as wild type controls. Mice were followed by echocardiography for 14 days post‐fistula to assess in vivo left ventricular (LV) structure and function. Wild type mice showed LV dilatation at 10 and 14 days post‐fistula, which did not occur in NK‐1 KO mice. LV posterior wall thickening was evident at 14 days post‐fistula in NK‐1 KO mice, but not in wild type, suggesting an adaptive response to increased wall stress in the NK‐1 KO. Ejection fraction decreased 3 days post‐fistula in both groups, returning to normal at 14 days post fistula in NK‐1 KO mice, but not in wild type. Thus, deletion of the NK‐1 receptor in mice exposed to volume overload prevented adverse myocardial remodeling in the form of LV dilatation, and restored systolic function. This study demonstrates for the first time the potential critical role for substance P, acting via the NK‐1 receptor, in adverse myocardial remodeling.
INTERACTION AND REGULATION OF FOCAL ADHESION KINASE BY CARDIAC MYOSIN
Authors: Santos Aline M. , Schechtman Deborah , Marin Talita M. , Franchini Kleber G.
Published: June 2021

Aim

Focal Adhesion Kinase (FAK) has been implicated in signaling and hypertrophy to mechanical stress in cardiomyocytes. We investigated whether C‐terminal sarcomeric myosin (C‐MHC) controls FAK quiescence and its function in cardiomyocytes.

Methods

FAK/MHC association was detected by pulldown assay. Peptides were rationally designed aligning ligants of C‐MHC with the FAK FERM domain. FERM derived peptide (R1) and control peptide (R2) were conjugated to TAT47‐57 carrier. Neonatal rat ventricular myocytes (NRVMs) were stretched in silicone plates (1 Hz, 10%). FAK distribution was assessed by immunofluorescence. FAK depletion was obtained with siRNA.

Results

Pulldown assays showed C‐MHC/FAK physical interaction mediated by FERM domain. FAK autophosphorylation reduced its ability to bind C‐MHC. Stretching NRVMs or treating them with R1 reduced FAK/C‐MHC association in parallel with increasing FAK phosphorylation at Tyr397 (4 fold) and translocation to the nucleus. Treatment with R1 (24hs) induced hypertrophic morphology in NRVMs. These effects were accompanied by activation of AKT/mTOR/S6K pathways. Treatment with rapamycin or depletion of FAK with siRNA abolished the hypertrophy induced by R1.

Conclusion

Our results indicate that the interaction of FAK with C‐MHC may have a role in the regulation of FAK activity and hypertrophic effects in cardiomyocytes.
Effects of zinc protoporphyrin and NO synthase inhibitor on cyclic guanosine monophosphate content in penile tissue of rats
Authors: Qin Wen‐Bo , Ma Wen‐Bin , Kang Yu‐Ming , Wei Xiao‐Dong , Wang Shu‐Qing , Wang Shu‐Qiu , Chi Bao‐Jin , Wang Xian‐Gang , Liu Jun‐Xing
Published: June 2021

The present study was undertaken to investigate the influence of carbon monoxide (CO) synthase inhibitor zinc protoporphyrin (ZnPP) and nitrous oxide (NO) synthase inhibitor L‐NAME on cGMP content in penile tissue of rats and the relation of them to erectile dysfunction. Thirty male Sprage‐Dawley rats were divided into 3 groups (normal control group, ZnPP group and L‐NAME group) and given the physiological saline, ZnPP (45µmol/kg) and L‐NAME (10mg/kg) each group, then the rats sacrificed 3 days later. The penis were made into homogenate to determine the content of nitric oxide synthase(NOS), NO, CO and cGMP. Compared with control group, the content of CO, NOS, NO and cGMP decreased in the ZnPP group and L‐NAME group, and the statistical difference was significant. The results suggest that the CO synthase inhibitor ZnPP and NO synthase inhibitor L‐NAME can reduce the content of CO, NO and cGMP that contain in the rat penis, however, the concrete mechanism still needs further research.
Contractile function of fibroblast fiber in activation with calf serum and thromboxane A2
Authors: Hiromi Nobe , Nobe Koji , Paul Richard J , Honda Kazuo , Ishida Yukisato
Published: June 2021

Wound repair processes involve epithelialization, connective tissue deposition and contraction. Contraction is one of the important steps, inasmuch as it draws the margins of open wounds together. We used fibroblast fibers reconstituted in a three‐dimensional collagen matrix with NIH 3T3 fibroblast cells characterize nonmuscle contractility and calcium signaling. Calf serum (CS:3~30%) and thromboxane A2 (TXA2:1~1000nM) elicited dose‐dependent increases in isometric force with the similar time courses. The maximal force to TXA2 was 65% of that to CS. CS‐induced contraction was dependent on an intracellular calcium stores. While, the TXA2 response depended on both calcium release from intracellular stores and calcium influx from the extracellular medium. The Rho kinase inhibitor suppressed TXA2‐ and CS‐induced responses. In contrast, inhibition of protein kinase C only reduced the TXA2 response. Moreover, addition of TXA2 during the CS contracture further enhanced force and calcium signal. TXA2‐induced responses are not differential mechanism in the CS‐induced contraction. We considering the healing process, TXA2 functions not only as a blood coagulation factor at the initial stage of wound repair but also as a contractile factor for fibroblasts in the later repair processes.