bluuebunny/crossref_metadata_2025_split huggingface dataset:
Modulating GABAA receptors that contain alpha 2/3 subunits corrects respiratory disturbances in a mouse model of Rett syndrome
Authors: Bissonnette John M , Knopp Sharon J 
Published: June 2021

We have recently shown that augmenting γ‐aminobutyric acid (GABA) A inhibition in a mouse model of Rett syndrome corrects periodic apneas and restores cycle regularity to that of wild type (2008 EB abstracts [on CD‐ROM], Abstract#1232.12). These mice are deficient in α2 subunits of GABAA receptors (J Neurophysiol 99:112, 2008). Here we show that allosteric modulation of GABAA receptors with a compound that is relatively specific for receptors that contain α2/3 subunits (L‐838,417, Nature Neurosci 3:587, 2000) is equally efficient as a broad‐spectrum benzodiazepine in correcting the respiratory disorder. Studies were performed in heterozygous methyl‐CpG‐binding protein 2 (Mecp2) deficient female mice with continuous monitoring of pleural pressure by telemetry. Following ip injection of L‐838,417 (20 mg/Kg) or vehicle respiratory pattern was examined for 3 hrs.Adjusting for differences in wake/sleep pattern from animal activity (Physiol Genomics 28:232, 2006) did not alter the effect of L‐838,417.Support: MOD #6‐FY‐314 and IRSF #0802
Mechanical Tissue Resuscitation (MTR) Reduces Myocardial Reperfusion Injury
Authors: Jordan James E , Morykwas Michael J , Mays Jennifer J , Thompson Edreca A , Argenta Louis C 
Published: June 2021

Treating injured tissue with negative pressure re‐establishes physiologic homeostasis and improves tissue survival. Injury sustained following reperfusion of ischemic myocardium involves processes similar to those seen after burns and other wounds. This study explores the possibility of using a mechanical device to achieve local resuscitation of myocardial tissue following acute myocardial infarction.Anesthetized swine were subjected to 75 min of left coronary artery occlusion and 3 hours of reperfusion. Animals were assigned to 1 of 3 groups: (1) control, treatment with (2) 50 mmHg or (3) 125 mmHg of MTR during reperfusion. All three groups were subjected to equivalent ischemic stress [area of risk (AAR) =12.9± 1.2%, 11.8±1.2% and 11.9±1.4%, respectively]. The application of MTR directly to the injured myocardium for 180 minutes significantly (p<0.001) reduced infarct size in both treatment groups compared to control: 9.3±1.8% (50 mm Hg) vs 26.4±2.1% (control); and 11.9±1.2% (125 mm Hg) vs 26.4±2.1% (control).Application of a controlled vacuum device directly to the injured myocardium for a defined period of time can achieve MTR and reduce tissue death after ischemia and reperfusion. This treatment strategy offers a simple, effective, clinically relevant, non‐pharmacologic approach to reducing myocardial injury following ischemia and reperfusion.
Age‐dependent cardiovascular responses to severe haemorrhage in conscious lambs
Authors: Samhan Mohamed A , Qi Wei , Smith Francine G 
Published: June 2021

The present study was designed to investigate cardiovascular responses to severe haemorrhage (exceeding the critical point at which compensatory mechanisms fail) in two age groups of conscious lambs (Group I: 1‐2 weeks, N=5; group II: 6‐7 weeks, N=6). Mean arterial pressure (MAP), systolic and diastolic pressures (SAP, DAP), heart rate (HR) and renal blood flow (RBF) were measured for 20 min before (Control, C) and after a fixed haemorrhage of 30% of calculated blood volume. After haemorrhage in group I lambs, MAP decreased from 75±5 [SD] (C) to 55±8 mmHg at 5 min, returning to 61±6 mmHg at 20 min, while in group II lambs, MAP decreased further from 74±4 (C) to 38±7 mmHg at 5 min, returning to 55±6 mmHg at 20 min. The fall in SAP 5 min after haemorrhage was also greater in group II lambs (104±8 (C) to 65±15 mmHg) than in group I lambs (110±14 (C) to 91±8 mmHg), whereas DAP followed changes in MAP in both groups. HR increased in group I lambs from 164±43 (C) to 193±45 at 5 min, decreasing thereafter, whereas in group II lambs, HR was unaltered from 119±20 (C). After haemorrhage, RBF fell to zero at 5 min in both groups, returning towards C at 20 min. These results provide new evidence that the cardiovascular responses to severe haemorrhage are developmentally regulated. Mechanisms underlying these differential responses are not yet known.This research was supported by the Canadian Institutes of Health Research (CIHR).
Comparison of plasma volume assessments following hemorrhagic shock in pigs
Authors: Colvin Shavaughn Kristin , Dubick Michael A , Martini Wenjun Z 
Published: June 2021

Indocyanine green (ICG) dye is frequently used for plasma volume (PV) measurements. However, the accuracy of ICG measurements following severe hemorrhage (H) is unclear due to limited knowledge of its metabolism when liver function is altered by H. This study assessed changes of PV in pigs after H and compared the ICG results with Hct and total protein. Sixteen pigs were divided into controls (n=4) or H with either LR (n=6) or saline (n=6) resuscitation. H was induced by withdrawing 60% of the estimated blood volume from the femoral artery, followed by resuscitation with LR or saline. ICG (25mg/10ml) was injected iv to assess PV at baseline and 6 h after H and resuscitation. Blood samples were taken before each injection for blood gas and chemistry. PV was also calculated based on the changes of Hct and total protein for comparison. There were no changes in PV in controls throughout the study. At 6 h following H and resuscitation, PV by ICG was 121±3% and 120±4% of baseline in the LR and saline groups, respectively. PV calculated by changes of Hct showed 119±1% and 119±2% of baseline in these groups, respectively. Similarly, PV calculated by changes of total protein was 121±6% and 124±3 % of baseline in these groups, respectively. In conclusion, these data suggest that following severe H, ICG remains a reliable method for measurements of PV.This work was supported by US Army Medical Research and Medical Command.
Sheep red cell survival (RCS) can be followed using 4 different densities of biotin label
Authors: Matthews Nell I , Mock Donald M , Strauss Ronald G , Burmeister Leon F , Schmidt Robert , Widness John A 
Published: June 2021

Anemia is a serious problem in critically ill neonates. To investigate anemia, to assess responses to red blood cell (RBC) transfusions and erythropoietin therapy, and to compare sources of RBCs and storage methods, simultaneous measurement of RCS would be useful. Here we report the validation in sheep (a common model for perinatal studies of physiology and erythropoiesis) of a method for independent measurement of RCS using 4 populations of RBCs labeled at distinct biotin densities.MethodsBy varying the biotinylating reagent from 12 to 96 ?g/mL RBC, the density of biotin on sheep RBCs was incrementally increased to produce 4 discrete populations. Biotinylated RBCs (BioRBCs) were reinfused, and blood was sampled over the next 4 months. RCS was determined using flow cytometric enumeration and by scintillation counting.ResultsBioRBCs produced parallel disappearance curves with life spans from the linear phase that were similar to the reference 14C‐cyanate method (mean potential lifespan = 66±2 vs 62 d).ConclusionRCS can be measured accurately and simultaneously in sheep using BioRBCs without radiolabeling. NIH P01 HL046925; Thrasher Research Foundation 02825‐3.
Characterization of ATP uptake into isolated pancreatic zymogen granules
Authors: Haanes Kristian Agmund , Novak Ivana 
Published: June 2021

Our previous bioimaging studies on pancreatic acinar cells indicated that ATP is stored in granules. Present study aimed to quantify ATP content and characterize ATP uptake into Zymogen Granules (ZG). ZGs were isolated from rat pancreas and stained with Rab3D antibody to confirm identity as described by Chen et al. (Mol. Cell. Proteomics 5(2):306, 2006). ATP and protein were measured using Luciferin/Luciferase and Bradford assays. ATP concentrations in ZG were calculated assuming that ZG protein concentration is 592 mg/ml, as determined by Burnham et al. (Biochem. J. 227(3):743, 1985). Results have shown that the freshly isolated ZGs contained an ATP concentration of 55 ± 7 μM (n=6). Furthermore, ATP uptake into ZGs was characterized using incubation with different ATP concentrations in a buffer (4 mM KCl, sucrose, MES EGTA, pH 6). A Km of 1.4 ± 0.6 mM and a Vmax of 280 ± 60 μM/granule/30 min was calculated using a Eadie‐Hofstee plot (n=5). Loading of 4 mM ATP resulted in ZG ATP concentration of 211 ± 17 μM/30 min. However, when 100 μM DIDS was added, ATP uptake was abolished, i.e. 0 ± 10 µM/granule/30 min (n=7). Bafilomycin, CCCP and Valinomycin did not appear to have significant effects in these assay conditions. In conclusion, this study shows the first quantitative measurements of ATP concentration in zymogen granules and it characterizes a DIDS‐sensitive ATP uptake mechanism.Support: DNSRC 272‐05‐0420
Modulation of folic acid uptake into BeWo cells by cannabinoids
Authors: Araújo João Ricardo , Gonçalves Pedro , Martel Fátima 
Published: June 2021

The aim of this study was to investigate the influence of cannabinoids (CB) on the uptake of folic acid (FA) by the choriocarcinoma cell line (BeWo cells). Acutely, anandamide caused a 15% decrease in 3H‐FA uptake (pH 7.5). Moreover, tetrahydrocannabinol (THC) caused a 30% increase, and AM630 a 15% decrease (pH 6.5). Neither the inhibitory effect of anandamide nor the stimulatory effect of THC were changed by the CB receptor type 1 or type 2 antagonists (AM251 and AM630, respectively). Chronically (48 h), THC and AM251 decreased by 20% the uptake of 3H‐FA, and anandamide and AM630 increased it by 30% (pH 7.5). Moreover, CP 55,940 increased uptake of 3H‐FA by 30% (pH 6.5). RT‐PCR analysis showed that the mRNA levels of the Reduced Folate Transporter 1 increased by 9% after chronic treatment with AM630, and the mRNA levels of the Proton‐coupled Folate Transporter decreased by 17% and increased by 18% after chronic treatment with THC and AM251, respectively. In conclusion, uptake of 3H‐FA by BeWo cells is significantly, although not very markedly, changed by several distinct CB receptor agonists and antagonists, either after acute and after chronic exposure of the cells. The acute effects of CB receptor agonists do not seem to be CB receptor‐mediated, and with a few exceptions the chronic effects do not seem to be related to changes in the expression levels of FA transporters.Supported by FCT (PTDC/SAU‐FCF/67805/2006)
SMALL INTESTINAL GLUCOSE ABSORPTION AFTER GASTRIC BYPASS SURGERY.
Authors: Shah Hiral , Bal Bikram , Frederick Finelli , Koch Timothy R. 
Published: June 2021

In small intestine (SI), a Na‐dependent glucose transporter is at apical epithelial cells; a diffusion glucose transporter is at the basolateral membrane. Obese Zucker rats have decreased glucose absorption in SI compared to lean rats (J Nutrition, 1993). After gastric bypass surgery (GBS), small intestinal bacterial overgrowth (SIBO) induces thiamine deficiency (Nutrition Res, 2008). Our hypothesis is that SIBO alters glucose transport in the morbidly obese.METHODSWe reviewed GBS patients from 1999‐2008 with glucose breath testing (GBT). SIBO is defined by: fasting breath H2 or CH4 at time 0 or rise of >10 ppm </= 30 minutes after 50 grams oral glucose; colonic bacteria defined by: breath H2 or CH4 rise of >10 ppm at >/= 45 minutes after glucose.RESULTSStudy includes 64 female & 5 male patients, ages 22‐67 years (mean: 48 years). 54 of 69 patients (78%) had a rise in H2 or CH4 at >/= 45 minutes. In these 54, 37 (69%) are H2 producers and 17 (31%) are CH4 producers. Only 31 (57%) of the 54 patients had evidence for SIBO.CONCLUSIONSThe delayed rise in breath H2 or CH4 in 78% of GBS patients is due to fermentation of glucose by colonic bacteria. This supports our hypothesis that morbidly obese patients have decreased SI glucose transport. We can not differentiate between a defective glucose transporter at an apical or basolateral location. SIBO does not explain this defect, and so it may represent a homeostatic mechanism.
hGLUT9 as a novel urate transporter: its role in liver urate handling and functional study of SLC2A9 SNPs
Authors: Witkowska Kate , Smith Kyla , Karpinski Ed , Young James , O'Neill Debbie , Cheeseman Chris 
Published: June 2021

In humans, urate is the final product of purine breakdown in the liver which releases urate into the circulation and the intestine and kidney regulate its excretion. Humans have a high circulating level of urate and small deviations are associated with a variety of metabolic diseases. A genome‐wide association scan correlated SNPs within the SLC2A9 gene, for the hexose transporter GLUT9, with hyperuricemia. This resulted in the identification of this protein as a high capacity urate transporter, which can exchange intracellular urate for extracellular glucose (1). Urate is an organic anion and voltage clamp experiments with GLUT9 expressed in Xenopus oocytes showed that its transport is electrogenic. Immunohistochemistry showed that all of the GLUT9 SNP mutants were expressed in the oocyte plasma membrane and flux studies indicate that all are functional. GLUT9 is expressed in the hepatocyte basolateral membrane and these data support the hypothesis that GLUT9 mediates the efflux of urate down its electrical gradient. Further, in the absence of direct functional effects of the SNPs, it appears that more complex effects of these mutations, perhaps on cytoskeletal interactions or yet to be identified regulatory mechanisms, affect the activity of GLUT9.1. Mark J. Caulfield, et al (2008) SLC2A9 is a high capacity urate transporter in man. PLoS Medicine 5(10):e197.Supported by the Canadian Breast Cancer Foundation.
Elevated Hydrostatic Pressure Activates Sodium‐Hydrogen Exchanger‐1 in Rat Optic Nerve Head Astrocytes
Authors: Mandal Amritlal , Shahidullah Mohammad , Delamere Nicholas A , Teran Marcos A 
Published: June 2021

ObjectiveOptic nerve head (ONH) astrocytes are thought to involve in the degeneration of ganglion cells and remodeling of surrounding extracellular matrix in glaucoma. Elevated intraocular pressure (IOP) is the main glaucoma risk factor. Here we report altered sodium hydrogen exchange (NHE) activity in cells exposed to elevated hydrostatic pressure .MethodsHydorstatic pressure (HP) of 15 mmHg was applied to the cells in a 20 cm glass cylinder filled with the culture medium equilibrated with 5% CO2/95% air for 2h. Proteins were analyzed by Western blot and cytoplasmic pH (pHi) was measured by BCECF ratiometry.ResultsElevated HP causes phosphorylation of ERK1/2, ribosomal S6 kinase (p90RSK), and Na‐H exchanger‐1 (NHE1). The MEK/ERK inhibitor UO126 abolished phosphorylation of NHE1 and p90RSK as well as ERK1/2. Although baseline pHi was unaltered in pressure treated cells, the rate of pHi recovery from ammonium chloride acidification was four fold higher. Dimethylamiloride (DMA), an NHE inhibitor, abolished and U0126 partially inhibited the increased rate of pHi recovery in pressure treated astrocytes.ConclusionsThe findings suggest elevated HP caused NHE‐1 activation through phosphorylation following activation of ERK1/2 and p90RSK. These events may lead to the long term changes of protein expression known to occur in pressure‐stressed astrocytes.FundingNIH Grant EY014069
Involvement of Na+/HCO3‐ Cotransporter in Intracellular pH Regulation and Anion Secretion in Cultured Rat Epididymal Epithelia
Authors: Zuo Wulin , Li Sheng , Zhang Geng , Ruan Yechun , Zhou Wenliang 
Published: June 2021

Intracellular pH(pHi) plays a critical role in many intracellular processes. Several ion transporters, including Na+/H+ exchanger (NHE), Na+/HCO3‐ cotransporter(NBC) and vacuolar H+‐ATPase(H+‐ATPase), contribute to pHi regulation in different types of cells. The present study examined the mechanisms of pHi regulation by NBC in cultured caput epididymal epithelia by using the fluorescent pH sensitive probe, SNARF‐4F. In the HEPES buffered Krebs‐Henseleit(KH) solution, a pHi recovery from NH4Cl induced acidification was completely inhibited by amiloride, the inhibitor of NHE. Then immediate change of the KH solution from HEPES buffered to HCO3‐ buffered would cause another pHi recovery. The pHi recovery in HCO3‐ buffered KH solution was inhibited by 4, 4'diisothiocyanatostilbene‐2, 2′‐disulfonic acid (DIDS) or removal of extracellualr Na+. However, the pHi recovery was not affected by removal of extracellular Cl‐. In addition, the short circuit current measurement showed that adding DIDS to the basolateral side of epididymal epithelial monolayer could inhibit adrenalin induced anion secretion. In summary, these results indicate that not only does NBC play a functional role in pHi regulation of epididymal epithelia, but also regulate the epididymal fluid microenvironment by affecting anion secretion.This work was supported by the National Natural Science Foundation of China (No.30770817)
Extracellular ATP synthesis regulates ATP release and bicarbonate secretion in rat duodenum
Authors: Watanabe Chikako , Akiba Yasutada , Nakano Takanari , Kaunitz Jonathan D 
Published: June 2021

The pH of the alkaline microclimate overlying duodenal enterocyte brush border is regulated by an ecto‐purinergic signaling; intestinal alkaline phosphatase (IAP), extracellular ATP and P2Y receptors regulate duodenal bicarbonate secretion (DBS). IAP inhibition increases DBS and non‐lytic ATP release into the lumen by the mechanism unknown. Ecto‐F1F0‐ATP synthase has been localized to plasma membranes of several cell types. We hypothesized that ecto‐ATP synthase generates extracellular ATP, regulating DBS.We measured DBS with flow‐through pH and CO2 electrodes with perfusate ATP content measured by luciferin‐luciferase bioassay. We perfused the competitive AP inhibitor glycerol phosphate (GP) with ATP synthase inhibitors oligomycin (Om), piceatannol (Pic) or resveratorol (Res), or the uncoupler CCCP.GP increased DBS and ATP output. Pic or Res inhibited GP‐induced augmented DBS and ATP output. GP, Pic or Res with GP had no effect on LDH release, confirming no cellular injury. Om or CCCP inhibited GP‐induced ATP output with increased LDH release, consistent with cell injury.Luminal ATP release unmasked by IAP inhibition is dependent on ecto‐ATP synthase activity, consistent with the presence of ecto‐ATP synthase on the brush border. Ecto‐ATP synthesis and ATP degradation by IAP may coordinately regulate ATP‐P2Y signaling for DBS as part of a novel ecto‐purinergic signaling system.VA, NIH‐NIDDK
Cysteines in Extracellular Loop 3 of NBCe1‐A Form Intra‐ and Inter‐Molecular Disulfide Bonds
Authors: Zhu Quansheng , Azimov Rustam , Kao Liyo , Pushkin Alexander , Kurtz Ira 
Published: June 2021

NBCe1‐A cotransports sodium and bicarbonate across the basolateral membrane of the proximal tubule. Four cysteines in the putative NBCe1‐A extracellular loop 3 are conserved among all the SLC4 family sodium coupled bicarbonate transporters. To determine the structural importance of these conserved cysteines, we explored the potential disulfide bonding among the 4 cysteine residues. NBCe1‐A constructs bearing various cysteine mutations were expressed in HEK‐293 cells and examined for the presence of disulfide bonds with the cysteine accessibility method (CAM). Initially we analyzed the aqueous accessibility of the 5 endogenous cytoplasmic cysteines. The results revealed that C120 and C399 are not exposed to the aqueous medium, whereas C389, C992 and C1035 are highly exposed, suggesting that the N‐terminal cytoplasmic region of NBCe1‐A is structured. Mutation of the cytoplasmic cysteines to serines eliminated any free aqueous accessible cysteines indicating that cysteines 583, 585, 630 and 642 in extracellular loop 3 partake in either intra‐ or inter‐monomeric disulfide bonding. Mutation of both cysteine 583 and 585 to serine did not free any cysteines, however, upon treatment with 5mM DTT, free cysteines were detected. Further analysis on mutants where 3 of the 4 cysteines were replaced with serine: 583/585/630 or 583/585/642 showed that the remaining cysteine (642 or 630) in each construct could only be freed after DTT treatment. In conclusion, our data suggests that cysteine 583 and 585 form an intra molecular disulfide bond, whereas cysteine 630 and 642 each form disulfide bonds with the same residue on the opposite monomer, and therefore appears to play an important role in the oligomerization of the cotransporter.Supported by NIH
Stoichiometry and Inhibitory Pharmacology of Electrogenic Sodium Bicarbonate Cotransporter NBC4c (NBCe2‐C) Expressed in HEK‐293 Cells
Authors: Shao Xuesi , Kao Liyo , Abuladze Natalia , Kurtz Ira 
Published: June 2021

NBC4c is an electrogenic sodium bicarbonate cotransporter encoded by SLC4A5. To examine the electrophysiologic properties of NBC4c in a mammalian system, HEK‐293 cells permanently expressing the cotransporter were whole‐cell patch‐clamped. When the patch pipette and bath solutions contained 0 mM HCO3−, increasing [HCO3−]o from 0 to 25 mM induced a mean outward current of 48.4 pA at +45 mV that was blocked by DIDS and tenidap. The inhibitory profiles of DIDS and tenidap were fit to the equation I/I0=1/(1+(C/Ki)n), where Ki is the inhibitory constant, C is the concentration and n is the pseudo Hill coefficient. Tenidap was more potent than DIDS. Tenidap inhibition was voltage dependent with reduced inhibition at more positive voltages. At known intracellular and extracellular Na+ and HCO3− concentrations, HCO3− to Na+ stoichiometry (q) could be calculated with the equation: Erev = RT/(F(q‐1))ln(([Na+]i/[Na+]o)([HCO3−]i/[HCO3−]o)q) where reversal potential Erev (VI=0) was determined from the DIDS‐inhibitable I‐V curves from ‐95 mV to +45 mV. Erev was measured in two independent experiments where [HCO3−]i and [HCO3−]o were equal (25 mM), therefore Erev depended only on [Na+]i/[Na+]o. [Na+]i/[Na+]o values were chosen so that Erev would be significantly different when q = 2 or q = 3 predicted with the above equation. In the first protocol, using [Na+]i/[Na+]o = 40/80 mM, Erev = ‐22.2 ± 3.87 mV. In the second protocol using [Na+]i/[Na+]o = 25/135 mM, Erev = ‐40.5 ± 5.12 mV. These Erev values are close to the calculated values of ‐17.8 and ‐43.3 mV, respectively, assuming q = 2. Our data indicate that NBC4c expressed in HEK‐293 cells transports sodium bicarbonate electrogenically with a stoichiometric ratio of 2 HCO3−: 1 Na+. NBC4c is blocked by DIDS and tenidap with different mechanisms.Supported by NIH
Nox2 is required for amiloride‐sensitive H+ efflux in a rat medullary thick ascending limb cell line
Authors: O'Connor Paul M , Fang Yi , Feng Di , Liu Yong , Liang Mingyu , Ferreri Nicholas , Cowley Allen W 
Published: June 2021

We have recently demonstrated that an amiloride (NMA)‐sensitive outward H+ current is present in mTAL of salt‐sensitive (SS) but not salt‐resistant (SS.13BN) rats and that when activated, this stimulates the production of excess superoxide (O2−). The identity of the H+ transporter remains unclear, however it displays many of the characteristics of a nox2 associated H+ current characterized in macrophages. The aim of this study was to determine which subunits of NAD(P)H oxidase are expressed in a rat mTAL cell line and whether a similar H+ transport pathway exists in these cells. mTAL cells were cultured, transfected with nox2, loaded with BCECF dye, imaged on a inverted microscope and intracellular pH (pHi) recovery determined following an NH4Cl prepulse in 0 Na+ solution with BaCl2. pHi recovery following an NH4Cl prepulse was absent in control transfected mTAL (δpHi/sec ‐1.9±2*10−5) however, in mTAL transfected with nox2, amiloride‐sensitive pHi recovery could be detected (δpHi/sec 7.9±3.2*10−5 vs ‐1.6±2*10−5 with NMA; P<0.03). Cellular mRNA expression of endogenous NAD(P)H oxidase subunits are shown in the table below. We conclude that although nox4 is the primary gp91 homolog expressed in mTAL cells, nox2 over‐expression is required to produce the outward NMA‐sensitive H+ current observed in SS mTAL.mTAL copy # per 106 18S RNA (n=4)