bluuebunny/crossref_metadata_2025 huggingface dataset:
Physiological changes in plasma [K+] affect NCC function via Cl‐ inhibition of WNK4
Authors: Terker Andrew , Yang Chao‐Ling , Ellison David
Published: June 2021

We have shown that dietary K+ modulates NCC via plasma K+ ([K+]p). [K+]p determines distal convoluted tubule (DCT) membrane potential and therefore, intracellular chloride ([Cl‐]i) (estimated to be near 20 mM). Extracellular [K+] modulates NCC in HEK cells via endogenous WNK1, but HEK [Cl‐]i is higher than DCT [Cl‐]i. WNK4 DCT abundance and redistribution in response to K+ intake suggests that it plays a dominant role in vivo, but this model requires that Cl‐ affects WNK4 at low, physiological [Cl‐]i.

To test this, we compared WNK1 and WNK4 kinase activities on SPAK. Although Cl‐ inhibited both WNK1 and WNK4, the half maximal inhibiting [Cl‐] was much lower for WNK4 than WNK1.

To confirm that WNK4 Cl‐ sensitivity affects SPAK and NCC activity in cells, we mutated the WNK4 Cl‐‐sensing domain (WNK4 LL‐FF) that is homologous to the WNK1 domain. Although WT WNK4 reduced pSPAK in HEK cells, WNK4 LL‐FF increased it. Kinase‐dead WNK4 LL‐FF returned pSPAK to WT WNK4 levels. Furthermore, WNK4 LL‐FF increased pNCC compared to WT WNK4.

To confirm the physiological dominance of this pathway, we tested effects of multiple mouse models, including dietary manipulations (both Na+ and K+), drug treatments (amiloride), and genetic manipulations (MR knockout). A tight relationship between [K+]p and pNCC was observed (pNCC=42.95[K+]‐3.1, R2=0.77), which was linear in the physiological range of [K+]p. Between 5 mM and 3.5 mM, for every 0.1 mM decrease in [K+]p, pNCC increased by 20%.

Lastly, dietary K+ titration (0% to 5%) produced a linear increase in [K+]p from 2.9 mM to 3.85 mM. [K+]p and pNCC were also highly correlated (pNCC=‐0.63[K+]+3.2, R2=0.63).

WNK4 kinase activity is Cl‐‐sensitive in the range of DCT [Cl‐]i, while WNK1 inhibition requires higher [Cl‐]. pNCC tightly correlates with [K+]p throughout the physiological range in multiple models.
Long‐term Telemetric Blood Pressure Measurement in a Translational Model of Non‐human Primate Spontaneous Hypertension
Authors: Osborn J , Rhoads M , Collett J , Beierschmitt A , Dascanio J , Knot H
Published: June 2021

A non‐human primate model of spontaneous hypertension has potential for understanding mechanisms of cardiovascular disease but requires a novel method for telemetric blood pressure measurement. Stellar Telemetry solid‐state pressure sensors were surgically implanted into the abdominal aorta of feral, male monkeys (Chlorocebus aethiops sabaeus). Systolic (SBP), diastolic (DBP) and mean (MBP) arterial pressures, core body temperature and heart rate were determined using different data acquisition parameters to assess system precision and reliability in this physically active non‐human primate. Animals were pre‐phenotyped for normotension (NT) or hypertension (HT) using forearm plethysmography. Various recording parameters yielded consistent blood pressure in NT (MBP=104±4.35 mmHg) and HT (MBP=126±7.57 mmHg) animals. Ultrasonography documented effective placement of sensors within the aorta lumen. Consistent aortic blood pressure recordings were obtained for 6 months post catheter implantation. Necropsy revealed no sensor induced blood or organ pathologies. The Stellar Telemetry system effectively monitors conscious blood pressure, heart rate and body temperature in active primates allowing for physiological assessments during natural social interactions and species specific behaviors. This model of hypertension in an old world, non‐human primate is translational to human essential hypertension and allows for new directions in a genetically similar species.

Supported by Biomedical Sciences Research Group, LLC; TSE Systems, Inc.; Behavioural Sciences Foundation
Induced Pan‐nephron Mineralocorticoid Receptor Knockout Causes Na+ Wasting and K+ Retention
Authors: Terker Andrew , Lazelle Rebecca , yang Chao‐Ling , Ellison David
Published: June 2021

The mineralocorticoid receptor (MR) plays a key role in Na+ and K+ homeostasis. Constitutive MR deletion is lethal, but deletion along the connecting tubule (CNT) and collecting duct (CD) has relatively mild effects. To determine if this difference reflects MR actions along other nephron segments, we deleted MR along the entire nephron.

Using the doxycyline‐inducible Pax8‐rtTA system, we deleted MR in the nephron of adult mice; RT‐PCR confirmed substantial mRNA reduction. MR knockout (MR KO) mice survived, but displayed weight loss, mild hyperkalemia, Na+ wasting, and elevated plasma aldosterone at baseline. Although the abundance of ENaC was not affected, total and phosphorylated NCC (pNCC) abundance were slightly reduced.

Low Na+ diet (0.02% Na+) enhanced the Na+‐wasting phenotype with dramatic weight loss, hyponatremia, and severe hyperkalemia (6.57 mM). Although Na+ deprivation increased pNCC in WT, it strikingly reduced pNCC in MR KO. In contrast, Na+ deprivation did not affect β ENaC in WT, but increased it in MR KO.

High K+ diet (5% K+) also produced weight loss, hyponatremia, and hyperkalemia (7.58 mM) with increased β ENaC abundance and decreased pNCC abundance in MR KO. It also produced significantly more volume contraction (Δ Hct) in MR KO than in WT.

Induced pan‐nephron MR KO mice display a more severe phenotype than CNT/CD MR KO, but a less dramatic one than constitutive total body KO. Though unchanged at baseline, increased β ENaC abundance following diet changes likely reflects MR‐independent regulation; the phenotype suggests that MR is required for activation, not synthesis. Plasma K+ appears to drive changes in NCC. Thus, baseline reductions of NCC in MR KO appear to be secondary to hyperkalemia, rather than direct MR effects in the DCT.
Differential Effects of Endoplasmic Reticulum Stress on Dipsogenic and Blood Pressure Responses to DOCA‐Salt
Authors: Sigmund Curt , Jo Fusakazu , Jo Hiromi , Hilzendeger Aline , Cassell Martin , Rutkowski Thomas , Davisson Robin , Grobe Justin
Published: June 2021

Endoplasmic reticulum (ER) stress in the subfornical organ has been identified as an important mechanism mediating the hypertensive effects of angiotensin II (Ang‐II). We examined the role of ER stress in the polydipsic and pressor responses to DOCA‐salt. ICV delivery of the ER stress‐reducing chemical chaperone TUDCA had no effect on the magnitude of hypertension, but markedly decreased saline intake to DOCA‐salt. Saline intake increased one day after TUDCA was stopped. Similarly, decreased saline intake was observed after ICV 4‐phenylbutyrate, another chemical chaperone. CCAAT‐enhancer‐binding protein homologous protein (CHOP, a marker of chronic ER stress) immunoreactivity was increased in the SFO of DOCA‐salt mice, but the signal was absent in control and CHOP‐/‐ mice. Electron micrographic studies revealed abnormalities in ER structure (decrease in ER membrane length, distended ER membranes, and decreased ribosome numbers) in the SFO consistent with the induction of ER stress. SFO‐targeted injection of AdGRP78, a molecular chaperone, decreased DOCA‐salt‐induced saline intake. Like the effects of TUDCA, the increase in saline intake in response to DOCA‐salt was blunted in CHOP‐/‐ mice. However, CHOP‐/‐ mice exhibited a similar hypertensive response to DOCA‐salt. These data mechanistically implicate ER stress in saline intake, but not blood pressure responses to DOCA‐salt, and further suggest that CHOP may play a functional role in DOCA‐salt‐induced saline intake. The results suggest a distinction between the importance of ER stress in mediating effects of DOCA‐salt on polydipsia and hypertension.
Generation of Mice Expressing RFP‐tagged Sodium Phosphate Cotransporter NaPi‐IIa
Authors: Ruminska Justyna , Patti Monica , Vollero Alessandra , Bourgeois Soline , Forster Ian , Hummler Edith , Ponce de Leon Veronica , Biber Jürg , Hernando Nati , Wagner Carsten
Published: June 2021

Homeostasis of inorganic phosphate (Pi) is essential for various physiologic functions. The sodium phosphate cotransporter NaPi‐IIa is located in the brush border membrane (BBM) of the renal proximal tubule and represents the major renal Pi transporter. The plasma concentration of Pi is determined by factors regulating NaPi‐IIa abundance at the BBM. We are developing a novel mouse model expressing NaPi‐IIa tagged with red fluorescent protein (RFP) to analyze the regulation of the cotransporter at the BBM in real time and space and at the molecular level. We have generated RFP‐NaPi‐IIa constructs containing the RFP at three different locations of the cotransporter, the very N‐terminus, within the N‐terminal tail and within the big extracellular loop. Control experiments in opossum kidney cells and in Xenopus laevis oocytes to analyze the cotransporter polarization, PTH sensitivity, Na+‐dependent cotransport activity and kinetic characterization revealed that the RFP within the N‐terminal tail did not impair any of the analyzed parameters. Upon identification of the proper insertion site, we generated three pairs of transcription activator‐like effector nucleases (TALENs) predicted to cut the Slc34a1 (NaPi‐IIa) gene within the desired sequence. The pair with the highest cutting efficiency was chosen for the generation of RFP‐NaPi‐IIa mice. Consequently, we produced RFP donor DNA containing 5' and 3' homologous arms, as well as the RFP coding sequence. TALENs and the RFP donor were injected into one‐cell stage embryos to generate transgenic mice.

This work was supported by NCCR Kidney.CH
AMPK Regulates the Vacuolar H+‐ATPase via 14‐­3­‐3 Proteins
Authors: Al‐bataineh Mohammad , Li Hui , Marciszyn Allison , Bhalla Vivek , Hallows Kenneth , Pastor‐Soler Nuria
Published: June 2021

The vacuolar proton‐ATPase (V‐ATPase) is abundantly expressed at the apical pole of type A intercalated cells and proximal tubule cells. This pump mediates H+ transport across membranes against a gradient. Defects in V‐ATPase function lead to renal tubular acidosis with kidney and systemic complications. In kidney cells we showed that the metabolic sensor AMP‐activated kinase (AMPK) phosphorylates the V‐ATPase A subunit at S384. AMPK can regulate transport proteins by promoting their ubiquitination and degradation, mechanisms that are likely important for inhibition of membrane transport during cellular metabolic stress. We noticed that S384 exists within a 14‐3‐3 binding motif. We identified another site (Site III) in the A subunit that may play a role in the interaction with 14‐3‐3 proteins (14‐3‐3s). Dimeric 14‐3‐3s bind to phosphorylated sites on target proteins and modulate protein function and protein‐protein interactions. We thus hypothesized that phosphorylation by AMPK and an additional kinase modulates A subunit binding to 14‐3‐3s and may promote V‐ATPase A subunit ubiquitination. We detected increased co‐immunoprecipitation of 14‐3‐3s with the A subunit in cells treated with the AMPK activator AICAR as compared with untreated cells. However, the phosphorylation‐deficient Ser‐to‐Ala A‐subunit mutants at either site (S384 and Site III) showed no significant changes in binding to 14‐3‐3s with AICAR treatment. Furthermore, AICAR increased A subunit ubiquitination. We propose that A subunit binding to 14‐3‐3s and ubiquitination are mediated by S384 and Site III phosphorylation thus linking V‐ATPase regulation to AMPK during metabolic depletion.
The Effect of WNK4 on the Na+:Cl‐ Cotransporter is modulated by Intracellular Chloride
Authors: Bazua‐Valenti Silvana , Chavez‐Canales PhD Maria , Rojas‐Vega Lorena , Gonzalez‐Rodriguez Xochiquetzal , Vazquez Norma , Rodriguez‐Gama Alejandro , Argaiz Eduardo , Plata Consuelo , Garcia‐Valdes Jesus , Ellison David , Hadchouel Juliette , Gamba Gerardo
Published: June 2021

Introduction

The effect of WNK4 on the renal Na+:Cl‐ cotransporter, NCC is controversial since several studies in vitro and in vivo have shown negative or positive effects. We hypothesize that both effects coexist and are modulated by the intracellular chloride concentration ([Cl‐]i). Piala et al. (2014) showed that L‐WNK1 is a Cl‐‐sensitive kinase and identified two leucine residues forming the Cl‐‐binding site. These leucines are conserved in WNK isoforms.

Materials and Methods

We used the functional expression system of Xenopus oocytes and HEK293 cells. NCC activity was measured by 22Na+ uptake and/or amino acid threonine phosphorylation in the absence or presence of wild type or mutant WNK4, in control conditions, or after [Cl‐]i depleting maneuvers. WNK4 autophosphorylation was also assessed in these conditions.

Results

At baseline [Cl‐]i ∼ 45 mM, WNK4 autophosphorylation was undetectable and NCC activity was either inhibited or unaffected. [Cl‐]i reduction (25‐30 mM) promoted autophosphorylation transforming WNK4 to an activator of NCC in a kinase‐dependent manner. The L322F substitution transformed WNK4 into a constitutively autophosphorylated kinase that activated NCC without chloride depletion.

Conclusion

WNK4 is Cl‐ sensitive kinase exerting differential effects on NCC depending on the [Cl‐]i. It is possible that small changes in [Cl‐]i in distal convoluted tubule cells may have a crucial role determining WNK4 effect on NCC, suggesting that diverse physiological processes could converge in the Cl‐modulation of WNK's.
Transcriptional Regulation Of The SLC52A3 Gene: Molecular Characterization Of The Minimal Promoter In Human Intestinal Cells
Authors: Ghosal Abhisek , Sabui Subrata , Said Hamid
Published: June 2021

The human the SLC52A3 gene encodes the riboflavin (RF) transporter‐3 (RFVT‐3), a membrane protein that plays an important role in intestinal RF absorption. There is little known about the regulation at the transcriptional and post‐transcriptional levels. Here we address the former regulation and for that we identified the minimal SLC52A3 promoter region, the regulatory element(s) involved in its activation, and confirmed promoter activity and establish physiological relevance in vivo in transgenic mice. Using transiently transfected human intestinal epithelial HuTu 80 cells and 5′‐deletion analysis, the minimal SLC52A3 promoter was found to be between ‐199 and +8 bp. Bioinformatics analysis predicted several cis‐regulatory elements in this region but only the stimulating protein‐1 (Sp1) (at position ‐74/‐71 bp) was found to play key role in promoter function. Electro‐mobility shift, super‐shift assays and by ChIP analysis confirmed the binding of the nuclear factor Sp1 to the minimal promoter. The importance of the Sp1 nuclear factor was further confirmed in studies with Drosophila SL2 cells (which lack endogenous Sps expression) where significant induction in SLC52A3 promoter activity was observed upon transfection with exogenous Sp1. Finally, activity of the cloned SLC52A3 promoter fused to the luciferase reporter gene was confirmed in vivo in transgenic mice. We report on the identification and molecular characterization of the minimal SLC52A3 promoter, and on the role of Sp1 in regulating its activity in gut epithelial cells. [Supported by grants from the DVA and NIDDK]
Effects of NOD2 in intestinal epithelial cells: Cross‐talk between NHE and Na+‐K+‐ATPase activity
Authors: Cabral Jose , Grácio Daniela , Soares‐da‐Silva Patricio , Magro Fernando
Published: June 2021

NOD‐like receptors are a family of pattern recognition receptors that plays a role in the control of inﬂammation and have been associated with inflammatory bowel disease (IBD). Na+/H+ exchangers (NHE) are responsible for the net uptake of sodium, electrolyte homeostasis and decreases in pHi during inflammatory process.

Aim

Evaluate the effect of NOD2 upon NHE and Na+‐K+‐ATPase activity.

Methods

NHE activity, Na+‐K+‐ATPase activity and levels of intracellular calcium were measured using fluorimetric assays.

Results

Short‐term NOD2 activation stimulated NHE1 activity in a concentration dependent manner (100±3 to 129±4% in T84 and 100±4 to 133±2% in NCM460 cells). To understand the transduction signalling involved experiments were performed in the presence of inhibitors of Rip‐like interacting caspase‐like apoptosis‐regulatory protein kinase (RIP2), NF‐kβ, tyrosine‐protein kinase (SRC), transforming growth factor‐beta‐activated kinase1 (TAK 1), TNF receptor associated fator 6, adenylyl cyclase (AC), protein kinase A (PKA), phospholipase C (PLC), protein kinase C, Na+‐K+‐ATPase and potassium channels blockers. The NOD2‐mediated increase in NHE1 activity was prevented by inhibitors of RIP2, SRC, TAK1, AC, PKA, PLC, Na+‐K+‐ATPase and Ca2+‐dependent‐ATP‐sensitive K+ channels in both cell lines; this pathway was NF‐kβ independent. Increases in intracellular Ca2+ were observed after NOD2 activation (100±1 to 360±1% in T84 and 100±1 to 254±12% in NCM460 cells).

Conclusions

Activation of NOD2 promotes an increase in Na+‐K+‐ATPase and NHE1 activities as a result of opening of Ca2+‐dependent ATP‐sensitive K+ channels. A better understanding of this pathway is considered for the development of novel therapies for IBD.
Gout‐Causing Q141K Mutation in ABCG2 Leads to Defective Nucleotide‐Binding Domain Function
Authors: Woodward Owen , Moosvi Zain
Published: June 2021

ABCG2 is a secretory efflux uric acid transporter of the kidney proximal tubule and gut that plays a key role in uric acid excretion. Genetic defect in ABCG2 leads to significant increases in serum urate levels (SUA) causing hyperuricemia and gout. A single common variant, Q141K, is responsible for the majority of ABCG2 associated gout risk. The Q141K mutation is a loss of function mutation associated with a severe reduction in protein abundance and transport function. Previously we hypothesized the Q141K mutation leads to instability in the nucleotide‐binding domain and here we present the specific molecular mechanism of the defect and a proof of concept of its correction using small molecules. We found using a ABCG2 structural model and targeted amino acid substitutions that the Q141K mutations leads to a localized disruption in packing that affects abundance and can be partially rescued with a secondary substitution at H155A. However the use of the signature motif suppressor mutation, G188E, provides full rescue of the Q141K abundance by stabilizing the NBD dimer sandwich, a finding consistent with a Q141K defect in NBD dimer formation. Finally we show that a small molecule, VRT‐325, known to bind directly to the NBD of ABC transporters can rescue both abundance and function of Q141K ABCG2.
Calreticulin Up‐Regulates VEGFs Expression in Neuroblastoma Cell Lines
Authors: Lin Kuan‐Hung , Weng Wen‐Chin , Wu Pei‐Yi , Lu Yi‐Chien , Wang Bo‐Jeng , Liao Yung‐Feng , Hsu Wen‐Ming , Lee Hsinyu
Published: June 2021

Neuroblastoma (NB) is a childhood cancer with a low survival rate and great potential for metastasis. Calreticulin (CRT), an ER chaperone protein, is one of the biomarkers of NB. In gastric cancer cells, we have investigated that CRT strongly enhances angiogenesis by promoting the expression of vascular endothelial growth factors (VEGFs). In this study, we aimed to investigate the correlation between the expression of CRT and VEGFs in NB. It was found that over‐expression of CRT up‐regulated VEGF expression at both the mRNA and protein levels in SK‐N‐DZ and SH‐SY5Y cells. CRT RNAi efficiently suppresses CRT expression resulting in down‐regulation of VEGFs.By using ELISA analysis, CRT was shown to significantly enhance VEGF‐A expression in conditioned medium. The CRT‐inducible stNB‐V1 cell line has been established by the lentivirus transduction system We further demonstrated that NB cell apoptosis was not affected by CRT induction in stNB‐V1 cells. Nevertheless, over‐expressing CRT suppressed cell proliferation and enhanced cell differentiation in stNB‐V1 cells, whereas blockage of VEGFR‐1 markedly suppressed expressions of neuron specific markers such as GAP43, NSE, NFH and TrkA. In xenograft experiments, we found that xenograft tumors with induced CRT expression had much smaller sizes. VEGF‐A had a much higher expression level in the tumor samples compared to control samples. These findings suggest that VEGF‐A is involved in CRT‐related neuronal differentiation in NB. Our work may provide important information for developing a new therapeutic strategy to improve the outcome of NB patients.
BMPR2 mutation in cardiomyocytes impairs insulin‐dependent trafficking of CD36
Authors: Talati Megha , Funke Mitchell , Penner Niki , West James , Hemnes Anna
Published: June 2021

In PAH, insulin resistance is associated with impaired RV function. In mouse model of PAH with mutant BMPR2, we have shown impaired fatty acid oxidation and lipid accumulation in RV cardiomyocytes. We hypothesize that in cardiomyocytes with mutant BMPR2 insulin dependent translocation of CD36, fatty acid transporter molecule, is impaired and may contribute to lipid accumulation.

H9C2 cardiomyocytes were stably transfected with empty vector (control) or plasmid containing mutant BMPR2 (mutant). CD36 protein was analyzed by western and confocal microscopy at baseline (BL) and insulin stimulation (IS) at 15 minutes (m), 30m, 1 hour (h), 24h. Key intracellular insulin signaling pathway intermediates analyzed by western.

At BL, CD36 was 2fold up in mutant compared to control and increased 2fold in controls at 1h of IS. In mutant, IS lead to 50% increase in CD36 from 15m to 24h. Immunolocalization studies recapitulated these findings. IS increased CD36 in the cytoplasm and plasma membrane (PM) in mutant compared to control. At BL, pAkt Ser473, a key intermediate, was 2fold up in mutant compared to control. Within 15m of IS there was 4fold increase in pAkt Ser473 in control, and droping to BL by 24h. In mutant, there was similar increase in pAkt Ser473 for 1h, before dropped to BL at 24h. In control and mutant, levels of pAkt Ser473 substrate were similar at BL. In control, IS resulted in 2 and 5fold increase in pAS160 and AS160 from 15 and 30m to 24h respectively. In mutant pAS160 increased 2fold at 15m and AS160 protein increased only slightly compared to BL.

Using cardiomyocytes with mutant BMPR2, we show impaired insulin‐mediated Akt‐AS160‐CD36 trafficking resulting in enhanced CD36 translocation to PM. This altered axis may contribute to increased lipid import and cardiac lipotoxicity.

1 P01 HL 108800‐01A1
Sec10 and the Exocyst are Necessary for Renal Epithelial Monolayer Homeostasis and Ciliogenesis In Vitro and In Vivo
Authors: Polgar Noemi , Lee Amanda , Lui Vanessa , Fogelgren Ben
Published: June 2021

The eight‐protein exocyst complex regulates targeted delivery of exocytic vesicles. A previous report demonstrated that shRNA‐knockdown of Sec10 (Sec10‐KD), a central subunit of the exocyst, in Madin‐Darby canine kidney (MDCK) cells disrupted primary cilia assembly and 3D cyst formation. To further study the mechanisms by which Sec10 and the exocyst regulate epithelial polarity, morphogenesis, and homeostasis, we used three dimensional collagen cultures of MDCK cells. Sec10‐KD cysts initially showed undisturbed lumen formation, even though subsequently displayed significantly fewer and shorter primary cilia than controls. Later in cystogenesis, while Sec10‐KD cysts showed numerous apoptotic cells extruded basally into the collagen matrix, control cells maintained normal homeostasis. Sec10‐KD MDCK cells were also more sensitive to apoptotic triggers than controls. Apico‐basal polarity appeared normal in Sec10‐KD cysts, while a planar cell polarity defect was suggested by the differences in mitotic spindle angles between knockdowns and controls. Furthermore, analysis of renal tubules in our recently generated kidney‐specific Sec10 knockout mouse model revealed significant defects in primary cilia assembly and in the targeted renal tubules, abnormal epithelial cell extrusion was also observed, supporting our in vitro findings. We hypothesize that in Sec10‐KD cells, the disrupted exocyst activity leads to an increased apoptotic sensitivity through disrupted primary cilia signaling, and in combination with an increased basal cell extrusion rate, it affects epithelial barrier integrity and homeostasis.

Supported by: the NIH, March of Dimes, UH RCMI‐BRIDGES and HRFDCC at University of Alabama.
In Vitro Hypoxia/Reoxygenation Modeling of Ischemia/Reperfusion Injury Using Glucose Oxidase in Human Cells
Authors: Barr Johnny , Dubick Michael , Bowman Phillip
Published: June 2021

Ischemia/reperfusion injury (I/RI) is associated with trauma and various diseases. This research investigated an in vitro system of hypoxia/reoxygenation that may be used as a screen for drugs designed to reduce I/RI. Subjecting cells to reduced oxygen (O2) by purging the atmosphere with nitrogen is a slow process that does not reproduce the rapid local reduction in O2 that occurs in vivo during ischemia. Glucose oxidase (GOX) rapidly oxidizes glucose and produces a very low oxygen level (< 0.4 µM within 5 min) with an insignificant loss of glucose. Human umbilical vein endothelial cells (HUVEC) and HeLa cells were subjected to hypoxia for 10 min and returned to normoxia for up to 6 hrs in complete medium. Noticeable cell death was detected by one hour and most died by 6 hrs. At 3.5 hrs a set of cells were homogenized and assayed for total antioxidant potential (TAP) and activities of select antioxidant enzymes. In HUVECs, TAP was 73% higher in cells made anoxic (A) compared to controls (C). Catalase and xanthine oxidase activities were 2‐fold and 70% higher, respectively, in A cells than C while superoxide dismutase or glutathione peroxidase activities were not significantly different between A and C cells. Similar responses were observed in HeLa cells. Catalase could prevent cell death implicating hydrogen peroxide as an important contributor to this injury. This in vitro system can be used to understand the response to IRI and allow testing of potential therapeutics. Funded by US Army Medical Research Materiel Command.
Presence of Octopamine Receptors in Heart of the Bivalve Crassostrea viginica
Authors: Bess Fiana , Harris Ave , Welsh Christopher , Catapane Edward , Carroll Margaret
Published: June 2021

Octopamine (OA), a biogenic amine first identified in octopus, is well studied in arthropods and gastropods where it functions as a neurotransmitter and hormone. OA rarely has been reported in bivalves. Using HPLC we found OA in ganglia, gill, heart, palps and hemolymph of the oyster Crassostrea virginica and using immunohistofluorescence visualized OA in ganglia, gill and heart. Our physiological studies found OA is cardioactive for C. virginica and Mytilus edulis hearts. We hypothesize OA receptors are present in heart of C. virginica. To test this we did Western Blot (WB) using and pan TAAR (trace amine‐associated receptor) 1̊ antibodies, which are reactive with OA, beta‐phenylethylamine (b‐PEA), p‐tyramine (p‐TYR) and tryptamine receptors, but unresponsive to classical biogenic amines and histamine receptors. For WB, heart tissue lysate was prepared in NP‐40 detergent buffer containing protease inhibitor, followed by centrifuging to obtain supernatant with solubilized membrane proteins. Up to 30 µg of solubilized protein was subjected to SDS‐PAGE with 10% acrylamide gels and electroblotted onto nitrocellulose. Pan TAAR receptor immunoreactivity was revealed by 1̊ antibodies followed by HRP‐conjugated 2̊ antibodies. WB showed an 85 kD band corresponding to OA receptors in heart. The present project, coupled with our earlier studies, confirms the presence of OA receptors and furthers understanding of a physiological role for OA in C. virginica.
Thermoregulatory Scope Associated with Underwater Exercise in a Polar Seal
Authors: Hindle Allyson , Horning Markus , Mellish Jo‐Ann
Published: June 2021

A weakness in predicting habitat change impacts in polar regions is that little is known about thermoregulatory energetics in high‐latitude seals. To better understand thermoregulatory range associated with the partitioned air‐water habitat of ice seals, we collected skin surface heat flux data from 42 free‐ranging Weddell seals over 1‐13 day deployments. We modeled total body heat dissipation from heat flux data to address the thresholds of basal versus activity‐derived heat loss over a range of swim speeds. As body size/condition affect heat loss and may manifest as altered thresholds for thermal homeostasis, we compared 4 life history categories—pups, juveniles, non‐reproductive adult females and post‐weaning females. Preliminary evidence indicates obligate heat dissipation, reflecting thermoregulatory costs, is elevated in water versus air, and locomotory heat generation offsets these costs. Dive activity‐heat loss relationships were compared among the 4 life history stages to detect differences in baseline obligate heat loss and threshold swim speeds for thermal substitution. Group differences were apparent under non‐steady state conditions such as transition from haul‐out to water, where post‐weaning females lost more heat than good condition skip breeder females (+40% W/m2 heat loss). Juveniles, with limited blubber insulation, also lost more heat than pups on initial water entry. Continued efforts to quantify and model thermoregulatory thresholds will provide important insights into the ability of polar pinnipeds to respond to changing ice conditions.
Paradoxical Anaerobism in Desert Pupfish
Authors: van Breukelen Frank , Hillyard Stanley
Published: June 2021

Oxidation of glucose through aerobic pathways is ~15X more efficient than is anaerobic metabolism. Pupfish of the genus Cyprinodon experience variable periods of up to nearly 5h of virtually no oxygen consumption. This paradoxical anaerobism occurs in the presence of oxygen. The longest continuous bout was 149 min although more typical bouts are only a few minutes in length. Fish randomly cycle between periods of paradoxical anaerobism and stable oxygen consumption patterns suggesting no long‐term damage or alternations to mitochondria. There is no evidence for compensatory oxygen use. Fish produce ethanol as an apparent alternate end product of anaerobic metabolism. Exposure to exogenous ethanol induces rapid onset of paradoxical anaerobism.
Physiological Tradeoffs Underlie the Evolution of Hypoxia Tolerance and Exercise Performance in Fish
Authors: Scott Graham , Crans Kyle , Pranckevicius Nicole
Published: June 2021

There is a frequently observed tradeoff between hypoxia tolerance and exercise performance in fish, even though both of these traits are often associated with a high O2 transport capacity. We examined the physiological basis for this tradeoff in four species of bass and sunfish from the family Centrarchidae. Hypoxia tolerance was greatest in rock bass, intermediate in pumpkinseed sunfish and bluegill sunfish, and lowest in largemouth bass, based on measurements of critical O2 tension (Pcrit) and the O2 tension at loss of equilibrium. The least hypoxia‐tolerant species had the highest critical swimming speed (Ucrit) and maximal O2 consumption rate during swimming, and suffered the greatest decrease in Ucrit during exercise in hypoxia, consistent with there being a tradeoff between hypoxia tolerance and exercise performance. Some traits appeared to contribute to both hypoxia tolerance and exercise performance, as reflected by the traits that were highest in both rock bass and largemouth bass, such as the gas‐exchange surface area of the gills and the enzyme activities of lactate dehydrogenase and the gluconeogenic enzyme phosphoenolpyruvate carboxykinase in the liver. Some other traits were uniquely associated with hypoxia tolerance (pyruvate kinase and lactate dehydrogenase activities in the heart) or exercise performance (capillarity and fibre size of the axial swimming muscle). Therefore, the cumulative influence of a variety of respiratory and metabolic traits likely underlies the physiological tradeoffs associated with the evolution of hypoxia tolerance and exercise performance in fish. (Supported by NSERC of Canada)
Neuronal and neuroendocrine mechanisms of social rank and stress coping in teleost fish
Authors: Winberg Svante , Thörnqvist Per‐Ove , Dahlbom Josefin , Roman Erika , Höglund Erik
Published: June 2021

Fights for social dominance are stressful and results in an activation of the brain serotonergic system. Subordinate animals in an established dominance hierarchy are characterized by a chronic elevation of brain serotonergic activity, an effect that seems to mediate the behavioural inhibition observed in these animals. By contrast, social dominance has been associated with elevated dopaminergic activity, and dopamine (DA) has behavioural effects to some extent opposing those of serotonin (5HT). In addition to effects of the social environment, brain monoaminergic functions are controlled by genetic factors. For instance, zebrafish (Danio rerio) classified as bold, showing a propensity for being social dominant, display higher expression of DA2b receptor mRNA than shy conspecifics. In rainbow trout (Oncorhynchus mykiss) and Baltic salmon (Salmo salar) bold and shy individuals differ in the expression of 5HT1A receptor mRNA. In salmonids, bold and shy fish also show divergent effects of stress on brain 5HT neurotransmission. Personality traits are to a large extent genetically controlled. However, teleost fish are extremely plastic and it is not clear to what extent “personality related” differences in monoaminergic, and peptidergic, systems is related to heritable factors or early social interaction.
Baroreflex Modulation by Physical Training: Involvement of nNOS in the Paraventricular Nucleus of Hypothalamus (PVN)
Authors: Raquel Hiviny , Masson Gustavo , Barna Barbara , Michelini Lisete , Martins‐Pinge Marli
Published: June 2021

We evaluated the baroreflex and involvement of nNOS in the PVN in sedentary (Gsed) or trained animals (Gtr). Wistar male rats of Gsed or Gtr underwent guide cannulas implantation to PVN and catheterization of the femoral artery and vein to record mean arterial pressure (MAP) and heart rate (HR). Phenylephrine and sodium nitroprusside were infused before and after bilateral microinjection of nNOS inhibitor in the PVN. The nNOS expression in the PVN was analyzed by Real Time qPCR and the content of the isoforms PVNmedial (PVNm) and posterior (PVNp) by immunohistochemistry. At rest, the Gtr had bradycardia and increased baroreflex gain. The Gtr showed higher variation of MAP (Gsed: 53 ± 2 vs. Gtr: 66 ± 4 mmHg) and HR (Gsed: ‐84 ± 1 vs. Gtr: ‐94 ± 0,9bpm) with phenylephrine. After nNOS inhibition, the differences were abolished (ΔPAM: Gsed: 55 ± 1 vs. Gtr: 54 ± 1 mmHg) and (ΔFC: Gsed: ‐99 ± 6 vs. Gtr: ‐99 ± 6 bpm). Variations in MAP and HR with sodium nitroprusside were higher in Gtr before (ΔPAM: Gsed: ‐22 ± 2 vs. Gtr: ‐44 ± 1 mmHg / ΔFC: Gsed: 108 ± 3 vs. Gtr: 142 ± 3 bpm) and after (ΔPAM: Gsed: ‐24 ± 3 Gtr: ‐42 ± 3 mmHg / ΔFC: Gsed: Gtr 100 ± 3: 150 ± 3 bpm) nNOS inhibition. The training induced higher nNOS gene expression and increased the amount of immunoreactive cells in PVNm. Our data indicate that swimming training modulates baroreflex gain due in part to nNOS increase in the PVN.
Hypoxia and Hypercapnia Impairs Cardiac Vagal Control: the Role of Orexin
Authors: Dergacheva Olga , Mendelowitz David
Published: June 2021

Patients with obstructive sleep apnea (OSA) have an increased risk of cardiovascular diseases and altered plasma levels of orexin. REM sleep further increases the incidence of adverse cardiovascular events. The mechanisms underlying REM sleep‐related reduction in parasympathetic activity include activation of inhibitory input to cardiac vagal neurons (CVNs) in the brainstem originating from the lateral paragigantocellular nucleus (LPGi), a nucleus which plays a role in REM sleep control. This study tests the hypotheses that (1) chronic intermittent hypoxia and hypercapnia (CIHH) inhibits CVNs due to exaggeration of GABAergic pathway from the LPGi to CVNs; (2) orexinergic signaling in CVNs is diminished by CIHH; and (3) identified orexin neurons (orexin‐EGFP‐2A‐Cre rat) are inhibited by acute hypoxia/hypercapnia. GABAergic neurotransmission to CVNs evoked by electrical stimulation of the LPGi was examined using whole‐cell patch‐clamp recordings in rats exposed to CIHH and control rats. GABAergic synaptic events were enhanced following 4 weeks of CIHH. Orexin‐A elicited inhibition of LPGi‐evoked GABAergic current in control animals but evoked no significant changes in CIHH rats. Acute hypoxia/hypercapnia inhibited the firing activity of orexin neurons and diminished glutamatergic neurotransmission to orexin neurons. In summary, repetitive episodes of hypoxia/hypercapnia would diminish activity of orexin neurons, including the orexinergic neurotransmission to CVNs. Diminished orexinergic signaling, along with exaggerated inhibitory neurotransmission from the LPGi to CVNs would reduce cardio‐protective parasympathetic activity and increase risk of cardiovascular diseases.

Support: Christian Gillin Research Grant 004GN12 to O.D. and NIH grants HL49965, HL59895 and HL72006 to D.M.
Impact of DOCA‐Salt Hypertension on Gene Expression in the Dorsal Pons: a Microarray Analysis
Authors: Hernandez Morgan , Travis Thomas , Hayward Linda
Published: June 2021

We had previously reported that induction of DOCA/salt hypertension altered the cardiovascular response to stress and this was associated with altered gene expression in the amygdala. Since a major source of afferent input to the amygdala originates from the dorsal pons, the present study was undertaken to evaluate how DOCA/salt hypertension alters gene expression in this brain region. Differential gene expression was measured by high‐density cDNA microarray analysis (Affymetrix Rat Gene Chip) and validated with real‐time PCR in DOCA‐salt hypertensive versus normotensive controls (n=4/group). Microanalysis identified 934 genes that were up‐regulated, including genes related to catecholamine synthesis and transport, inflammation, taste and smell. Solute carrier family 6 (dopamine neurotransmitter transporter) and tyrosine hydroxylase (TH) were the two most highly upregulated genes in DOCA/salt vs normotensive controls. Real‐time PCR analysis confirmed a 5.3 fold increase in dopamine transporter and a 10 fold increase in TH gene expression in the dorsal pons of the DOCA/salt treated animals vs controls. There was also evidence of up‐regulation of GFAP and IL‐10 and down regulation of glutamate receptors. Since changes in catecholamine release in both the amygdala and parabrachial nucleus can alter salt intake and catecholamine receptor activation in the lateral hypothalamus has been linked to salt intake gratification, our results raise the new possibility that alterations in catecholamine synthesis in the dorsal pons plays a significant role in DOCA driven salt intake.
Hypercapnia but Not Hypoxia Attenuates Cardio‐Respiratory Uncoupling Caused by Photo‐Evoked Bradycardia in Mice Expressing Channelrhodopsin‐2
Authors: Bloom Jacob , Garcia Alfredo , Ramirez Jan
Published: June 2021

Hypoxia and hypercapnia are common stimuli for cardio‐respiratory physiology and encountered in several conditions such as sleep disordered breathing and sudden infant death syndrome. This study examines how hypoxia and hypercapnia affects evoked parasympathetic tone over heart rate and cardio‐respiratory coupling. In vivo experiments were conducted in anesthetized freely breathing mice expressing channelrhodopsin‐2 under the regulation of the ChAT promoter. The nucleus ambiguus (NA) was photo‐stimulated (λ=470nm) with continuous or train (1‐20Hz) pulses while freely breathing 100% O2, 10% O2/90% N2 (hypoxia), or 95% O2/5% CO2 (hypercapnia). While respiratory rate was unaffected by photo‐stimulation of the NA in all three conditions, in 100% O2 and hypoxia photo‐stimulation of the NA caused profound bradycardia (R‐R interval increased: 146 ± 25.9%, n=12; and 193 ± 67.6%, n=5, respectively). In contrast, hypercapnia attenuated photo‐evoked bradycardia (R‐R interval increased: 27±6.1%, n=8). Administration of propanol (3.5 mg/kg) did not prevent hypercapnic attenuation of photo‐evoked bradycardia (n=4). These findings suggest that hypercapnia attenuates parasympathetic tone over heart rate through a β‐receptor independent mechanism. Moreover, we show that gas environment and parasympathetic tone are significant factors to consider when examining cardiac sympatho‐vagal balance and cardio‐respiratory coupling.
Central inflammation induced by high fat diet sensitizes angiotensin II hypertension
Authors: Xue Baojian , Yu Yang , Guo Fang , Beltz Terry , Felder Robert , Johnson Alan
Published: June 2021

Obesity is considered to be a chronic low‐grade inflammatory state. High fat diet (HFD) has been shown to promote the inflammation in the hypothalamus and to increase sympathetic activity. Our previous studies demonstrated that administration of either central tumor necrosis factor (TNF)‐α or a subpressor dose of angiotensin (ANG) II sensitizes subsequent ANG II‐elicited hypertension. The present study tested the hypothesis that feeding of HFD also induces the sensitization of the ANG II‐elicited hypertensive response. Male rats were prepared for telemetry recording of blood pressure (BP) and were given 1) normal fat diet (NFD, 10% fat) or 2) HFD (60% fat) or 3) HFD plus the TNF‐α synthesis inhibitorpentoxifylline (10 μg/h, ICV) for three weeks. The rats were then administrated ANG II (120 ng/kg/min, SC) for 2 weeks. HFD itself did not increase BP during the feeding period, but rats that received the HFD pretreatment responded with an enhanced hypertensive response to ANG II when compared to those that received the NFD (Δ39.4±3.7 mmHg vs. Δ25.4±2.8 mmHg). The TNF‐α synthesis inhibitor abolished sensitization produced by the HFD Δ24.9±4.8 mmHg). RT‐PCR analysis of tissue from the lamina terminalis and the paraventricular nucleus indicated that the HFD up‐regulated mRNA expression of several components of the renin‐angiotensin system and proinflammatory cytokines including renin, angiotensin type 1 receptor, angiotensin converting enzyme, TNF‐α and interleukin‐6. The results indicate that HFD‐induced sensitization of ANG II‐elicited hypertension is mediated by upregulation of central proinflammatory cytokines.
Caenorhabditis elegans displays different adaptations to single vs. multigenerational growth during spaceflight
Authors: Etheridge Tim , Higashibata Akira , Szewczyk Nathaniel , Higashitani Atsushi
Published: June 2021

Muscle atrophy is observed across species during spaceflight, however the sequence of molecular events that cause this phenomenon are not known. We flew liquid‐cultured C. elegans to the International Space Station, and assessed populations for movement rate before freezing on‐orbit at 4 and 8 d of flight. Post‐flight samples were analysed for genomic, proteomic, fat accumulation and targeted molecular changes versus 1G‐centrifuged flight samples. After 4 d spaceflight (first generation), animals displayed reduced movement rates versus 1G controls (1.53±0.64 vs. 2.61±0.12 Hz, respectively; P<0.01) and lowered whole‐body fat accumulation. Gene/protein expression profiles at 4 d flight showed reductions in several components involved in: a) the muscle contractile apparatus; b) cytoskeletal adhesion, and; c) mitochondrial function. This was accompanied by upregulation of daf‐16/foxo‐1 and sirt‐2.1/sirt‐1 and concurrent downregulation of sirtuin effectors abu‐6, abu‐7 and pqn‐5 (transmembrane components). However, second generation animals (8 d flight) displayed a return to 1G expression levels for all indices altered in the first generation. The exception was mitochondrial DNA content and associated gene expression, which remained depressed after 8 d flight. These results suggest that spaceflight‐sensitive mechanisms (insulin‐ and attachment‐mediated signalling) regulate an early metabolic downregulation, which recover in subsequent generations with lowered mitochondrial function remaining as a chronic adaptive response to microgravity. Such sustained mitochondrial impairment could reduce cellular energy sufficiently to account for chronic declines in muscle mass and function.
Cerebral Vascular Changes in Space Mice Calvaria
Authors: Siamwala Jamila , Macias Brandon , Healey Robert , Pichika Rajeshwari , Hargens Alan
Published: June 2021

Spaceflight induces headward fluid shifts which may remodel calvarial bone. Previous microCT studies from our lab showed that mice exposed to 15 days of microgravity had increased bone volume density and cortical thickness of calvaria compared to ground control mice. Bone formation is initiated by angiogenesis during normal bone development. VEGF, a key angiogenic molecule, interacts with BMP2 in bone formation and bone healing. Our hypothesis was that bone formation in space mice calvaria is accompanied by vascular changes.

Coronal sections from ground control (GC) and spaceflight (SF) mice (n=2) calvaria were stained with eosin and haematoxylin. Unstained sections were processed using a series of dehydration steps with xylene followed by rehydration with ethanol. The slides were then stained with lectin which binds to blood vessels. The vessels were counted manually from 6 sections (10 fields/slide, n=3). BMP‐2, VEGFA and iNOS gene expressions were quantified using real time PCR.

Our results document no difference in the number of vessels between GC and SF mice. However the diameter of the vessels (4 vessels/slide) measured using image pro was significantly lower in SF compared to GC mice (34.1 bfl vs 60.3 bfl, p<0.0001, t test). BMP‐2 expression increased 11 fold in SF (n=6, p<0.01, t test). However there was no significant change in VEGFA. Further there was iNOS gene expression in SF (0.3) compared to GC mice (1). These results corroborate that vascular changes observed in the space flight mice maybe partly responsible for increased bone observed in the SF mice. Future studies are required to study the mechanisms of bone‐vascular interaction and bone growth. Supported by Wood Whelan fellowship (JHS), NASA grants NNX09AP11G and NNX13AJ12G (ARH), NSBRI through NASA NCC 9‐58 (BRM).
Striking Gender‐based Differences in Bone Mineral Density in a Resource‐Limited Setting
Authors: Nell Theo , Kruger Maritza
Published: June 2021

The determination of bone health status is largely absent in South Africa due to the lack and cost of equipment required for the assessment of bone mineral density (BMD). Dual‐energy x‐ray absorptiometry (DEXA) ‐ also the gold standard evaluation, is too expensive for widespread implementation. In light of this, the current study evaluated an ultrasound bone densitometer (Sonost 3000 Co Ltd.) as a novel screening tool for BMD in a resource‐deprived setting such as South Africa. We screened the heel bones of 88 female and 38 male students (21‐23 years age) at Stellenbosch University by employing the Sonost 3000 that passes non‐radiating ultrasound through the calcaneus bone. Weight and height, together with age, gender and ethnicity were recorded to accurately determine bone health status. Similar degrees of osteopenia in male and female students (13% and 14%, respectively) were noted. In addition, age, broadband ultrasound attenuation (BUA – indicative of bone structure) and sun exposure did not differ between the two groups. However, male students displayed a significantly increased speed of sound (SOS; marker of bone density and elasticity), bone quality index (BQI), as well as T‐scores vs. females. Furthermore, males led a relatively active lifestyle (58% vs 41%) and lifted more weights daily (26% vs 10%) compared to females. This study demonstrates that male students displayed improved bone health status versus females, and that this is likely due to the pursuance of a more active lifestyle. Our findings also show that the Sonost 3000 offers early promise as a preliminary screening method to assess BMD in especially resource‐limited settings.